Medical Physiology, 3rd Edition

Physiology of Voltage-Gated Channels and Their Relatives

A large superfamily of structurally related membrane proteins includes voltage-gated and related channels

Voltage-gated Na+ channels, Ca2+ channels, and K+ channels are part of a superfamily of channel proteins called the voltage-gated–like (VGL) ion channel superfamily (see Table 6-2). This superfamily also includes structurally related channels that are not strictly activated by voltage. Figure 7-9 shows a dendrogram based on an analysis of evolutionary relationships of the minimal pore regions of 143 human channels belonging to the VGL superfamily. Major branches of the tree define groups of related channel genes present in the human genome. In this section, we discuss how structural relationships among these proteins determine their physiological functions.


FIGURE 7-9 Family tree of hypothetical evolutionary relationships among voltage-gated cation channels represented in the human genome, based on sequences of the pore domain. This dendrogram of the superfamily of VGL channels (see Table 6-2) shows distinct branches colored to indicate various tetrameric K+ channels (Kv, KCa, Kir, K2P) in red, CNG and HCN channels in pink, and TRP channels in green. CaM, calmodulin; CNBD, cyclic nucleotide–binding domain. A separate branch includes the pseudotetrameric Cav and Nav channels in blue. (Data from Yu FH, Catterall WA: The VGL-chanome: A protein superfamily specialized for electrical signaling and ionic homeostasis. Sci STKE 2004:re15, 2004.)

Initial progress toward biochemical characterization of the voltage-gated ion channels responsible for the action potential began with the discovery of naturally occurring, specific, high-affinity neurotoxins such as TTX and STX and their use as biochemical probes. Tritium-labeled derivatives of TTX and STX were prepared chemically and used in radioligand-binding assays to directly measure the number of voltage-gated Na+ channels in excitable tissues.

The electroplax organ of the electric eel Electrophorus electricusimageN7-9 proved to be a convenient source of tissue for the first successful biochemical purification of the Na+ channel protein by William Agnew and coworkers in 1978. These Na+ channels consist of a large glycosylated α subunit of ~200 kDa that contains the TTX-binding site. Reconstitution experiments revealed that the α subunit—by itself—is the channel-forming protein that mediates ionic selectivity for Na+, voltage-dependent gating, and pharmacological sensitivity to various neurotoxins. Similar approaches with skeletal muscle and brain led to the identification of analogous mammalian Na+ channel α subunits, which are protein products of related genes.


Electroplax Organ of the Electric Eel

Contributed by Ed Moczydlowski

The electroplax organ of the electric eel (Electrophorus electricus) is composed of specialized cells called electrocytes that are an evolutionary adaptation of skeletal muscle cells. The innervated membrane face of the electrocytes contains a high density of both nicotinic acetylcholine receptors and voltage-gated Na+ channels. Thus, this tissue is a rich source of both proteins. Indeed, for both proteins, this tissue played a key role in the purification, biochemical characterization, reconstitution into lipid membranes, and physiological characterization.

In addition to the α subunit, the functional complex of the rat skeletal muscle Na+ channel contains a 38-kDa subunit, and the rat brain Na+ channel contains both a 33- and a 36-kDa subunit. These smaller β subunits of mammalian Na+ channels contain a single transmembrane span and appear to play an accessory role in modulating channel gating or channel expression. In humans, four genes (SCN1B to SCN4B) encode β subunits—β1 to β4—that preferentially associate with different α subunits in different tissues.

Molecular biological studies of voltage-gated channels began in 1984 with the cloning of the Electrophorus Na+ channel α subunit by the laboratory of Shosaku Numa. These investigators used antibodies raised against the purified α subunit to screen a complementary DNA (cDNA) library, and they isolated the cDNA encoding the electroplax Na+ channel. In addition, direct sequencing of channel peptides provided partial amino-acid sequence information for confirmation. Similar strategies led to the purification and cloning of voltage-gated Ca2+ channel proteins from skeletal muscle and brain tissue. The primary sequence of the α1 subunit of the Ca2+ channel is structurally homologous to the α subunit of the Na+ channel.

Whereas a biochemical approach was used to discover Na+ and Ca2+ channels, the initial breakthrough in the molecular biology of K+ channels came with the study of Shaker mutants of the fruit fly Drosophila. These mutants are called Shaker because their bodies literally shake under the influence of ether anesthesia. This phenotype is due to defective voltage-gated K+ channels. The laboratory of L.Y. Jan and Y.N. Jan, and those of O. Pongs and M. Tanouye, used molecular genetic techniques to identify and clone the first K+ channel genes in 1987.

For voltage-gated K+ channels, plots based on the hydropathy index of each amino acid (see Table 2-1) typically reveal six distinct peaks of hydrophobicity (Fig. 7-10A), corresponding to transmembrane segments S1 to S6—a conserved structural feature of all voltage-gated K+ channels. Transmembrane segments S1 to S6 have an α-helical secondary structure and are connected by cytoplasmic and extracellular linker regions (see Fig. 7-10B).


FIGURE 7-10 Membrane topology model of a single subunit of a voltage-gated K+ channel. A, This voltage-dependent K+ channel, a member of the Shaker family (Kv1.1), has six transmembrane segments (S1 to S6) with a high hydropathy index. Each of these six segments (highlighted in green or yellow) is presumed to traverse the membrane completely. In addition, the channel also has a smaller region (highlighted in red) with a somewhat lower hydropathy index, termed the P region. B, This topology model is based on the hydropathy data in A. The six membrane-spanning segments are assumed to be α helices. The S4 segment (highlighted in yellow) has a large number of positively charged lysine and arginine residues and is part of the voltage-sensing domain that comprises the entire S1 to S4 region. S5 and S6—as well as the intervening P region—comprise the S5-P-S6 pore domain (see Fig. 7-11). imageN7-13 (Data from Shen NV, Pfaffinger PJ: Conservation of K+ channel properties in gene subfamilies. In Peracchia C [ed]: Handbook of Membrane Channels: Molecular and Cellular Physiology. New York, Academic Press, 1994, pp 5–16.)

Extensive mutagenesis studies on cloned channel genes have associated various channel functions and binding properties with particular domains. The amino-terminal part of the channel, including the S1 to S4 transmembrane segments, forms a voltage-sensing domain (see Fig. 7-10B). The S4 segment has four to seven arginine or lysine residues that occur at every third S4 residue in voltage-gated K+, Na+, and Ca2+channels. The positively charged S4 segment acts as the voltage sensor for channel activation by moving outward when the membrane depolarizes. This movement causes the four S6 helices—which form the inner lining of the pore—to bend away from the pore axis, thereby opening the channel.

The extracellular linker region between the S5 and S6 segments is termed the P region (for pore region) and contains residues that form the binding sites for toxins and external blocking molecules such as TEA. The pore domain formed by S5-P-S6 is the minimal structure required to form an ion-conducting pore for this class of channels (see Fig. 7-10B). The P region also contains conserved residues that form the selectivity filter, which determines the ionic selectivity for permeant cations. imageN7-10


Selectivity Filter

Contributed by Ed Moczydlowski

Evidence from mutational and structural analysis has revealed the basic mechanism by which voltage-gated K+, Na+, and Ca2+ channels discriminate permeant ions. In the case of Kv K+ channels, four selective binding sites for K+ are formed within cages of eight oxygen atoms from the peptide backbone and hydroxyl groups of Thr (see Table 2-1 for amino-acid code). In Ca2+ channels, the carboxyl groups from the side chains of four negatively charged Glu residues—called the EEEE locus—form two binding sites for Ca2+. Na+ channels have a selectivity filter similar to that of Ca2+ channels, but contain a conserved DEKA locus, consisting of Asp, Glu, Lys, and Ala residues.

Since the discovery and recognition of diverse genes belonging to the voltage-gated channel superfamily, structural-biological studies have substantially advanced our understanding of the three-dimensional structure of certain channel proteins. imageN7-11 In 1998, a major breakthrough in elucidating the structure of ion channel proteins occurred when MacKinnon and colleagues reported the crystal structure of a bacterial K+ channel protein called KcsA. This work revealed the three-dimensional structure of a protein that contained segments analogous to the S5-P-S6 part of voltage-gated channels, which forms the ion conduction pathway. For his work on the structural biology of ion channels, Roderick MacKinnon shared the 2003 Nobel Prize in Chemistry. imageN7-12


Crystal Structures of Ion Channel Proteins

Contributed by Ed Moczydlowski

The accompanying table summarizes data for 12 ion channel proteins that have been crystallized.

Crystal Structures of Ion Channel Proteins


Summary Notes

PDB Structure File




GenBank Accession


1. KcsA

First crystal structure of a tetrameric K+ channel pore domain revealing the basis of K+ selectivity. Recognized with the 2003 Nobel Prize in Chemistry.


Doyle et al: Science 280:69–77, 1998

3.2 Å



Streptomyces lividans

KcsA high resolution

Higher-resolution KcsA structure complexed with Fab antibody fragments showing the location of 7 binding sites for K+.


Zhou et al: Nature 414:43–48, 2001

2.0 Å




KcsA full length

Structure of full-length KcsA showing the cytoplasmic 4-helix bundle that was missing in previous structures.


Uysal et al: Proc Natl Acad Sci U S A 106:6644–6649, 2009

3.8 Å




2. KirBac1.1

Crystal structure of a tetrameric prokaryotic inward-rectifier K+ channel. Shows the location of negatively charged residues in the cytoplasmic C-terminal vestibule domain that play a role in intracellular block by Mg2+ and polyamines.


Kuo et al: Science 300:1922–1926, 2003

3.65 Å



Burkholderia pseudomallei

3. KirBac1.3-Kir3.1 chimera

Structure of a tetrameric inward-rectifier K+ channel chimera with a prokaryotic pore domain and a cytoplasmic vestibule domain of Kir3.1 from mouse. Shows location of positively charged residues at the intracellular membrane interface that play a role in PIP2 lipid activation.


Nishida et al: EMBO J 26:4005–4015, 2007

2.20 Å



Burkholderia xenovorans (KirBac1.3) and Mus musculus (Kir3.1)

4. Kir2.2

Kir channel from chicken is 90% identical to human ortholog. Possible binding sites for Mg2+ are identified from crystals grown in the presence of various inorganic cations. Additional structures show location of PIP2 lipid-binding sites.


Tao et al: Science 326:1668–1674, 2009
Hansen et al: Nature 477:495–498, 2011

3.1 Å



Gallus gallus

5. Kv1.2

First structure of a tetrameric mammalian voltage-gated K+ channel showing the structure of the voltage-sensor domains and the cytoplasmic β subunit.


Long et al: Science 309:897–903; 309:903–908, 2005

2.9 Å



Rattus norvegicus

6. KvAP

Structure of a tetrameric voltage-gated K+ channel from a thermophilic archaebacterium with voltage sensors in a non-native conformation.


Jiang et al: Nature 423:33–41, 2003

3.2 Å



Aeropyrum pernix


Additional structure of KvAP with information on variable orientation of the pore domain to the loosely adherent voltage-sensor domains.


Lee et al: Proc Natl Acad Sci U S A 102:15441–15446, 2005

3.9 Å




7. MthK

Structure of a tetrameric prokaryotic Ca2+-activated K+ channel showing the intracellular gating ring composed of two RCK domains.


Jiang et al: Nature 417:515–522; 417:523–526, 2002

3.3 Å



Methanobacterium thermoautotrophicum

8. BKCa channel

Structure of intracellular gating ring of the large-conductance human Ca2+-activated K+ channel (HSlo, BKCa) provides insight into the mechanism of Ca2+ activation.


Yuan et al: Science 329:182–186, 2010
Wu et al: Nature 466:393–397, 2010

3.0 Å




9. NaK

Structure of a tetrameric prokaryotic Na+,K+-nonselective cation channel with a TVGDG (named for the five single-letter amino-acid codes) selectivity filter resembling that of cyclic nucleotide–gated channels.


Shi et al: Nature 440:570–574, 2006

2.8 Å



Bacillus cereus


High-resolution structures of NaK channel in the open state revealing changes involved in gating of the pore. Complexes with different inorganic cations reveal mechanisms involved in ion selectivity.


Alam & Jiang: Nat Struct Mol Biol 16:30–34; 16:35–41, 2009

1.8 Å




10. TRAAK (TWIK-related arachidonic acid–stimulated K+ channel)

Structure of a dimeric two-pore K+-leak channel activated by arachidonic acid and mechanical deformation of the membrane. Structure shows a unique 35-Å-tall helical cap topping the extracellular entrance to the pore creating a bifurcated entryway for K+.


Brohawn et al: Science 335:436–441, 2012

3.8 Å



Homo sapiens

11. NaAb

Structure of a prokaryotic tetrameric homolog of voltage-gated Na+-selective channels shows features of the selectivity filter that distinguish Nav and Cav channels from K+ channels. Additional structures show changes associated with inactivation gating.


Payandeh et al: Nature 475:353–358, 2011
Payandeh et al: Nature 486:135–139, 2012

2.7 Å



Arcobacter butzleri

12. NavRh

Structure of bacterial tetrameric homolog of voltage-gated Na+ channels showing details of ion selectivity and inactivation gating.


Zhang et al: Nature 486:130–134, 2012

3.05 Å



alpha proteobacterium, Rickettsiales species

PDB, Protein Data Bank.


Roderick MacKinnon

For more information about Roderick MacKinnon and the work that led to his Nobel Prize, visit (accessed October 2014).

In 2005, the MacKinnon laboratory solved the structure of a mammalian voltage-gated K+ channel containing both the S1 to S4 voltage-sensing domain and the S5-P-S6 pore domain (Fig. 7-11). imageN7-13


FIGURE 7-11 Crystal structure of the mammalian K+ channel, Kv1.2, at a resolution of 2.9 Å. A, Four α subunits of the channel, each in a different color, viewed from the extracellular side. A K+ ion is shown in the central open pore. B, Side view of the four α and four β subunits of the channel, each in a different color, with extracellular solution on the top and intracellular solution on the bottom. The transmembrane domain (TM) of each α subunit is preceded by an –NH2 terminus (T1 domain). The T1 domain is located over the intracellular entryway to the pore but allows access of K+ ions to the pore through side portals. The T1 domain is also a docking platform for the oxidoreductase β subunit. Each β subunit is colored according to the α subunit it contacts. C, Side view of one α subunit and adjacent β subunit. Transmembrane segments are labeled S1 to S6. Tetramers of segments S5, pore helix, and S6 constitute the conduction pore in the shape of an inverted “teepee.” The selectivity filter lies in the wide portion (extracellular end) of the teepee. Helices S1 to S4 constitute the voltage sensors that are connected by a linker helix (S4-S5) to the pore. The PVP sequence (Pro-Val-Pro) on S6 is critical for gating. (From Long SB, Campbell EB, MacKinnon R: Crystal structure of a mammalian voltage-dependent Shaker family K+channel. Science 309:897–903, 2005.)


Crystal Structures of a Vertebrate K+ Channel

Contributed by Ed Moczydlowski

In 2005, the MacKinnon laboratory solved the crystal structure of a rat voltage-gated K+ channel called Kv1.2, which is homologous to the Drosophila Shaker channel and human Kv1 K+ channels that function in repolarization of nerve and muscle action potentials. This structure, which shows the channel in an open state, reveals that the S1 to S4 domain containing the voltage-sensing S4 element is spatially separated from the K+ pore domain (S5-P-S6). The tetrameric Kv1.2 channel has a pinwheel shape when viewed from the extracellular surface (see Fig. 7-11A). The central square portion of the Kv1.2 pinwheel is the pore—formed by the assembly of four S5-P-S6 pore domains, one from each monomer—and closely resembles the entire bacterial KcsA imageN7-22 channel. The four wings of the pinwheel correspond to the four S1 to S4 voltage-sensor domains. The four Kv1.2 monomers (yellow, green, blue, and red in Fig. 7-11A) form an interlinked assembly in which the S1 to S4 voltage-sensing domain of any given monomer lies closest to the S5-P-S6 domain of an adjacent monomer.

A lateral view of Kv1.2 shows an intracellular T1 domain formed by the four N-terminal segments of the channel (see Fig. 7-11B). The T1 domain of Kv channels is also called the tetramerization domain because it helps assemble and maintain the tetrameric structure of the channel. This view also shows four separately attached intracellular β subunits (see p. 183). These β subunits of Kv channels are part of a separate gene family of soluble accessory proteins with structural homology to oxidoreductase enzymes. Certain variants of both the T1 domain and β subunits may contain an N-terminal inactivation peptide that produces the rapid N-type inactivation (ball-and-chain mechanism) of some Kv channels by plugging the intracellular entrance to the pore.

Figure 7-11C shows a lateral view of a single Kv1.2 monomer in an open configuration as well as a single β subunit. On depolarization, the S4 segment moves within the membrane toward the extracellular side of the membrane. This mechanical movement of the S4 segment shifts an α-helical S4-S5 linker, causing a bending of the S6 transmembrane α helix from a linear configuration in the closed state to a curved configuration in the open state of the channel shown. Thus, voltage-dependent channel activation is an electromechanical coupling mechanism.

Other important features of K+ channel function are revealed by crystal structures (not shown) of a Kir2.2 inward-rectifier K+ channel from chicken and the C-terminal intracellular gating ring of the BKCa(KCa1.1) large-conductance Ca2+-activated K+ channel. Kir2.2 structures show the location of the PIP2 lipid-binding site in the inner leaflet of the lipid bilayer at the interface between the pore domain and the cytoplasmic domain. PIP2 stabilizes the interaction of these two protein domains to favor opening of the channel, corresponding to the mechanism of activation of Kir channels by PIP2.

Crystal structures (not shown) of the intracellular gating ring domain of the human BKCa channel show a tetrameric assembly of two tandem C-terminal regulator of K+ conductance domains (RCK1 and RCK2 domains). A region of the RCK2 domain rich in Asp residues called the calcium bowl forms four Ca2+ binding sites, one from each BKCa channel subunit. Binding of Ca2+ to these sites located at the interface between RCK domains facilitates conformational changes of the gating ring that favor channel opening. Detailed structural analyses of channel proteins help us to understand how genetic defects and drugs affect channel function.


Hoshi T, Lahiri S. Oxygen sensing: It's a gas!. Science. 2004;306:2050–2051.

Schnermann J, Chou C-L, Ma T, et al. Defective proximal tubular fluid reabsorption in transgenic aquaporin-1 null mice. Proc Natl Acad Sci U S A. 1998;95:9660–9664.

Williams SEJ, Wootton P, Mason HS, et al. Hemoxygenase-2 is an oxygen sensor for a calcium-sensitive potassium channel. Science. 2004;306:2093–2097.


Crystal Structure of the KcsA K+ Channel

Contributed by Ed Moczydlowski

In 1998, the laboratory of Roderick MacKinnon at Rockefeller University used x-ray diffraction to solve the three-dimensional crystal structure of a membrane protein known as KcsA. KcsA is the protein product of a gene from the actinomycete bacterium Streptomyces lividans. KcsA is homologous to the S5-P-S6 region of the Shaker K+ channel and is known to function as a K+ channel in planar bilayer membranes. KcsA lacks the S1 to S4 voltage-sensing region and consists of a pore-forming domain equivalent to that of the vertebrate inward-rectifier K+ channel gene family (Kir), which we discuss in this chapter. As is the case for the Shaker-type K+ channels, KcsA is a homotetramer. The P-region sequence of KcsA is very similar to the P region of the Shaker K+ channel, which contains amino-acid residues critical for K+ selectivity, as well as extracellular sensitivity to blockade by TEA and charybdotoxin. The accompanying eFigure 7-1 shows a ribbon diagram representation of the structure of KcsA, in one view looking down from the top of the membrane (see eFig. 7-1A) and in a second view looking from the side (see eFig. 7-1B). Each of the four monomer subunits of the protein is shown in a different color. Starting from the intracellular N terminus, the first transmembrane span (“outer helix,” corresponding to Shaker S5) forms an α helix that serves as the periphery of the channel. After crossing the membrane to the extracellular side, the peptide backbone then forms a loop that corresponds to the P region. The first half of this loop is a short α helix that folds back a short distance into the plane of the membrane and then immediately exits the extracellular side of the membrane. In the tetrameric complex that constitutes the channel protein, this latter portion of the P loop forms a narrow tunnel-like region called the ion selectivity filter. After exiting the extracellular face of the membrane, the peptide backbone turns again to form a third α helix (“inner helix,” corresponding to Shaker S6) that crosses the membrane to the intracellular side. The four inner helices of the tetramer form the scaffold of the ion channel pore. These four inner helices are tilted in a remarkable flower-like configuration that has also been compared to four poles of an inverted teepee tent dwelling.

The KcsA structure reveals the molecular basis for the K+ selectivity of K+ channel pores. The selectivity filter region is lined not by the side chains of amino acids, but rather by four rings of carbonyl oxygen atoms contributed by the peptide backbone of four amino-acid residues in the P region. K+ ions in the 12-Å-long selectivity filter (near the extracellular surface of the channel) are bound in a cage by coordination to oxygen atoms contributed by each of the four subunits. This cage is just the right size for a K+ ion. A smaller Na+ ion would fit too loosely, so its binding in the cage would not be energetically favorable in comparison to its binding to water in its normal hydrated state. eFigure 7-1C is a cutaway surface view of the pore showing the location of three K+ ions in the crystal structure. Up to seven distinct binding sites for K+ have been identified in high-resolution studies of the KcsA pore. The presence of multiple K+ ions in the pore is consistent with the results of many electrophysiological studies, which suggest that multiple K+ ions move through the channel in single file.

For his work on the structural biology of ion channels, Roderick MacKinnon shared the 2003 Nobel Prize in Chemistry. imageN7-12


EFIGURE 7-1 Structure of the Streptomyces K+ channel (KcsA). A, KcsA is a homotetramer. Each monomer is represented in a different color and contains only two membrane-spanning elements, which is analogous to the S5-P-S6 portion of Shaker-type K+channels. B, The side view more clearly shows the P region, which is very similar to the P region of the Shaker K+ channel. The P region appears to form the selectivity filter of the channel. C, This cut-away view of the pore shows three K+ ions. The top two K+ions are bound in a tight cage that is formed by the peptide backbones of the P regions of each of the four channel subunits. (Data from Doyle DA, Morais Cabral J, Pfuetzner RA, et al: The structure of the potassium channel: Molecular basis of K+ conduction and selectivity. Science 280:69–77, 1998.)

Figure 7-12 shows a comparison of the predicted membrane-folding diagrams of three families of voltage-gated channels: Na+, Ca2+, and K+ channels. The channel-forming subunit of each type of channel is called the α subunit for Na+ and K+ channels and the α1 subunit for Ca2+ channels. Accessory subunits include β1 and β2 for Na+ channels; α2, δ, β, and γ, for Ca2+ channels; and β for K+ channels.


FIGURE 7-12 Subunit structure and membrane-folding models of voltage-gated channels. A, A voltage-gated Na+ channel (Nav) is made up of a pseudo-oligomeric α subunit as well as membrane-spanning β1 and β2 subunits. B, A voltage-gated Ca2+ channel (Cav) is made up of a pseudo-oligomeric α1 subunit as well as an extracellular α2 subunit, a cytoplasmic β subunit (not shown in left panel), and membrane-spanning γ and δ subunits. Note that the domains I to IV of the Nav and Cav α subunits are homologous to a single subunit of a voltage-gated K+ channel (see C). C, A voltage-gated K+ channel is made up of four α subunits as well as four cytoplasmic β subunits (not shown in left panel). (Data from Isom LL, De Jongh KS, Catterall WA: Auxiliary subunits of voltage-gated ion channels. Neuron 12:1183–1194, 1994.)

The α subunit of Na+ channels (see Fig. 7-12A) and the α1 subunit of Ca2+ channels (see Fig. 7-12B) consist of four internally homologous repeats—domains I, II, III, and IV—each containing an S1 to S6 motif composed of the S1 to S4 voltage-sensing domain and the S5-P-S6 pore domain. In contrast, voltage-gated K+ channels (see Fig. 7-12C) are homotetramers of four identical α subunits (see p. 162), whereas Na+ and Ca2+ channels are pseudotetramers. Molecular evolution of the pseudotetrameric I to IV domain structure of Na+ and Ca2+ channels is believed to have occurred by consecutive gene duplication of a primordial gene encoding a structure similar to the basic S1 to S6 motif of K+ channels.

Na+ channels generate the rapid initial depolarization of the action potential

Because the equilibrium potential for Na+ and Ca2+ is in the positive voltage range for normal cellular ionic gradients, channels that are selectively permeable to these ions mediate electrical depolarization. However, prolonged cellular depolarization is an adverse condition inasmuch as it results in sustained contraction and rigor of muscle fibers, cardiac dysfunction, and abnormally elevated levels of intracellular Ca2+, which leads to cell death. Thus, it is critical that Na+ and Ca2+ channels normally reside in a closed conformation at the resting membrane potential. Their opening is an intrinsically transient process that is determined by the kinetics of channel activation and inactivation.

The primary role of voltage-gated Na+ channels is to produce the initial depolarizing phase of fast action potentials in neurons and skeletal and cardiac muscle. The selectivity of Na+ channels for Na+ is much higher than that for other alkali cations. The permeability ratio of Na+ relative to K+ (PNa/PK) lies in the range of 11 to 20 under physiological conditions. Voltage-gated Na+ channels are virtually impermeable to Ca2+ and other divalent cations under normal physiological conditions.

Although Na+ channels do not significantly conduct Ca2+ ions across the cell membrane, the voltage dependence of Na+ channel gating is nevertheless dependent on the extracellular Ca2+ concentration ([Ca2+]o). If [Ca2+]o is progressively increased above the normal physiological level, the voltage activation range of Na+ channels progressively shifts to more positive voltages. In Figure 7-13 this change is represented as a shift in the Po versus Vm relationship. Similarly, if [Ca2+]o is decreased, the voltage activation range is shifted to more negative voltages. This phenomenon has important clinical implications because a negative shift corresponds to a reduced voltage threshold for action potential firing and results in hyperexcitability and spontaneous muscle twitching. Similarly, a positive voltage shift of Na+ channel gating corresponds to decreased electrical excitability (i.e., the threshold is now farther away from resting Vm), resulting in muscle weakness. Thus, metabolic disorders that result in abnormal plasma [Ca2+], such as hypoparathyroidism (low [Ca2+]) and hyperparathyroidism (high [Ca2+]), can cause marked neurological and neuromuscular symptoms. The mechanism of this voltage shift in Na+ channel gating by extracellular divalent cations such as Ca2+ is thought to involve an alteration in the transmembrane electrical field that is sensed by the channel protein. Presumably, this effect is caused by Ca2+ binding or electrostatic screening of negative charges at the membrane surface.


FIGURE 7-13 Effect of extracellular Ca2+ concentration on Na+ channel activation. High [Ca2+]o shifts the Po versus Vm to more positive voltages (i.e., less excitable). Thus, hypocalcemia leads to hyperexcitability.

Humans have 10 homologous genes (Table 7-1) that encode the pore-forming α subunit of voltage-gated Na+ channels (Navs). The isoforms encoded by these genes are expressed in different excitable tissues and can be partially discriminated on the basis of their sensitivity to TTX. Four of the isoforms (Nav1.1, 1.2, 1.3, and 1.6) are differentially expressed in various regions of the brain. Nav1.4 and Nav1.5 are the major isoforms in skeletal and cardiac muscle, respectively. A divergent Na+ channel gene Nax (SCN7A) functions as a homeostatic sensor of plasma [Na+] in circumventricular organs of the brain (see pp. 284–285).


Na+ Channel α Subunits







CNS, PNS, heart








CNS, PNS, heart




Skeletal muscle, heart




Heart, denervated skeletal muscle

Insensitive, 10−6







PNS (nociception)




PNS (nociception)

Insensitive, 10−6



PNS (nociception)

Insensitive, 10−6



CNS/circumventricular organs (Na+ sensor)


PNS, peripheral nervous system.


Erythromelalgia or Primary Erythermalgia

Contributed by Ed Moczydlowski

Certain defects in the human gene SCN9A, which encodes the peripheral nerve Na+ channel Nav1.7, result in a variety of syndromes that alter pain perception. The absence of functional expression of this channel by nonsense mutation results in complete insensitivity to pain. Various single amino-acid replacements due to missense mutations of the channel gene result in gain-of-function syndromes that lead to heightened and severe sensitivity to pain known as primary erythermalgia or erythromelalgia (from the Greek erythros [red] + melos [limb] + algos [pain]) and paroxysmal extreme pain disorder. These findings suggest that Nav1.7 may be a good target in the search for new drugs for the treatment of pain.


Drenth JPH, Waxman SG. Mutations in sodium-channel gene SCN9A cause a spectrum of human genetic pain disorders. J Clin Invest. 2007;117:3606–3609.

Fischer TZ, Gilmore ES, Estacion M, et al. A novel Nav1.7 mutation producing carbamazepine-responsive erythromelalgia. Ann Neurol. 2009;65:733–741.

Wikipedia. s.v. Erythromelalgia. [Accessed July 12, 2015].

Na+ channels are blocked by neurotoxins and local anesthetics

Studies of the mechanism of action of neurotoxins have provided important insight into channel function and structure. The guanidinium toxins TTX and STX (see Fig. 7-5C) are specific blocking agents of Na+channels that act on the extracellular side of the cell membrane.

TTX is produced by certain bacteria and is accumulated in tissues of various marine invertebrates, amphibians, and fish. The viscera of the Fugu pufferfish consumed in Japan often contain lethal amounts of TTX. The flesh of such fish must be carefully prepared to prevent food poisoning.

STX is produced by specific species of marine dinoflagellates that are responsible for “red tide” in the ocean as well as by freshwater cyanobacteria, which can poison ponds and rivers. It is the agent responsible for paralytic shellfish poisoning, which is caused by human ingestion of toxic shellfish that have accumulated STX-producing plankton. Death from TTX and STX intoxication, which ultimately results from respiratory paralysis, can be prevented by the timely initiation of mechanical ventilation.

Venomous marine snails of the Conus genus produce a variety of peptides that target ion channels. The µ-conotoxinsimageN7-14 block muscle Na+ channels by binding near the external binding site for TTX and STX.


Effects of µ-Conotoxin

Contributed by Ed Moczydlowski

The µ-conotoxins are specific blockers of the subtype of voltage-gated Na+ channels that are present in adult skeletal muscle (Nav1.4). This conclusion can be verified by performing a simple electrophysiological experiment on a “nerve-muscle” preparation consisting of a motor nerve and the attached skeletal muscle fibers. The approach is to record the membrane potential of a muscle fiber membrane while artificially stimulating the preparation with a brief electrical depolarization applied either to the nerve or directly to the muscle. In a normal preparation, either stimulus is able to evoke a muscle action potential. However, in a preparation exposed to µ-conotoxin, one observes no response when stimulating the muscle fiber directly but observes a graded postsynaptic potential in the end-plate region when stimulating the nerve directly. This latter response demonstrates that µ-conotoxin does not affect either the motor nerve or the neuromuscular junction (e.g., the nicotinic acetylcholine receptor at the motor end plate).

TTX, STX, and µ-conotoxins are important pharmacological probes because they can be used to functionally discriminate among several distinct isoforms of Na+ channels (see Table 7-1). Other important neurotoxins that act on Na+ channels include batrachotoxin (a steroidal alkaloid from certain tropical frogs and birds), various plant alkaloids (veratridine, grayanotoxin, aconitine), natural plant insecticides (pyrethrins), brevetoxins (cyclic polyethers from dinoflagellates), α and β scorpion peptide toxins, and peptide toxins from tarantulas. Some of these neurotoxins are stimulatory, acting primarily by altering gating kinetics, so that the Na+ channels are open at voltages in which Na+ channels are normally closed or inactivated. Others block the channel by stabilizing the voltage sensor in the resting (i.e., channel-closed) conformation.

Local anesthetics are a large group of synthetic drugs that are generally characterized by an aromatic moiety linked to a tertiary amine substituent via an ester or amide linkage (Fig. 7-14A). Drug development of local anesthetics began in the late 1800s with the recognition by Karl Koller and Sigmund Freud that the plant alkaloid cocaine numbs sensation in the tongue and suppresses eye movement during ophthalmological procedures, in addition to having psychoactive effects on the central nervous system (CNS). Attempts to synthesize safer alternatives to cocaine led to procaine, which mimics the local anesthetic effect of cocaine without the CNS effects.


FIGURE 7-14 Effect of local anesthetics. A, The three clinically useful local anesthetics shown here are synthetic analogs of the plant alkaloid cocaine. B, In the presence of lidocaine, the relative Na+ current decays with time during repetitive stimulation. However, the inhibition becomes more pronounced as the rate of stimulation increases from 1/s to 8/s; these findings demonstrate use-dependent inhibition. (Data from Hille B: Local anesthetics: Hydrophilic and hydrophobic pathways for the drug-receptor reaction. J Gen Physiol 69:497–515, 1977.)

Local anesthetics that are used clinically, such as procaine, lidocaine, and tetracaine, reversibly block nerve impulse generation and propagation by inhibiting voltage-gated Na+ channels. The action of these drugs is “use dependent,” which means that inhibition of Na+ current progresses in a time-dependent manner with increasing repetitive stimulation or firing of action potentials (see Fig. 7-14B). Use dependence occurs because the drug binds most effectively only after the Na+ channel has already opened. This use-dependent action of the drug further enhances inhibition of nerve impulses at sites where repetitive firing of action potentials takes place. Local anesthetics are widely used to control pain during dental procedures, many types of minor surgery, and labor in childbirth.

Box 7-1

Genetic Defects of Voltage-gated Na+ Channels

A diverse spectrum of human genetic diseases has been mapped to inheritable defects in the α-subunit genes for voltage-gated Na+ channels (see Table 7-1). Genetic diseases have been linked to five different Nav genes: SCN4A (Nav1.4) expressed in skeletal muscle, SCN5A expressed in the heart (Nav1.5), and three genes—SCN1A (Nav1.1), SCN2A (Nav1.2), and SCN9A (Nav1.7)—expressed in central and peripheral neurons.

One disorder of skeletal muscle resulting from mutations in Nav1.4 is called hyperkalemic periodic paralysis (HYPP or HyperPP) because muscle weakness is triggered by an elevation in serum [K+] that may occur after vigorous exercise or ingestion of foods rich in K+. A second Nav1.4-linked muscle disorder is called paramyotonia congenita (PC). This form of periodic paralysis may be induced in affected individuals by exposure to cold temperature and results in symptoms of myotonia (muscle stiffness) associated with abnormal repetitive firing of muscle action potentials.

Long QT syndromes comprise a diverse number of inherited defects in heart rhythm that can lead to sudden death from cardiac arrhythmias, including ventricular fibrillation. Long QT refers to lengthening of the duration of the cardiac action potential as measured by the QT interval on the electrocardiogram (see p. 506). One form of these syndromes, classified as LQT3, is the result of a deletion of three amino acids, ΔKPQ, in the linker region between repetitive domains III and IV of the Nav1.5 heart Na+ channel. Many other LQT3 mutations of Nav1.5 have been mapped in affected families. Genetic defects in various cardiac K+ channels also cause long QT syndromes as described in Box 7-3.

Mutations in two different Nav channels expressed in the brain result in syndromes called generalized epilepsy with febrile seizures plus (GEFS+; Nav1.1) and infantile epileptic encephalopathy (Nav1.2).

Certain defects in Nav1.7 in peripheral nerves result in a variety of syndromes that alter pain perception (nociception). Various single amino-acid replacements in Nav1.7 result in syndromes of heightened and severe sensitivity to pain—primary erythermalgia (PE) or familial erythromelalgiaimageN7-21 (from the Greek erythros [red] + melos [limb] + algos [pain]) and paroxysmal extreme pain disorder (PEPD). Conversely, the absence of functional expression of this channel by nonsense mutation prevents action potential firing in sensory afferent nociceptive neurons, resulting in a complete inability to perceive pain—channelopathy-associated insensitivity to pain (CIP). Thus, the development of drugs that selectively block Nav1.7 channels could lead to a new class of pain medications.

Figure 7-15 illustrates the location of various Nav mutations mapped onto a membrane topology diagram of a generic Nav channel. Most of these Nav mutations cause hyperexcitability of nerve or muscle due to altered voltage-dependent gating kinetics, resulting in excessive or prolonged channel opening.


FIGURE 7-15 Na+ channel mutations in human genetic diseases. The topology diagram shows a few examples of locations of mutations mapped in three Na+ channel genes for six human genetic channelopathy diseases. Symbols show sites on a generic Na+channel α subunit. All these disease mutations are missense mutations resulting in amino-acid changes or deletions except for channelopathy-associated insensitivity to pain, which is due to nonsense mutations that result in lack of functional Nav1.7 expression. (Data from Catterall WA: Cellular and molecular biology of voltage-gated sodium channels. Physiol Rev 72:S15–S48, 1992; Ashcroft FM: Ion Channels and Disease, Academic Press, 2000; and Drenth JPH, Waxman S: Mutations in sodium-channel gene SCN9A cause a spectrum of human genetic pain disorders. J Clin Invest 117:3606–3609, 2007.)

Ca2+ channels contribute to action potentials in some cells and also function in electrical and chemical coupling mechanisms

Ca2+ channels play important roles in the depolarization phase of certain action potentials, in coupling electrical excitation to secretion or muscle contraction, and in other signal-transduction processes. Because [Ca2+]o is ~1.2 mM, whereas resting [Ca2+]i is only ~10−7 M, a huge gradient favors the passive influx of Ca2+ into cells. At the relatively high [Ca2+]o that prevails under physiological conditions, voltage-gated Ca2+channels are highly selective for Ca2+, with permeability to Ca2+ being ~1000-fold greater than permeability to Na+. Other alkaline earth divalent cations such as Sr2+ and Ba2+ also readily permeate Ca2+ channels and are often used as substitute ions for recording the activity of Ca2+ channels in electrophysiological studies. However, if [Ca2+]o is experimentally reduced to a nonphysiological level of <10−6 M with the use of chelating agents, Ca2+ channels can also conduct large currents of monovalent alkali cations, such as Na+ and K+. Thus, in terms of its intrinsic ionic selectivity, the Ca2+ channel is functionally similar to the Na+channel, except that high-affinity binding of Ca2+ in the pore effectively prevents permeation of all other physiological ions except Ca2+.

The mechanism of this extraordinary selectivity behavior is based on ion-ion interactions within the selectivity filter of the pore. imageN7-10 For the Ca2+ channel to conduct current, at least two Ca2+ ions must bind simultaneously. This general mechanism, referred to as multi-ion conduction, also applies to many other classes of ion channels, notably K+ channels. Multi-ion conduction generally plays an important role in determining the permeation properties of channels with a high ionic selectivity, such as Ca2+ and K+ channels.

Voltage-gated Ca2+ channels contribute to the depolarizing phase of action potentials in certain cell types. Notably, the gating of voltage-gated Ca2+ channels is slower than that of Na+ channels. Whereas Na+channels are most important in initiating action potentials and generating rapidly propagating spikes in axons, Ca2+ channels often give rise to a more sustained depolarizing current, which is the basis for the long-lived action potentials in cardiac cells, smooth-muscle cells, secretory cells, and many types of neurons.

The exquisite selectivity of Ca2+ channels under physiological conditions endows them with special roles in cellular regulation. When activated by a depolarizing electrical stimulus or a signal-transduction cascade, these Ca2+ channels mediate an influx of Ca2+ that raises [Ca2+]i. Thus, in serving as a major gateway for Ca2+ influx across the plasma membrane, Ca2+ channels not only contribute to membrane depolarization but also play a role in signal transduction because Ca2+ is an important second messenger (see p. 60).

Ca2+ channels also play a pivotal role in a special subset of signal-transduction processes known as excitation-contraction coupling and excitation-secretion coupling. Excitation-contraction (EC) coupling refers to the process by which an electrical depolarization at the cell membrane leads to cell contraction, such as the contraction of a skeletal muscle fiber. In EC coupling of skeletal muscle (see p. 229), one class of plasma-membrane Ca2+ channel that is located in the transverse tubule membrane of skeletal muscle serves as the voltage sensor and forms a direct mechanical linkage to intracellular Ca2+-release channels that are located in the sarcoplasmic reticulum (SR) membrane. In contrast, Ca2+ channels play a different role in EC coupling in cardiac muscle, where Ca2+ channels in the plasma membrane mediate an initial influx of Ca2+. The resultant increase in [Ca2+]i triggers an additional release of Ca2+ stored in the SR by a process known as Ca2+-induced Ca2+ release (see pp. 242–243).

Excitation-secretion (ES) coupling is the process by which depolarization of the plasma membrane causes release of neurotransmitters in the nervous system and the secretion of hormones in the endocrine system. Such processes require an increase in [Ca2+]i to trigger exocytosis of synaptic and secretory vesicles.

In keeping with the diverse roles of Ca2+ channels, the human genome contains 10 distinct genes (Table 7-2) for the channel-forming α1 subunit of voltage-gated Ca2+ channels (Cavs). Molecular studies have also identified four accessory subunits of Ca2+ channels: α2, δ, β, and γ (see Fig. 7-12B). A single gene gives rise to the α2 and δ subunits, which are separated by proteolytic cleavage. Coexpression studies show that these accessory subunits can greatly influence the kinetics, voltage sensitivity, and peak currents exhibited by various α1 channel subunits. Table 7-2 shows that the genetic diversity is mirrored by a diversity of Ca2+ currents in various cell types.


Properties and Classification of Ca2+ Channel α Subunits










Long duration


Intermediate to long duration

Intermediate to long duration

Intermediate duration

Voltage activation

High threshold (> −30 mV)

Low threshold (< −30 mV)

High threshold (> −30 mV)

High threshold (> −30 mV)

High threshold (> −30 mV)


Blocked by DHPs

Less sensitive to DHPs

Insensitive to DHPs, blocked by ω-conotoxin GVIA

Insensitive to DHPs, blocked by ω-agatoxin IVA

Insensitive to DHPs, ω-conotoxin GVIA, and ω-agatoxin IVA


Heart, skeletal muscle, neurons, vascular smooth muscle, uterus, neuroendocrine cells

Sinoatrial node of heart, brain neurons

Presynaptic terminals, dendrites, and cell bodies of neurons

Cerebellar Purkinje's and granule cells, cell bodies of central neurons

Cerebellar granule cells, neurons


EC coupling in skeletal muscle, link membrane depolarization to intracellular Ca signalling

Repetitive firing of action potentials in heart and many neurons

Exocytotic neurotransmitter release

Exocytotic neurotransmitter release

Exocytotic neurotransmitter release

Channel protein (gene)

Cav1.1 (CACNA1S)
Cav1.2 (CACNA1C)
Cav1.3 (CACNA1D)
Cav1.4 (CACNA1F)

Cav3.1 (CACNA1G)
Cav3.2 (CACNA1H)
Cav3.3 (CACNA1I)

Cav2.2 (CACNA1B)

Cav2.1 (CACNA1A)

Cav2.3 (CACNA1E)

Ca2+ channels are characterized as L-, T-, P/Q-, N-, and R-type channels on the basis of kinetic properties and inhibitor sensitivity

An example of the functional diversity of Ca2+ channels is illustrated in Figure 7-16, which shows two different types of voltage-gated Ca2+ channels that have been identified in cardiac ventricular cells by the patch-clamp technique. If the cell-attached patch, initially clamped at −50 mV, is suddenly depolarized to +10 mV, currents appear from a large-conductance (18 to 25 pS), slowly inactivating Ca2+ channel (see Fig. 7-16A). However, if the same patch is initially clamped at −70 mV and depolarized to only −20 mV, currents appear instead from a small-conductance (8 pS), rapidly inactivating Ca2+ channel (see Fig. 7-16B). These two types of Ca2+ channel are, respectively, named L-type (for long-lived) and T-type (for transient) channels. T-type channels are activated at a lower voltage threshold (more negative than −30 mV) than are other types of Ca2+ channels and are also inactivated over a more negative voltage range. These characteristics of T-type channels permit them to function briefly in the initiation of action potentials and to play a role in the repetitive firing of cardiac cells and neurons. Other types of Ca2+ channels, including L-, N-, P/Q-, and R-type channels, which are activated at a higher voltage threshold (more positive than −30 mV), mediate the long-lived plateau phase of slow action potentials and provide a more substantial influx of Ca2+ for contractile and secretory responses. N-, P/Q-, and R-type Ca2+ channels appear to mediate the entry of Ca2+ into certain types of presynaptic nerve terminals and thus play an important role in facilitating the release of neurotransmitters.

Box 7-2

Consequences of Genetically Defective Ca2+ Channels

Given their significance in excitation-contraction and excitation-secretion coupling, mutations resulting in dysfunctional voltage-gated Ca2+ channels would be expected to have many adverse physiological consequences. Here we describe Cav defects that perturb cardiac and neurological function. Cav mutations and related autoimmune disorders that specifically affect muscle are the focus of Box 9-1.

The L-type Cav1.2 channel is highly expressed in the heart but also in many other tissues. Mutations in the human CACNA1C gene for Cav1.2 are linked to two distinct genetic channelopathies: Timothy syndrome (LQT8) and a form of Brugada syndrome. Timothy syndrome is inherited in an autosomal dominant pattern with common features of syndactyly (fused fingers), autism, and long QT arrhythmia. The arrhythmia apparently reflects a gain of function resulting from defective Cav inactivation. Brugada syndrome, which also carries a risk of sudden cardiac death, is recognized by an abnormal pattern on electrocardiogram characterized by an elevated ST segment and shortened QT interval (see Fig. 21-7), or so-called short QT. Defects in at least six different human genes are linked to Brugada-like symptoms. However, loss-of-function Brugada mutations in the Cav1.2 channel cause defective trafficking of this channel, reducing Ca2+-current density in the heart.

The Cav1.4 L-type channel is highly expressed in the retina and is genetically linked to visual disorders. One form of X-linked congenital stationary night blindness is due to mutations in the CACANA1F gene that result in nonfunctional Cav1.4 channels. The consequence is reduced sustained transmitter release by photoreceptor cells in the dark (see Fig. 15-11).

Amino-acid variations in the human CACNA1H gene for the Cav3.2 T-type Ca2+ channel are associated with various forms of epilepsy. These symptoms appear to reflect a gain of function that enhances Ca2+current. Genetic syndromes linked to defects in Cav3.2 include a variety of idiopathic generalized epilepsies such as juvenile myoclonic epilepsy, generalized tonic-clonic seizures, and generalized epilepsy with febrile seizures.

The P/Q-type Cav2.1 channel, expressed in cerebellar Purkinje cells, is the culprit in diverse CNS movement disorders (ataxia) and migraine syndromes. Mutations in the CACNA1A gene for Cav2.1 variously cause episodic ataxia type 2 (EA2), spinocerebellar ataxia type 6, and familial hemiplegic migraine. Symptoms of EA2 result from Cav2.1 mutations that decrease Ca2+ current, whereas the other two disorders stem from mutations that increase Ca2+ entry. Discovery of the genetic origin of such diseases has led to the realization that delicate perturbations of Ca2+ channel activity can have profound consequences on proper function of the human nervous system.


FIGURE 7-16 Current records from two types of Ca2+ channel. A, Data from an experiment using cell-attached patches on guinea pig ventricular myocytes are shown. These currents are carried by Ba2+ through L-type Ca2+ channels, which conduct Ba2+ even better than Ca2+. Shown in the middle panel are seven single channel current records obtained during and after a shift of the cytosolic voltage from −50 to +10 mV. Channel activity (i.e., downward deflections) begins only after depolarization and continues more or less at the same level throughout the depolarization. The lower panel shows the average of many records that are similar to those shown in the middle panel. B, The experiments yielding the data shown for T-type Ca2+ channels are identical in design to those in A, except for the depolarizing step. Again, channel activity begins only after depolarization (middle panel). However, channel activity is transient, waning during a sustained depolarization, as confirmed by the average current shown in the lower panel. (Data from Nilius B, Hess P, Lansman JB, Tsien RW: A novel type of cardiac calcium channel in ventricular cells. Nature 316:443–446, 1985.)

In addition to discrimination on the basis of gating behavior, Ca2+ channel isoforms can also be distinguished by their sensitivity to different drugs and toxins (see Table 7-2). Ca2+ channel blockers are an important group of therapeutic agents. Figure 7-17 shows the structures of representatives of three different classes of Ca2+ channel blockers: 1,4-dihydropyridines (DHPs), phenylalkylamines, and benzothiazepines. These synthetic compounds are available for treatment of cardiovascular disorders such as angina pectoris (see Box 24-1), hypertension, and various arrhythmias and also have potential applications in the treatment of CNS conditions such as cerebral vasospasm and epileptic seizure.


FIGURE 7-17 Antagonists and agonists of L-type Ca2+ channels. A, 1,4-Dihydropyridines. One DHP, nitrendipine, is an antagonist; another, Bay K8644, is an agonist. B, Phenylalkylamines. Verapamil is an antagonist. C, Benzothiazepines. Diltiazem is an antagonist.

DHPs such as nitrendipine selectively block L-type Ca2+ channels. Phenylalkylamines (e.g., verapamil) and benzothiazepines (e.g., diltiazem) also inhibit L-type Ca2+ channels; however, these other two classes of drugs act at sites that are distinct from the site that binds DHPs. Particular DHP derivatives, such as Bay K8644, actually enhance rather than inhibit Ca2+ channel currents. DHPs can have the contrasting effects of either inhibitors (antagonists) or activators (agonists) because they act not by plugging the channel pore directly but by binding to a site composed of transmembrane helices S5 and S6 in domain III and S6 in domain IV. Drug binding in this region probably induces various conformational changes in channel structure and thereby perturbs Ca2+ permeation and gating behavior.

Other molecules useful in discriminating Ca2+ channel isoforms are present in the venom of the marine snail Conus geographus and the funnel web spider Agelenopsis aperta. The snail produces a peptide called ω-conotoxin GVIA, which selectively blocks N-type Ca2+ channels. The spider produces the peptide ω-agatoxin IVA, which selectively blocks P/Q-type Ca2+ channels. In contrast, an R-type neuronal Ca2+ channel is resistant to these two peptide toxins.

The summary of the basic properties of L-, T-, N-, P/Q-, and R-type Ca2+ channels contained in Table 7-2 indicates their presumed correspondence to 10 known genes that encode α1 subunits.

K+ channels determine resting potential and regulate the frequency and termination of action potentials

K+ channels are the largest and most diverse family of voltage-gated ion channels. Humans have at least 79 distinct genes encoding K+ channels, characterized by a K+-selective S5-P-S6 pore domain (see Fig. 7-10). Ion conduction through most types of K+ channels is very selective for K+ according to the permeability sequence K+ > Rb+ > image ≫ Cs+ > Li+, Na+, Ca2+. Under normal physiological conditions, the permeability ratio PK/PNa is >100, and Na+ can block many K+ channels. Some K+ channels can pass Na+ current in the complete absence of K+. This characteristic is analogous to the behavior of Ca2+ channels, which can pass Na+ and K+ currents in the absence of Ca2+.

Given such strong K+ selectivity and an equilibrium potential near −80 mV, the primary role of K+ channels in excitable cells is to oppose the action of excitatory Na+ and Ca2+ channels and stabilize the resting state. Whereas some K+ channels are major determinants of the resting potential, other K+ channels mediate the repolarizing phase and shape of action potentials, control firing frequency, and define the bursting behavior of rhythmic firing. Such functions are broadly important in regulating the strength and frequency of all types of muscle contraction, in terminating transmitter release at nerve terminals, in attenuating the strength of synaptic connections, and in coding the intensity of sensory stimuli. Finally, in epithelia, K+ channels also function in K+ absorption and secretion.

Before understanding the molecular nature of K+ channels, electrophysiologists classified K+ currents according to their functional properties and gating behavior, grouping macroscopic K+ currents into four major types:

1. Delayed outward rectifiers

2. Transient outward rectifiers (A-type currents)

3. Ca2+-activated K+ currents

4. Inward rectifiers

These four fundamental K+ currents are the macroscopic manifestation of five distinct families of genes (see Table 6-2, family No. 2):

1. Kv channels (voltage-gated K+ channels related to the Shaker family)

2. Small- and intermediate-conductance KCa channels (Ca2+-calmodulin–activated K+ channels), including SKCa and IKCa channels

3. Large-conductance KCa channels (Ca2+-activated BKCa channels and related Na+- and H+- activated K+ channels)

4. Kir channels (inward-rectifier K+ channels)

5. K2P channels (two-pore K+ channels)

In the next three sections, we discuss the various families of K+ channels and their associated macroscopic currents.

The Kv (or Shaker-related) family of K+ channels mediates both the delayed outward-rectifier current and the transient A-type current

The K+ current in the HH voltage-clamp analysis of the squid giant axon (see pp. 177–178) is an example of a delayed outward rectifier. Figure 7-18A shows that this current activates with a sigmoidal lag phase (i.e., it is delayed in time, as in Fig. 7-6C). Figure 7-18B is an I-V plot of peak currents obtained in experiments such as that presented in Figure 7-18A and shows that the outward current rises steeply at positive voltages (i.e., it is an outward rectifier).


FIGURE 7-18 Outwardly rectifying K+ channels. A, Note that in a voltage-clamp experiment, a depolarizing step in Vm activates the current, but with a delay. B, The current-voltage relationship is shown for a delayed outwardly rectifying K+ channel, as in A. C, This A-type K+ current is active at relatively negative values of Vm and tends to hyperpolarize the cell. In a spontaneously spiking neuron, a low level of the A-type current allows Vm to rise relatively quickly toward the threshold, which produces a relatively short interspike interval and thus a high firing rate. D, In a spontaneously spiking neuron, a high level of the A-type current causes Vm to rise relatively slowly toward the threshold, which produces a relatively long interspike interval and thus a low firing rate. E, Four different types of K+ channels (Kv1.1, Kv1.2, Kv1.3, and Kv1.4) from mammalian brain and expressed in Xenopus oocytes show activation and inactivation kinetics during steps of Vm from −80 mV to 0 mV. The left panels, at high time resolution, show that some of these channels activate more slowly than others. The right panels, at a longer time scale, show that inactivation gradually speeds up from Kv1.1 to Kv1.4. F, The left panel shows N-type inactivation, so called because the N or amino terminus of the protein is essential for inactivation. Each of the four subunits is thought to have an N-terminal “ball” tethered by a “chain” that can swing into place to block the pore. The right panel shows a variant in which certain β subunits can provide the ball and chain for Kv channel α subunits that themselves lack this capability at their N termini. (Data from Stühmer W, Ruppersberg JP, Schroter KH, et al: Molecular basis of functional diversity of voltage-gated potassium channels in mammalian brain. EMBO J 8:3235–3244, 1989.)

A second variety of K+ current that is also outwardly rectifying is the transient A-type K+ current. This current was first characterized in mollusk neurons, but similar currents are common in the vertebrate nervous system. A-type currents are activated and inactivated over a relatively rapid time scale. Because their voltage activation range is typically more negative than that of other K+ currents, they are activated in the negative Vm range that prevails during the afterhyperpolarizing phase of action potentials. In neurons that spike repetitively, this A-type current can be very important in determining the interval between successive spikes and thus the timing of repetitive action potentials. For example, if the A-type current is small, Vm rises relatively quickly toward the threshold, and consequently the interspike interval is short and the firing frequency is high (see Fig. 7-18C). However, if the A-type current is large, Vm rises slowly toward the threshold, and therefore the interspike interval is long and the firing frequency is low (see Fig. 7-18D). Because the nervous system often encodes sensory information as a frequency-modulated signal, these A-type currents play a critical role.

The channels responsible for both the delayed outward-rectifier and the transient A-type currents belong to the Kv channel family (where v stands for voltage-gated). The prototypic protein subunit of these channels is the Shaker channel of Drosophila (see Fig. 7-12C). All channels belonging to this family contain the conserved S1 to S6 core that is characteristic of the Shaker channel (see Fig. 7-10), but may differ extensively in the length and sequence of their intracellular N-terminal and C-terminal domains. The voltage-sensing element in the S4 segment underlies activation by depolarization; the S4 segment actually moves outward across the membrane with depolarizing voltage, thus increasing the probability of the channel's being open. imageN7-13

The Kv channel family has multiple subclasses (see Table 6-2, family No. 2). Individual members of this Kv channel family, whether in Drosophila or humans, exhibit profound differences in gating kinetics that are analogous to delayed-rectifier (slow activation) or A-type (rapid inactivation) currents. For example, Figure 7-18E shows the macroscopic currents of four subtypes of rat brain Kv1 (or Shaker) channels heterologously expressed in frog oocytes. All of these Kv1 channel subtypes (Kv1.1 to Kv1.4) exhibit sigmoidal activation kinetics when examined on a brief time scale—in the millisecond range (left side of Fig. 7-18E). That is, these channels display some degree of “delayed” activation. Different Kv channels exhibit different rates of activation. Thus, these currents can modulate action potential duration by either keeping it short (e.g., in nerve and skeletal muscle) when the delayed rectifier turns on quickly or keeping it long (e.g., in heart) when the delayed rectifier turns on slowly.

Box 7-3

Congenital and Drug-Induced Cardiac Arrhythmias Linked to K+ Channels

Congenital Long QT Syndromes

As discussed in Box 7-1, congenital cardiac abnormality in some people results in lengthening of the QT interval of the electrocardiographic signal—long QT syndrome—which corresponds to a prolonged cardiac action potential. Affected children and young adults can exhibit an arrhythmic disturbance of the ventricular heartbeat that results in sudden death. As we have already seen in Box 7-1, one form of a long QT syndrome—LQT3—involves gain-of-function mutations of the cardiac Na+ channel Nav1.5 (SCN5A) that prolong Na+ channel opening. However, at least six forms of long QT syndrome—LQT1, LQT2, LQT5, LQT6, LQT7, and LQT13—are caused by loss-of-function mutations in cardiac K+ channels (see Table 6-2, family No. 2) or their accessory proteins.

LQT1 is due to mutations in the KCNQ1 gene encoding KvLQT1, a 581-residue protein belonging to the Kv family of voltage-gated K+ channels. Another form of this disease, LQT2, involves mutations in the KCNH2 gene encoding HERG (for human ether-à-go-go) which is related to the gene defective in the ether-à-go-go Drosophila mutation, in which flies convulsively shake under ether anesthesia. Both KvLQT1 and HERG K+ channels participate in repolarization of the human cardiac action potential (see p. 488). KvLQT1 mediates the slowly activating delayed-rectifier component (IKs) of cardiac action potential repolarization; HERG mediates the rapidly activating repolarization component (IKr). Both LQT1 and LQT2 result from loss-of-function effects associated with decreased K+ channel expression in cardiac myocytes.

KvLQT1 associates with minK, a small, single-span membrane protein encoded by the KCNE1 gene. minK modulates the gating kinetics of KvLQT1, and mutations in minK cause LQT5. Three other human proteins closely related to minK are known as MiRP1, MiRP2, and MiRP3 (minK-related proteins)—the products of the genes KCNE2, KCNE3, and KCNE4, respectively. MiRP1 associates with HERG, and mutations in MiRP1 are linked to LQT6.

Two other K+ channel genes also cause long QT syndromes. Mutations in Kir2.1, encoded by the gene KCNJ2, cause LQT7, whereas mutations in GIRK4, encoded by KCNJ5, cause LQT13.

Acquired Long QT Syndrome

The HERG channel is notorious for its sensitivity to blockade by many classes of therapeutic drugs, including antihistamines (e.g., terfenadine), antipsychotics (e.g., sertindole), and gastrointestinal drugs (e.g., cisapride). Blockade of HERG can readily mimic the genetic condition of LQT2. The promiscuous drug sensitivity of the HERG K+ channel appears to result from particular structural features of the internal aspect of the channel pore that favor binding of many hydrophobic small molecules. People who have natural variations in ion channel genes that cause a subclinical propensity for long QT intervals or who have deficiencies in drug-metabolizing enzymes appear to be especially at risk. Many drugs have been banned or limited for therapeutic use because of the risk of HERG channel block. Today all new drugs proposed for clinical use must first undergo screening for HERG blockade in order to prevent deaths by acquired long QT syndrome.

Kv1 channels also differ markedly in their inactivation kinetics when observed over a long time scale—in the range of seconds (right side of Fig. 7-18E). Kv1.1 exhibits little time-dependent inactivation (i.e., the current is sustained throughout the stimulus). On the other hand, the Kv1.4 channel completely inactivates in <1 second. Kv1.2 and Kv1.3 show intermediate behavior.

How are Kv channels inactivated? The structural basis for one particular type of K+ channel inactivation, known as N-type inactivation, is a stretch of ~20 amino-acid residues at the N terminus of some fast-inactivating Kv channels. This domain acts like a ball to block or to plug the internal mouth of the channel after it opens; the result is inactivation (see Fig. 7-18F). Thus, this process is also known as the ball-and-chain mechanism of K+ channel inactivation. Particular kinds of β subunits that are physically associated with some isoforms of Kv channels have structural elements that mimic this N-terminal ball domain and rapidly inactivate K+ channel α subunits that lack their own inactivation ball domain (see Fig. 7-11). Many K+ channels also exhibit a second, slower C-type inactivation, which appears to involve a constriction of the outer mouth of the pore.

Various delayed-rectifier K+ channels are blocked by either internal or external application of quaternary ammonium ions such as TEA (see Fig. 7-5C). Many transient A-type K+ currents are inhibited by another organic cation, 4-aminopyridine (4-AP), which effectively shifts the action potential threshold in the negative direction and also prolongs action potentials by inhibiting repolarization. Both effects promote the propagation of action potentials through demyelinated regions of axons, which is why 4-AP has been approved for treatment of symptoms of multiple sclerosis (see Box 12-1). Two distinct families of peptide toxins—charybdotoxins of scorpion venom and dendrotoxins of mamba snake venom—discriminate particular subtypes of Kv and KCa channels, depending on the particular amino acids present in the P region. Such toxins cause nerve and muscle hyperexcitability, resulting in paralysis of envenomated victims.

Two families of KCa K+ channels mediate Ca2+-activated K+ currents

Ca2+-activated K+ channels—KCa channels—are present in the plasma membrane of cells in many different tissues. A particular type of KCa channel is shown in Fig. 7-19A; this is called BKCa (for “big” KCa) or the maxi-KCa channel because it has a large unitary conductance (~300 pS). In patch-clamp experiments, all KCa channels are easily recognized because the open probability of individual channels increases with increasing [Ca2+] on the intracellular surface of the membrane patch (see Fig. 7-19B for BKCa). In addition, the Po of BKCa increases with positive voltage (see Fig. 7-19C). imageN7-15Figure 7-19D shows how increasing [Ca2+]i causes a negative or leftward shift in the Po versus Vm plot for BKCa channels. Thus, at negative voltages, the binding of Ca2+ to the regulator of conductance of K+ (RCK) domains causes BKCachannels to be open when they otherwise would be closed.


FIGURE 7-19 Large-conductance Ca2+-activated K+ channels (BKCa). A, Topology of BKCa channels. B, Shown are data from an experiment on BKCa channels that are expressed in Xenopus oocytes and studied by using a patch pipette in an inside-out configuration. Vm is always held at +40 mV and the [Ca2+] on the cytosolic side of the patch varies from 1 to 10 to 100 µM. Note that channel activity increases with increasing [Ca2+]iC, The experiment is the same as in B except that when Vm is held at −60 mV, there is very little channel activity. On the other hand, when Vm is +80 mV, both channels in the patch are open most of the time. D, Combined effects of changing Vm and [Ca2+]i. Shown is a plot of relative open probability (Po) of the KCa channels versus Vm at three different levels of Ca2+. The data come from experiments such as that in B. (Data from Butler A, Tsunoda S, McCobb DP, et al: mSlo, a complex mouse gene encoding “maxi” calcium-activated potassium channels. Science 261:221–224, 1993.)


The BKCa Channel

Contributed by Ed Moczydlowski

The BKCa channel was discovered by cloning and sequencing the Slowpoke gene of Drosophila. The BKCa channel-forming α subunit—activated by intracellular Ca2+—consists of a conserved S1 to S6 motif, an extra S0 transmembrane segment at the amino terminus, and a unique C-terminal intracellular domain of ~850 residues, consisting of two RCK domains. The RCK domains contain Ca2+-binding sites involved in channel activation (see Table 6-2, family No. 2, as well as Fig. 7-19).

Because BKCa channels—like Kv channels—have a voltage-sensing domain that is analogous to S4, they are also activated by positive voltage.

Besides the plasma membrane, BKCa channels are also present in the inner mitochondrial membrane of cardiac myocytes, where they exert a cardioprotective role under conditions of ischemia. Hypoxia increases the open probability of this channel, which would lead to an efflux of K+ from the mitochondrial matrix into the cytosol; this efflux would tend to maintain a relatively high cytosolic [K+] and help to maintain a relatively negative resting potential.

In principle, KCa channels provide a stabilizing mechanism to counteract repetitive excitation and intracellular Ca2+ loading. KCa channels mediate the afterhyperpolarizing phase of action potentials (see Fig. 7-1A) in cell bodies of various neurons. They have also been implicated in terminating bursts of action potentials in bursting neuronal pacemaker cells. Thus, the gradual increase in [Ca2+]i that occurs during repetitive firing triggers the opening of KCa channels, which results in hyperpolarization and a quiescent interburst period that lasts until intracellular Ca2+ accumulation is reversed by the action of Ca pumps (see p. 118). KCa channels are also present at high density in many types of smooth-muscle cells, where they appear to contribute to the relaxation of tension by providing a hyperpolarizing counterbalance to Ca2+-dependent contraction. In a number of nonexcitable cells, KCa channels are activated during cell swelling and contribute to regulatory volume decrease (see pp. 131–132).

In addition to the BKCa channel and the related Na+- and H+-activated K+ channels, there is another K+ channel gene family that includes small- and intermediate-conductance Ca2+-activated K+ channels, respectively termed SKCa and IKCa (see Table 6-2, family No. 2). Unlike BKCa channels, the closely related IKCa and SKCa channels are voltage insensitive and are activated by the Ca2+-binding protein calmodulin (see p. 60). In some cells, SKCa and IKCa channels participate in action potential repolarization and afterhyperpolarization, thus regulating action potential firing frequency. IKCa also functions in the activation of lymphocytes.

The Kir K+ channels mediate inward-rectifier K+ currents, and K2P channels may sense stress

In contrast to the delayed rectifiers and A-type currents—which are outwardly rectifying K+ currents—the inward-rectifier K+ current (also known as the anomalous rectifier) actually conducts more K+ current in the inward direction than in the outward direction. Such inwardly rectifying, steady-state K+ currents have been recorded in many types of cells, including heart, skeletal muscle, and epithelia. Physiologically, these channels help clamp the resting membrane potential close to the K+ equilibrium potential and prevent excessive loss of intracellular K+ during repetitive activity and long-duration action potentials. In epithelial cells, these inwardly rectifying K+ currents are important because they stabilize Vm in the face of electrogenic ion transporters that tend to depolarize the cell (see Chapter 5).

The channel-forming subunits of the inward-rectifier (Kir) K+ channel family are relatively small proteins (~400 to 500 residues) that do not contain the complete S1 to S6 core domain found in the Kv and KCachannel families. However, the Kir channels do have a conserved pore domain similar to the S5-P-S6 segment of Kv channels (Fig. 7-20A). imageN7-13 The conserved P region is the most basic structural element that is common to all K+ channels. The absence of an S1 to S4 voltage-sensing domain in Kir channels is consistent with their lack of steeply voltage-dependent activation.


FIGURE 7-20 Inwardly rectifying K+ channels. A, This family of channels has only two membrane-spanning segments that correspond to the S5-P-S6 domain of the voltage-gated K+ channels. B, The GIRK1 channels—a particular type of Kir channel—were expressed in Xenopus oocytes and studied by use of a patch pipette in the inside-out configuration. Vm was clamped to values between −100 mV and +60 mV, and [Mg2+] was 2.5 mM on the cytosolic side. Note that channel activity increases at more negative voltages but the channel is virtually inactive at positive voltages. C, The I-V plot shows that there is inward rectification only in the presence of Mg2+ on the cytosolic side. In the absence of Mg2+, the I-V relationship is nearly linear or ohmic. D, As shown in the left panel, cytosolic Mg2+ occludes the channel pore and prevents the exit of intracellular K+. However, even in the presence of Mg2+, extracellular K+ can move into the cell by displacing the Mg2+. (Data from Kubo Y, Reuveny E, Slesinger PA, et al: Primary structure and functional expression of a rat G protein–coupled muscarinic potassium channel. Nature 364:802–806, 1993.)

Figure 7-20B shows a series of single channel currents from a particular type of Kir channel, with equal concentrations of K+ on both sides of the membrane and Mg2+ on the cytosolic side. Under these conditions, the channel conducts K+ current only in the inward direction. An I-V plot derived from such data (see Fig. 7-20C) shows typical inward rectification of the unitary current. At negative values of Vm, the inward current decreases linearly as voltage becomes more positive, and no outward current is present at positive values of Vm. However, when Mg2+ is omitted from the cytosolic side of the membrane, the channel exhibits a linear or ohmic I-V curve even over the positive range of Vm values. Thus, the inward rectification in this example is due to intracellular block of the channel by Mg2+. Selective inhibition of outward K+ current in the presence of intracellular Mg2+ results from voltage-dependent binding of this divalent metal ion. Positive internal voltage favors the binding of Mg2+ to the inner mouth of this channel (see Fig. 7-20D), as would be expected if the Mg2+ binding site is located within the transmembrane electrical field. Because Mg2+ cannot permeate the channel, it acts as a blocker of outward K+ current. However, negative values of Vm pull the Mg2+ out of the channel. Moreover, incoming K+ tends to displace any remaining Mg2+. Thus, the Kir channel favors K+ influx over efflux. Under physiological conditions, intracellular cationic polyamines such as spermine and spermidine imageN7-16 block outward movements through Kir channels in a fashion similar to Mg2+ and largely account for the Kir rectification. In skeletal muscle, the Kir2.1 channel (see Table 6-2, family No. 2) appears to function in K+ re-uptake from the T-tubule system during prolonged periods of muscle contraction associated with a rise in extracellular [K+] and muscle fatigue.


Spermine and Spermidine

Contributed by Ed Moczydlowski

Spermine and spermidine are important regulators of numerous processes associated with cell growth. These polyamines modulate the activity of many cation channels in addition to having the blocking effect on Kir channels described here.

The Kir family of K+ channels exhibits various modes of regulation. One Kir subfamily (the G protein–activated inwardly rectifying K+ channels or GIRKs) is regulated by the βγ subunits of heterotrimeric G proteins (see pp. 51–66). For example, stimulation of the vagus nerve slows the heartbeat because the vagal neurotransmitter acetylcholine binds to postsynaptic muscarinic receptors in the heart that are coupled to G proteins. The binding of acetylcholine to muscarinic receptors causes the release of G-protein βγ subunits, which diffuse to a site on neighboring GIRK channels to activate their opening. The resulting increase in outward K+ current hyperpolarizes the cardiac cell, imageN7-17 thereby slowing the rate at which Vm approaches the threshold for firing action potentials and lowering the heart rate. GIRK channels are also activated by the membrane phospholipid phosphatidylinositol 4,5-bisphosphate (PIP2). Thus, G protein–coupled receptors that activate phospholipase C lead to the release of PIP2, thereby activating GIRK channels.


Hyperpolarization by Activation of GIRKs

Contributed by Ed Moczydlowski

Even though Kir channels pass current better in the inward than outward direction, the membrane potential (Vm) is typically never more negative than EK. Thus, net inward K+ current does not occur physiologically. As a result, the activation of GIRK channels hyperpolarizes cardiac cells by increasing K+ conductance or outward K+ current.

Another subfamily of Kir K+ channels known as KATP channels are directly regulated by adenine nucleotides. KATP channels are present in the plasma membrane of many cell types, including skeletal muscle, heart, neurons, insulin-secreting β cell of the pancreas, and renal tubule. These channels are inhibited by intracellular ATP and activated by ADP in a complex fashion. They are believed to provide a direct link between cellular metabolism, on the one hand, and membrane excitability and K+ transport, on the other. For example, if cellular ATP levels fall because of oxygen deprivation, opening of such channels hyperpolarizes the cell to suppress Ca2+ influx and firing of action potentials, and further reduce energy expenditure. Pancreatic β cells respond to an increase in plasma [glucose] by elevating their uptake of glucose and, thus, glucose metabolism and the cytoplasmic ATP/ADP ratio. This increased ratio inhibits enough KATP channels to cause a small depolarization, which in turn activates voltage-gated Ca2+ channels, resulting in Ca2+-dependent insulin secretion (see pp. 1039–1041).

KATP channels are the target of a group of antidiabetic drugs called sulfonylureas that include tolbutamide and glibenclamide. Sulfonylureas are used in the treatment of type 2 (or non–insulin-dependent) diabetes mellitus because they inhibit pancreatic KATP channels and stimulate insulin release. Another chemically diverse group of drugs (e.g., pinacidil and cromakalim) called K+ channel openers activate KATPand BKCa channels. Such K+ channel openers may have therapeutic potential in light of their ability to relax various types of smooth muscle. The ability of sulfonylurea drugs to inhibit KATP channels depends on an accessory subunit called the sulfonylurea receptor (SUR). This protein is a member of the ATP-binding cassette family of proteins (see pp. 119–120), which include two nucleotide-binding domains.

The most recently discovered family of K+ channels is that of the two-pore or K2P channels, which consist of a tandem repeat of the basic Kir topology (see Fig. 6-20F). Because the monomeric subunit of K2P channels contains two linked S5-P-S6 pore domains, a dimer of the monomer contains four S5-P-S6 pores and thereby forms a functional channel. K2P channels have been implicated in the genesis of the resting membrane potential. K+ channels encoded by the 15 human genes for K2P channels may be activated by various chemical and physical signals including PIP2, membrane stretch, heat, high intracellular pH, and general anesthetics. These channels are thought to be involved in a wide range of sensory and neuronal functions.