1.1 Structure of the Cell Membrane
Cell membranes are made up of amphipathic phospholipids with polar (hydrophilic) heads and apolar (hydrophobic) fatty acid tails (Fig. 1.1). Amphipathic molecules tend to arrange themselves to minimize the contact of hydrophobic portions with water. This causes the spontaneous formation of a lipid bilayer. Cholesterol molecules in the lipid bilayer affect membrane fluidity.
Integral membrane proteins are embedded in the lipid bilayer and have contact with both the extracellular and intracellular fluid. Most have components that span the bilayer multiple times. Peripheral membrane proteins are associated with either the phospholipid (hydrophilic) heads or other embedded proteins.
Oligosaccharide residues combine with lipids and proteins on the outer cell membrane surface to form glycolipids and glycoproteins.
– Glycolipids and glycoproteins both contribute to structural stability of cell membranes.
– Glycoproteins are also important for cell recognition and immune response.
Tight junctions are attachments between epithelial cells.
– True “tight” junctions prevent the movement of dissolved molecules and water from one side to the other.
– “Leaky” tight junctions act as a pathway for solutes and water to cross epithelial cell layers.
Gap junctions are channels between cells that permit intercellular communication.
– Small molecules (ions, adenosine triphosphate [ATP], cyclic adenosine monophosphate [cAMP], etc.) can pass through gap junctions.
– Gap junctions electrically couple cells, so they act together as a functional syncytium (e.g., in the heart and smooth muscles of the gut).
1.2 Transport across Membranes
Selective transport of substances across cell membranes allows cells to regulate their internal content and to carry out crucial functions such as secretion and absorption, which are controlled by neural and hormonal activity.
Fig. 1.1 Structure of the cell membrane.
The cell membrane consists of a phospholipid bilayer. Each phospholipid molecule has a glycerol head (hydrophilic) and two fatty acid tails (hydrophobic). These hydrophobic tails are arranged so that they face each other in the bilayer. Integral proteins and cholesterol are embedded within the bilayer. Other proteins may lie peripherally. Carbohydrate moieties may bind to lipids and proteins on the extracellular surface of the membrane, forming glycolipids and glycoproteins.
Free diffusion is the migration of molecules from a region of higher concentration to one of lower concentration as a result of random motion (Fig. 1.2).
– Free diffusion does not require external energy and is therefore passive.
– Example: Oxygen (O2) and carbon dioxide (CO2) move across cell membranes down their concentration gradients by diffusion.
Fick’s first law of diffusion states that the net flow of a substance (J) is proportional to the membrane permeability (P), the concentration gradient (ΔC), and the available area for diffusion (A):
J = PA (ΔC)
J = net flow (mmol/s)
P = membrane permeability (cm/s)
A = area (cm2)
ΔC = concentration difference (mmol/cm3)
Membrane permeability. Membrane permeability is a variable in Fick’s law and is increased by
– ↑ lipid solubility of the solute
– ↓ membrane thickness
– ↓ size of the solute
Lipid-soluble, small, nonionized substances are most permeable (Fig. 1.3).
Fig. 1.2 Passive transport: free diffusion and uniport.
Free diffusion occurs when a substance moves across a membrane down its electrochemical gradient. When this process requires a transport (carrier) protein, it is known as uniport or facilitated diffusion. It is a conformational change in the transport protein that permits this membrane transport. Both of these forms of membrane transport are passive because they do not require energy.
Fig. 1.3 Permeability of membranes.
Small apolar and polar uncharged molecules can diffuse freely through cell membranes. Large molecules and charged molecules cannot diffuse freely and must be transported to cross a membrane.
Carriers are integral membrane proteins that transport substances that are hydrophilic or too large to cross the membrane by simple diffusion. They also permit faster transport of lipid-soluble substances than simple diffusion. Carrier proteins possess the following characteristics:
– Selectivity: Most carriers exhibit a preference for just one or a small class of solutes.
– Competition for binding sites: Structurally related compounds can compete for binding sites and inhibit the binding of the related solute to the carrier protein; for example, glucose and galactose compete with each other for absorption into enterocytes by Na+-dependent cotransport (SGLT1).
– Saturation of carrier proteins: The rate of carrier-mediated transport may show saturation at high solute concentrations, as the number of carrier proteins is finite, and the cycling of carrier proteins is limited.
Uniport (formerly called facilitated diffusion) is a carrier-mediated transport mechanism that moves solutes down their electrochemical gradients (Fig. 1.2).
– It does not use metabolic energy and is therefore passive.
– Example: In the transport of glucose into red blood cells, L-glucose cannot enter red blood cells by simple diffusion. D-glucose enters via a protein glucose transporter that transports other sugars poorly. D-glucose transport saturates when all the transporters are being used.
Primary Active Transport
Primary active transport is a carrier-mediated transport mechanism that moves molecules against their electrochemical gradients (Fig. 1.4).
– It requires metabolic energy and uses ATP as the direct energy source.
– Example: Na+−K+ ATPase (carrier protein) pumps Na+ out of the cell and K+ into the cell against their concentration gradients. It maintains a low intracellular [Na+] and high intracellular [K+] ratio. Three Na+ ions are transported out of the cell for every two K+ ions that are transported into the cell (Fig. 1.5).
– Example: Ca2+−ATPase transports Ca2+ back into Ca2+ stores in a muscle cell and out of a muscle cell after the influx of Ca2+ triggers muscle contractions.
H+−K+ ATPase (the proton pump)
H+−K+ ATPase is an integral transmembrane protein that is present in gastric parietal cells. It functions to actively transport H+ into the lumen of the stomach, against its electrochemical gradient, in exchange for K+ (one H+ is exchanged for one K+). The energy required to drive this exchange is derived from the hydrolysis of ATP. In the lumen, Cl− and H+ combine to form gastric acid. Proton pump inhibitors (e.g., omeprazole) inhibit the H+−K+ ATPase pump, thus inhibiting gastric acid secretion into the lumen of the stomach. These drugs are used to treat peptic ulcers and gastroesophageal reflux disease (GERD).
Digoxin is a cardiac glycoside that was once one of the first-line agents used in the treatment of heart failure. Its use is now reserved for cases when symptoms are not fully treated by standard therapies or in cases of severe heart failure while standard therapies are initiated. The therapeutic and toxic effects of digoxin are attributable to inhibition of Na+−K+ ATPase (the digitalis receptor) located on the outside of the myocardial cell membrane. When the pump is inhibited, Na+ accumulates intracellularly. The decreased Na+ gradient that results from this affects Na+−Ca2+ exchange, and Ca2+ accumulates intracellularly. Consequently, more Ca2+ (stored in the sarcoplasmic reticulum) is available for release and interaction with the contractile proteins in these cells during the excitation–contraction coupling process. At therapeutic doses of digoxin, there is an increase in contractile force. Toxicity to digoxin also relates to inhibition of Na+−K+ ATPase. Inhibition of the Na+−K+ pump affects the K+ gradient; this may lead to a significant reduction of intracellular K+, predisposing the heart to arrhythmias. Likewise, high levels of Ca2+ intracellularly may contribute to serious arrhythmias.
Fig. 1.4 Active transport.
Active transport occurs when a substance is transported across a membrane against its electrochemical gradient by transport proteins. This process requires energy in the form of adenosine triphosphate (ATP), therefore it is active. The transport protein (an ATPase) binds the substance on one side of the membrane, and ATP-dependent phosphorylation causes a conformational change that releases it on the other side of the membrane.
Secondary Active Transport
Secondary active transport is a carrier-mediated transport mechanism that uses the downhill movement of one substance to move another substance uphill. The flow of one species down its electrochemical gradient powers the actively transported species against its electrochemical gradient (Fig. 1.6). The electrochemical gradient for Na+ is usually maintained by the Na+−K+ ATPase pump.
– Symporters carry the substrate and cosubstrate in the same direction.
– Example: In Na+−glucose cotransport in the small intestine and kidney, Na+ transported into cells brings glucose with it.
– Antiporters carry the substrate and cosubstrate in opposite directions.
– Example: HCO3−−Cl− countertransport at red blood cell membranes.
– Example: 3Na+−Ca2+ countertransport at cardiac muscle cell membranes.
Fig. 1.5 Na+−K+ ATPase.
The Na+−K+ ATPase (Na+−K+ pump) is present in all cell membranes. It consists of two α subunits and two β subunits. The α subunits are phosphorylated, causing a conformational change that allows them to form the ion transport pathway. During one transport cycle, three Na+ ions are pumped out of the cell, and two K+ ions are pumped into the cell by the Na+−K+ ATPase using one molecule of adenosine triphosphate (ATP). Both Na+ and K+ ions are transported against their concentration gradients. (ADP, adenosine diphosphate)
Fig. 1.6 Secondary active transport.
Secondary active transport occurs when uphill transport of a substance via a carrier protein (e.g., sodium–glucose transport type 2 [SGLT2]) is coupled with the downhill transport of an ion (Na+ in this example) (1). In this case, the electrochemical Na+ gradient into the cell (maintained by Na+−K+ATPase) provides the driving force for the cotransport of glucose into the cell. The SGLT2 is an example of a symporter, as Na+ and glucose are transported in the same direction. Examples 2 and 3 also illustrate symport. Antiport occurs when the compound and driving ions are transported in opposite directions. For example, when an electrochemical Na+ gradient drives H+ in the opposite direction (4).
Transport of Water across Membranes
Osmosis is the net diffusion of water across a semipermeable (permeable to water but not solutes) membrane. The osmolarity of a solution is the concentration of osmotically active particles in the solution. The net movement of water across a semipermeable membrane is due to the concentration differences of the nonpenetrating solutes. Water diffuses from a low osmolarity solution (high water concentration, low solute concentration) to a high osmolarity solution (low water concentration, high solute concentration) in attempting to achieve equal water concentrations on both sides of the membrane (Fig. 1.7).
Fig. 1.7 Water output and intake from the cell by osmosis.
In a hypertonic environment, there is a higher concentration of solutes outside the cell than inside, so water moves out of the cell by osmosis. In a hypotonic environment, there is a higher concentration of solutes inside the cell, so extracellular water moves into the cell by osmosis.
Osmotic Pressure. The osmotic pressure of a solution is the theoretical hydrostatic pressure that would be required to just prevent the osmotic flow of water across a semipermeable membrane. Numerically, it is simply a constant multiplied by the osmolality.
The flow of water through a membrane is expressed by the van’t Hoff equation:
Jv = Kc × Δπ
Jv = water flow (mL/min)
Kc = hydraulic conductivity of the membrane (mL/mm/mmHg)
Δπ = osmotic pressure difference (mOsm/g water)
Reflection Coefficient. The reflection coefficient is a number between 0 and 1 that describes the ability of a membrane to prevent diffusion of a solute relative to water.
– If the reflection coefficient is 1, the solute is completely impermeable and will not pass through the membrane. Serum albumin has a reflection coefficient that is close to 1. This explains why albumin is retained in the vascular compartment and exerts an osmotic effect.
– If it is 0, the solute will pass through the membrane as easily as water and will not exert any osmotic effect (i.e., it will not cause water to flow).
1.3 Receptors and Signal Transduction
Types of Receptor
Ligand-gated Ion Channels
Ligand-gated ion channels are specialized membrane pores made up of multisubunit proteins. Binding of ligands (e.g., hormones or neurotransmitters) to these receptors opens or closes the pores, thus changing the permeability of the membrane to Na+, K+, Cl−, or other ions (Fig. 1.8).
Fig. 1.8 Ligand-gated ion channel.
An example of a ligand-gated ion channel is the nicotinic receptor of the motor end plate. When two acetylcholine (ACh) molecules bind to this receptor simultaneously (at the α subunits), the inner pore opens; Na+ then enters the cell, and K+ leaves the cell. This causes membrane depolarization.
− Examples: Nicotinic receptor for acetylcholine (see Fig. 2.2), the glutamate receptor, and the gamma-aminobutyric acid type A (GABAA) receptor
G-Protein Coupled Receptors. G proteins facilitate signal transduction that is initiated by ligand-receptor binding and culminates in a cellular response. The mechanisms of G-protein signal transduction are discussed on page 9.
Voltage-dependent Ion Channels
Voltage-dependent ion channels open or close in response to changes in the membrane potential.
– Example: Depolarization opens the activation gate of Na+ channels, allowing Na+ to flow into cells.
Calcium channel blockers
Calcium channel blockers (e.g., verapamil and nifedipine) inhibit Ca2+ entry into cells via voltage-dependent ion channels. In smooth muscle cells, this produces arterial vasodilation, which leads to reduced coronary artery spasm, decreased blood pressure, and reduced cardiac work. In cardiac muscle cells, these agents inhibit cardiac functions, causing decreased heart rate, atrioventricular conduction, and contractility. Nifedipine acts predominantly on smooth muscle cells to produce vasodilation and has almost no effect on cardiac function at therapeutic doses. Verapamil acts on both smooth muscle cells and heart muscle cells.
Enzyme-linked Membrane Receptors
When a ligand binds to this receptor, it causes an enzyme to become “switched on” intracellularly. This enzyme then catalyzes the formation of other signal proteins that ultimately lead to the drug’s effect.
– Example: Insulin “switches on” the tyrosine kinase activity of the insulin receptor to affect glucose uptake into cells (Fig. 1.9).
Fig. 1.9 Enzyme-linked membrane receptor.
Insulin binding to the receptor causes the enzyme, tyrosine kinase, to phosphorylate tyrosine residues in proteins. These proteins can then signal other proteins to be formed, thus exerting the physiological effect.
Fig. 1.10 Intracellular receptor.
Lipophilic substances, such as steroid hormones and thyroid hormones, can diffuse through the cell membrane and interact with receptors in the cytoplasm or nucleus. The hormone-receptor complex then alters gene transcription causing proteins that exert the physiological effect to be made. The hormone-receptor complex interacts with DNA in pairs that may be identical (homodimeric) or nonidentical (heterodimeric).
Lipid-soluble substances diffuse through cell membranes and bind either to receptors in the cellular cytosol or in the nucleus. Gene expression is altered, and protein synthesis is either increased or decreased, which causes the cellular response (Fig. 1.10).
– Examples: Steroid hormones, calcitriol, and thyroxine
G-Protein Coupled Receptors and Signal Transduction
Heterotrimeric G proteins couple to membrane receptors (e.g., α-adrenergic receptors). When the receptor binds a ligand, this causes the α-subunit of the G protein to split from the β and γsubunits. The now free subunits then interact with other proteins in the membrane that may produce second messengers (Fig. 1.11). These second messengers are cAMP, diacylglycerol (DAG), and inositol 1,4,5-triphosphate (IP3).
– Gs proteins activate adenylate cyclase.
– Gi proteins inhibit adenylate cyclase.
– Gq proteins activate phospholipase C, which then activates DAG and IP3.
Fig. 1.11 Signal transduction by G proteins.
A substance binding to a G-protein coupled receptor alters its conformation and causes the α subunit of the attached G protein to exchange guanosine diphosphate (GDP) for guanosine triphosphate (GTP) (1). The G protein then separates from the receptor and dissociates into an α subunit and a βγ subunit. In the case illustrated, the α subunit activates adenylate cyclase, which promotes cyclic adenosine monophosphate (cAMP) production (2). The cAMP then acts as a second messenger, activating protein kinase A, which, in turn, activates further proteins (see Fig. 1.12). The intrinsic GTPase activity of the α subunit hydrolyzes bound GTP to GDP, thereby terminating the effect of the G protein. (ATP, adenosine triphosphate; PPi, diphosphate)
When G proteins are activated, guanosine triphosphate (GTP) replaces guanosine diphosphate (GDP) on the α subunit. Following activation of G proteins, GTP is rapidly degraded to inactive GDP by the activity of the α-subunit GTPase.
Second Messenger Systems
Adenylate Cyclase System. Gs-activating substances bind to a receptor that activates Gs, which, in turn, stimulates adenylate cyclase to convert ATP to cAMP, which then activates protein kinase A. This phosphorylates proteins, resulting in the physiologic response. Following its activation, cAMP is degraded to 5′ AMP by phosphodiesterase (Fig. 1.12).
Gi-activating substances bind to a receptor that activates Gi, which inhibits adenylate cyclase (↓cAMP). Therefore, protein kinase A is not activated, and proteins are not phosphorylated.
DAG and IP3 System. The amplifier enzyme phospholipase C produces the second messengers IP3 and DAG from a single precursor (Fig. 1.13).
Hydrophilic IP3 diffuses from the membrane to organelles containing Ca2+ and releases it. The Ca2+ released can then cause physiologic effects in the following ways:
– Interaction with the cAMP system
– Activation of protein kinase C (with DAG) leading to the phosphorylation of proteins
– Binding to calmodulin with the resultant complex mediating further effects, for example, production of nitric oxide (Fig. 1.14)
Lipophilic DAG has two functions:
– Activation of protein kinase C (this process is Ca2+ dependent)
– Formation of arachidonic acid (an eicosanoid precursor) following its degradation by DAG lipase.
Phosphodiesterase inhibitors inhibit the degradation of cAMP and cyclic guanosine monophosphate (cGMP). Drugs that specifically inhibit phosphodiesterase type 5 (e.g., sildenafil citrate [Viagra]) cause prolonged vasodilation of penile arteries and are therefore used to treat erectile dys-function. Phosphodiesterase inhibitors type 3 (e.g., milrinone and inamrinone) increase cAMP levels in cardiac cells. This causes an increase in intracellular Ca2+, resulting in increased heart rate and force of contraction (positive chronotropic and inotropic effects). They also cause vasodilation of blood vessels. These agents are used as adjuvants in heart failure therapy. Side effects of phosphodiesterase inhibitors include headache and cutaneous flushing.
Fig. 1.12 Cyclic AMP.
Adenylate cyclase synthesizes cAMP by cleaving diphosphate (PPi) from ATP. Adenylate cyclase is regulated by G proteins (Gs and Gi), which are controlled by substances attaching to G-protein coupled 7-helix receptors. The cAMP activates protein kinase A (PKA), which then phosphorylates proteins, including enzymes, transcription factors, and ion channels.
Fig. 1.13 Diacylglycerol (DAG) and inositol 1,4,5-triphosphate (IP3).
Binding of a molecule to a G protein activates phospholipase C, which, in turn, activates DAG and IP3. Both DAG and IP3 act to phosphorylate effector proteins. DAG also forms eicosanoids. Ca2+ exerts further effects by forming a complex with calmodulin. (ECF, extracellular fluid; PIP2, phosphadidylinositol-4, 5-biphosphonate; cGMP, cyclic guanosine monophosphate)
Fig. 1.14 Nitric oxide (NO) as a transmitter substance.
Ca2+−calmodulin complex activates nitric oxide synthase, which catalyzes the formation of NO from arginine. NO is then able to diffuse to other cells, where it activates guanylate cyclase, which converts GTP to cyclic guanosine monophosphate (cGMP). The cGMP activates protein kinase G, which, in turn, decreases the intracellular Ca2+ concentration by blocking the IP3 receptors on Ca2+ stores. This culminates in vasodilation. (ECF, extracellular fluid; NADPH, reduced form of nicotinamide adenine dinucleotide phosphate; PIP2, phosphadidylinositol-4,5-biphosphonate, PLC, phospholipase C)
1.4 Membrane Potentials
Ion movement or flux is controlled by both concentration gradients and electrical gradients. If these gradients are equal but opposite in direction for a particular ion, then its total electrochemical potential is zero, and there is no net current flow. This is electrochemical equilibrium.
The equilibrium potential is the membrane potential for an ion that would exactly oppose the tendency of an ion to move down its concentration gradient. It is calculated using the Nernst equation:
E = −61/z × log [ion]out/[ion]in
E = equilibrium potential
z = the charge of the ion
– Example: The equilibrium potential for K+ is calculated as follows:
Resting Membrane Potential
The resting membrane potential (RMP) is the normal potential difference across a membrane (in millivolts). It is established as a result of the difference in concentration of permeable ions across the membrane (Fig. 1.15) as each of these ions tries to reach its equilibrium potential.
– Skeletal muscle cell membranes at rest are 30 times more permeable to K+ than Na+, so the RMP of −90 mV approaches the equilibrium potential for K+ of −96 mV.
Fig. 1.15 Resting potential.
The resting membrane potential results from the uneven distribution of positively and negatively charged ions inside and outside the cell. The Na+−K+ ATPase pump establishes concentration differences by pumping three Na+ ions out of the cell and two K+ ions into the cell. Some K+ ions flow back down this concentration gradient and leave the cell via K+ channels. The protein anions that predominate inside the cell cannot follow them. The result is a slight excess of positively charged ions outside the cell, with a slight excess of anions inside the cell. The force of attraction between positive and negative ions creates the resting membrane potential (RMP).
– Neural cells are only 6 times as permeable, so they have RMPs of −70 mV. Cl− distributes passively at the potential determined by primarily K+ and secondarily by Na+.
– The Na+−K+ ATPase pump is responsible for generating the concentration differences needed to cross the membrane.
– The RMP is stored energy (a battery) whose brief discharge generates an action potential that can transmit a signal rapidly for a long distance through the body.
1.5 Action Potentials
Table 1.2 defines terms that must be understood when considering action potentials.
Action Potential Events
Depolarization due to a graded potential reduces the RMP approximately −15 mV. Some fast voltage-gated Na+ channels are activated and open, allowing some Na+ to enter the cell (Fig. 1.16).
When the depolarization threshold is reached, the action potential is initiated. Inward current causes the membrane potential difference to move rapidly toward the Na+ equilibrium potential of +55 mV. During this rising phase of depolarization, the relative conductance of the membrane to Na+ increases to ~50 times the conductance of K+. In the overshoot phase of an action potential, the membrane potential passes zero and is reversed (positive).
Na+ channels close spontaneously within a millisecond after opening. This process is called inactivation. The conductance of K+ also increases. The decrease of Na+ conductance relative to K+ causes the membrane to repolarize back toward the RMP, which facilitates the repolarization.
The electrochemical gradient for Na+ is usually maintained by the Na+−K+ ATPase pump.
The membrane hyperpolarizes briefly at the end of the action potential as it passes the RMP. This is due to the greater than normal conductance of K+ during repolarization. The RMP is reestablished as Na+ and K+ conductances return to their resting states.
Fig. 1.16 Action potential and ion conductivity.
Following the binding of a neurotransmitter to an inotropic receptor on the postsynaptic membrane, the following steps occur that culminate in an action potential: (1) Voltage-gated Na+ channels open, and due to their high equilibrium potential, Na+ ions flow into the cell, causing depolarization. (2) The Na+ channels immediately close again, so the influx of positive charges is very brief. (3) Voltage-dependent K+ channels open, and K+ flows out of the cell. This results in repolarization of the membrane. (4) This briefly leads to the potential falling below resting membrane potential (RMP), and the membrane is hyperpolarized. The K+ channels then close, and if there are gap junctions, the neuron is ready for restimulation. The Na+−K+ATPase (labeled in the membrane) operates continuously to maintain the concentration gradient for Na+ and K+.
Absolute Refractory Period
The absolute refractory period is the period when the membrane cannot be stimulated to produce a second action potential regardless of the stimulus strength. It is the duration of the action potential and is due to the inactivation of the majority of membrane Na+ channels.
Relative Refractory Period
The relative refractory period occurs after the absolute refractory period. During this time, a significantly larger depolarization stimulus (inward flow) can initiate an additional action potential. This is due to a prolonged increase in K+ conductance during this time, which opposes depolarization as the membrane is farther from threshold.
Accommodation is a slight increase in threshold in response to prolonged subthreshold stimulation.
– It is caused by a continuous activation and inactivation of some Na+ channels and K+ channels, so fewer Na+ channels are available for activation. It may occur in smooth muscle cells of the gut.
Propagation of Action Potentials
When an action potential depolarizes one portion of a membrane, local currents (Na+ influx) depolarize adjacent areas of the membrane, bringing them to threshold. Thus, the action potentials are propagated.
The conduction velocity of action potentials moving along nerve axons is increased by
– ↑ axon radius: Thicker axons have lower intracellular resistance to local current flow, so conduction velocity is faster.
– Myelination: Conduction velocity is greatly increased in myelinated neurons due to the insulating effects of the myelin and because myelin allows for saltatory conduction. In saltatory conduction, Na+ currents can only flow at the nodes of Ranvier, where the myelin sheath is absent, so the action potential “jumps” from node to node (Fig. 1.17). This significantly increases conduction velocity and requires less energy.
Fig. 1.17 Continuous (1a, 1b) and saltatory (2) propagation of action potentials.
(1a) Na+ enters the nerve fiber, causing depolarization of the adjacent membrane. (1b) Na+ then enters the adjacent membrane. The region of depolarization moves. (2) Myelin insulation forces current to flow to the next node of Ranvier, causing depolarization there. Na+ then enters at the next node of Ranvier, ~1 mm along the nerve fiber.