Channels and Pores

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+
+
6.6
the luminal pH of the stomach can reach 0.8 (0.15 M H ). The cells transport H against a concentration gradient of 1 × 10 . Assuming there is no electrical component, the energy for H+ secretion under these conditions can be calculated from Eq. 5.1 and is 9.1 kcal mol–1 of HCl.
5.6— Channels and Pores
Channels and Pores in Membranes Function Differently
Membrane channels are differentiated from membrane pores on the basis of their degree of specificity for molecules crossing the membrane. Channels are selective for specific inorganic cations and anions, whereas pores are not selective, permitting inorganic and organic molecules to pass through the membrane. The Na+ channel of plasma membranes of eukaryotic cells, for example, permits movement of Na+ at a rate more than ten times greater than that for K+. This difference between channels and pores is due to differences in size of the aqueous area created in the protein structure as well as amino acid residues lining the channel area. Channels and pores are intrinsic membrane proteins and amino acid sequences in the proteins of many channels suggest existence of structurally related superfamilies of proteins in which similar amino acid sequences occur. A common motif is a structure formed by amphipathic a ­helices of associated protein subunits or from domains within a single polypeptide chain creating a central aqueous space as pictured in Figure 5.33. Exceptions to the a ­helical structure are the porins (see below) of Gram­negative bacteria, which have a ,b ­sheet structure lining the central pore. The opening and closing of membrane channels involve a conformational change in the channel protein.
Figure 5.33 Arrangement of protein subunits or domains to form a membrane channel.
Opening and Closing of Channels Are Controlled
As indicated in Table 5.4, the opening and closing of some channels can be controlled by changes in the transmembrane potential. These are referred to as voltage­gated channels. In the case of the Na+ channel, depolarization of the membrane leads to an opening of the channel. Voltage­gated channels for Na+, K+, and Ca2+ are present in the plasma membrane of most cells. Clinical Correlation 22.8 (p. 956) describes changes in voltage­gated channels in myotonic muscle disorders. Mitochondria have a voltage­dependent channel for anions. Binding of a specific agent, termed an agonist, can also control the opening of a channel. A channel opens in the nicotinic–acetylcholine receptor on binding of acetylcholine allowing the flow of Na+ into the cell. This mechanism is important to neuronal electrical signal transmission (see p. 928). In addition, some channels are controlled by cAMP (see p. 862); Clin. Corr. 5.3 describes the modification of the Cl– channel in cystic fibrosis. These forms of control for opening channels are very fast, permitting bursts of ion flow through the membrane at rates of over 107 ions s–1, which is near the diffusion rate of these ions in water. This rate is necessary because these channels are involved in nerve conduction and muscle contraction. A number of pharmacological agents that modulate these channels are used therapeutically.
Sodium Channel
Voltage­sensitive sodium channels mediate rapid increase in intracellular Na+ following depolarization of the plasma membrane in nerve and muscle cells. The channel consists of a single large glycopolypeptide and several smaller glycoproteins. The genes for some of the Na+ channels have been cloned and the amino acid sequences have been determined. There are four repeat homology units, each with six transmembrane a ­helices. A model for this trans­
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CLINICAL CORRELATION 5.3 Cystic Fibrosis and the Cl– Channel
Cystic fibrosis (CF), an autosomal recessive disease, is the commonest, fatal, inherited disease of caucasians, occurring with a frequency of 1 in 2000 live births. It is a multi­
organ disease, with a principal manifestation being pulmonary obstruction; thick mucous secretions obstruct the small airways allowing recurrent bacterial infections. Exocrine pancreatic dysfunction occurs early and leads to steatorrhea (fatty stool) in CF patients; see page 1059 for a discussion of the role of the pancreas in fat digestion and absorption. CF patients have reduced Cl– permeability, which impairs fluid and electrolyte secretion, leading to luminal dehydration. Diagnosis of CF is confirmed by a significant increase of Cl– in sweat of affected in comparison to normal individuals.
The gene responsible for CF was identified in 1989 and over 400 mutations leading to CF have been found. The most common mutation (about 70%) leads to a deletion of a single phenylalanine at position 508 on the protein, but missense, nonsense, frameshift, and splice­junction mutations (see p. 628) have also been reported. The CF gene product is the cystic fibrosis transmembrane conductance regulator (CFTR), which is a cAMP­dependent Cl– channel. CFTR is composed of 1480 amino acids with structural homology to a family of transport proteins termed the transport ATPases. The gene has been cloned (see p. 765) and a major effort is under way to treat the disease by gene therapy, using both viral and nonviral vectors including liposomes (see Clin. Corr. 5.1).
Alton, E. W., and Geddes, D. M. Gene therapy for cystic fibrosis; a clinical perspective. Gene Ther. 2:88, 1995; Frizzell, R. A. Functions of the cystic fibrosis transmembrane conductance regulator protein. Am. J. Respir. Crit. Care Med. 151:54, 1995; and Wagner, J. A., Chao, A. C., and Gardner, P. Molecular strategies for therapy of cystic fibrosis. Annu. Rev. Pharmacol. Toxicol. 35:257, 1995.
Figure 5.34 Possible model of the Na+ channel. (a) The single peptide consists of four repeating units with each unit folding into six transmembrane helices. (b) Proposed arrangement of the transmembrane sequences as viewed down on the membrane. Redrawn from M. Noda et al., Nature 320:188, 1986.
porter is presented in Figure 5.34a and a possible arrangement of the helices in the membrane as viewed down on a membrane is presented in Figure 5.34b. One transmembrane segment, labeled S4, has a positively charged amino acid at every third position and may serve as a voltage sensor. A mechanical shift of this region due to a change in the membrane potential may lead to a conformational change in the protein, resulting in the opening of the channel. The channel size created by the protein, however, cannot totally explain the specificity for Na+.
Nicotinic–Acetylcholine Channel (nAChR)
The nicotinic–acetylcholine channel, also referred to as the acetylcholine receptor, is an example of a chemically regulated channel, where the binding of acetylcholine (Figure 5.35) opens the channel. The dual name is used to differentiate this receptor from other acetylcholine receptors, which function in a different manner. Acetylcholine, a neurotransmitter, is released at the
Figure 5.35 Structure of acetylcholine.
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neuromuscular junction by a neuron when electrically excited. The acetylcholine diffuses to the skeletal muscle membrane where it interacts with the acetylcholine receptor, opening the channel and allowing selective cations to move across the membrane (see p. 928). The change in transmembrane potential leads to a series of events culminating in muscle contraction. The nicotinic–acetylcholine receptor consists of five polypeptide subunits, with two a subunits and one each of b , g, and d ; each a subunit is phosphorylated and glycosylated and two others contain covalently bound lipid. The channel opens when two acetylcholine molecules bind to subunits and cause a change in protein conformation; reclosure of the channel occurs within a millisecond due to hydrolysis of acetylcholine to acetate and choline and release of bound ligand. A desensitized state of the receptor has been reported that does not open when acetylcholine binds. In the open conformation, cations and small nonelectrolytes can flow through the channel but not anions; negatively charged amino acid residues in the channel are sufficient to repel negatively charged ions from passing.
The nicotinic–acetylcholine receptor is inhibited by a number of deadly neurotoxins including d­tubocurarine, the active ingredient of curare, and several toxins from snakes including a ­bungarotoxin, erabutoxin, and co­bratoxin, the latter from the cobra. Succinylcholine, a muscle relaxant, activates the channel leading to depolarization of the membrane; succinyl choline is used in surgical procedures because its activity is reversible due to the rapid hydrolysis of the compound after cessation of administration.
Examples of Pores Are Gap Junctions and Nuclear Pores
Plasma membrane gap junctions and nuclear membrane pores are relatively large aqueous openings in the membrane created by specific proteins. Gap junctions are clusters of membrane channels lined by proteins spanning two plasma membranes that create aqueous connections between two cells. They permit the exchange between cells of ions and metabolites but not large molecular weight compounds such as proteins. The diameter of the opening ranges from 12 to 20 Å. Oligomers of the gap junction polypeptide (32 kDa), referred to as connexin, form the channel. Twelve subunits, six from each cell, form a hexameric structure in each membrane as shown in Figure 5.36. The channels are normally open but increases in cytosolic Ca2+, a change in metabolism, a drop in transmembrane potential, or acidification of the cytosol cause closure. When the channel is open subunits appear to be slightly tilted
Figure 5.36 Model for a channel in the gap junction.