The primary structure of voltagegated ion channels

The substantial voltages generated by the electric organ of the electric eel depend on the additive discharge of a large number of cells that are derived embryonically from muscle (see Fig. 2.4g). Their electrical excitability involves an increase of sodium permeability in the usual way, and this type of electric organ therefore provided ideal material first for isolating and purifying the sodium channel protein, and then for enabling its amino acid sequence to be determined. The initial biochemistry was greatly facilitated by the fact that the protein could be labelled with high specificity by the Japanese puffer-fish poison tetrodotoxin (TTX). The sodium channel was shown to be a single large peptide with a molecular mass of about 260kDa, which is glycosylated at several points on incorporation in the membrane.

A team of scientists led by Numa and Noda at Kyoto University successfully cloned and sequenced the cDNA of the sodium channel both in the Electrophorus electric organ and in rat brain. This was quickly followed up elsewhere by the cloning of voltage-gated potassium channels, most notably the Shaker gene of the fruit fly Drosophila, and of voltage-gated calcium channels such as the dihydropyridine receptor in muscle (see p. 141). It has now become clear that there is a large family of membrane proteins selective for K+, Na+ or Ca2+ ions, and gated not only by voltage but also in a variety of other ways, whose primary structures are all closely related. Taking the Electrophorus sodium channel as the prototype, the protein is a large monomer containing 1820 amino acid residues. The a-subunit shown diagrammatically in Fig. 5.1 can, when expressed on its own, produce a normally functioning sodium channel. It consists of four homologous domains labelled I, II, III and IV that span the membrane, and which have closely similar amino acid sequences. The size and structure of calcium channels are much the same, as are potassium channels, except that they are tetramers built up of four identical domains.

Of the 20 possible amino acids that make up a protein, it may be seen in Table 5.1 that the residues of eight are non-polar, seven are polar but uncharged, two are acidic and carry a negative charge, and three are basic and positively charged. The non-polar residues are hydrophobic, and therefore tend to be located in the centre of the molecule in the lipid core of the membrane. The polar or charged residues are hydrophilic, and are more likely to be found in the aqueous environment of the cytoplasm or at the outer surface of

Fig. 5.1. Primary structures of the a- and ^1-subunits of a sodium channel illustrated as transmembrane folding diagrams. The bold lines are polypeptide chains with the length of each segment roughly proportional to its true length in a rat brain sodium channel. The cylinders represent probable transmembrane a-helices, and parts of the external links between transmembrane segments S5 and S6 are shown as tucked back into the membrane to form the external pore. Sites are indicated of experimentally demonstrated glycosylation cAMP-dependent phosphorylation (P in a circle), protein kinase C phosphorylation (P in a diamond), amino acid residues required for TTX binding (ScTx), and of the inactivation particle (h in a circle). From Catterall (1992) with permission of the American Physiological Society.

Fig. 5.1. Primary structures of the a- and ^1-subunits of a sodium channel illustrated as transmembrane folding diagrams. The bold lines are polypeptide chains with the length of each segment roughly proportional to its true length in a rat brain sodium channel. The cylinders represent probable transmembrane a-helices, and parts of the external links between transmembrane segments S5 and S6 are shown as tucked back into the membrane to form the external pore. Sites are indicated of experimentally demonstrated glycosylation cAMP-dependent phosphorylation (P in a circle), protein kinase C phosphorylation (P in a diamond), amino acid residues required for TTX binding (ScTx), and of the inactivation particle (h in a circle). From Catterall (1992) with permission of the American Physiological Society.

the membrane. From a study of what is known as the hydropathy index of different stretches of the amino acid chain it has been deduced that each of the homologous domains comprises six segments that are largely hydropho-bic and form a-helices crossing the membrane from one side to the other. These segments are represented as cylinders in Fig. 5.1. The amino acid sequences of segments S2, S3 and S4 in the voltage-gated sodium and potassium channels of some typical species are shown in Fig. 5.2. Those of voltage-gated calcium channels are very similar.

A specific requirement of the channels with which this chapter is concerned is that the structure should incorporate voltage-sensing elements that will respond to alterations in the electric field across the membrane. The best

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Fig. 5.2. The amino acid sequences of the charge-carrying S2, S3 and S4 transmembrane segments of all four domains of voltage-gated K+ channels, and of the individual domains of Na+ channels, for the squid Loligo opalescens, the fly Drosophila and rat brain. Positively charged arginine residues (R) and lysine residues (K) are in bold type and the stretches over which they are separated by two non-polar residues are underlined. Negatively charged residues of aspartate (D) and glutamate (E) are ringed. Data selected from Figs. 1, 3 and 4 of Keynes & Elinder (1999).

candidates to act as voltage-sensors are agreed to be the S4 segments whose sequences are shown in Fig. 5.2, which are capable of moving across the membrane to a limited extent in a screwlike fashion. Each one carries between four and eight positively charged arginine or lysine residues, always separated by a pair of non-polar or in a few cases uncharged polar residues. They operate as explained below in conjunction with segments S2 and S3, on which are located altogether three negatively charged aspartate or glutamate residues in fixed positions.

An inevitable limitation of cDNA sequencing studies is that although they provide a wealth of accurate information about the primary structure of membrane proteins, it is necessary to depend on indirect and often speculative arguments to decide exactly how the molecule is folded, and to elucidate the nature of the conformational changes that bring about the opening and closing of the channels.

An additional tool that is particularly valuable in the study of voltage-gated and ligand-gated ion channels is the ability to express the proteins whose primary structures have been determined, by the injection of the corresponding messenger RNA into the oocytes of the African clawed toad Xenopus. These are large cells that are about to develop into mature eggs. They possess the normal translation machinery, and will respond to the injection of messenger RNA by making the protein for which it codes and incorporating it in the membrane. After synthesizing the corresponding messenger RNA, the majority of the voltage-gated and ligand-gated channel proteins that have so far been isolated have been successfully expressed in such oocytes, and have been shown either by patch-clamping or by recording macroscopic single-cell currents to behave in an essentially normal fashion. An important extension of the technique is then to alter the sequence of the amino acid residues by the procedure known as site-directed mutagenesis to explore in detail the effect of artificial modifications of the protein structure.

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