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The primary structure of a voltage-dependent calcium channel from rabbit brain has been deduced by cloning and sequencing the complementary DNA. Calcium channel activity expressed from the cDNA is dramatically increased by coexpression of the alpha 2 and beta subunits, known to be associated with the dihydropyridine receptor. This channel is a high voltage-activated calcium channel that is insensitive both to nifedipine and to omega-conotoxin. We suggest that it is expressed predominantly in cerebellar Purkinje cells and granule cells.

The skeletal muscle dihydropyridine (DHP) receptor serves dual functions, as a voltage sensor for excitation-contraction coupling and as an L-type calcium channel. Biochemical analysis indicates the presence of two forms of the DHP receptor polypeptide in skeletal muscle, a full-length translation product present as a minor species and a much more abundant form that has a truncated carboxy-terminus. On the basis of these and other observations, it has been proposed that, in skeletal muscle, only the full-length DHP receptor can function as a calcium channel and that the truncated form can only function as a voltage sensor for excitation-contraction coupling. To resolve this issue, we have now constructed a complementary DNA (pC6 delta 1) encoding a protein corresponding to the truncated DHP receptor in skeletal muscle. Expression of pC6 delta 1 in dysgenic myotubes fully restores both excitation-contraction coupling and calcium current, consistent with the idea that a single class of DHP receptors performs both functions.

THE sodium channel, one of the family of structurally homologous voltage-gated ion channels1, differs from other members, such as the calcium and the potassium channels, in its high selectivity for Na+. This selectivity presumably reflects a distinct structure of its ion-conducting pore. We have recently identified two clusters of predominantly negatively charged amino-acid residues, located at equivalent positions in the four internal repeats of the sodium channel as the main determinants of sensitivity to the blockers tetrodotoxin and saxitoxin2. All site-directed mutations reducing net negative charge at these positions also caused a marked decrease in single-channel conductance2. Thus these two amino-acid clusters probably form part of the extracellular mouth and/or the pore wall of the sodium channel. We report here the effects on ion selectivity of replacing lysine at position 1,422 in repeat III and/or alanine at position 1,714 in repeat IV of rat sodium channel II (ref. 3), each located in one of the two clusters, by glutamic acid, which ccurs at the equivalent positions in calcium channels. These amino-acid substitutions, unlike other substitutions in the adjacent regions, alter ion-selection properties of the sodium channel to resemble those of calcium channels. This result indicates that lysine 1,422 and alanine 1,714 are critical in deter mining the ion selectivity of the sodium channel, suggesting that these residues constitute part of the selectivity filter of the channel.

MEMBRANE depolarization causes many kinds of ion channels to open, a process termed activation1. For both Na+ channels2–4 and Ca2+ channels5,6, kinetic analysis of current has suggested that during activation the channel undergoes several conformational changes before reaching the open state. Structurally, these channels share a common motif7: the central element is a large polypeptide with four repeating units of homology (repeats I-IV), each containing a voltage-sensing region, the S4 segment8–11. This suggests that the distinct conformational transitions inferred from kinetic analysis may be equated with conformational changes of the individual structural repeats8. To investigate the molecular basis of channel activation, we constructed complementary DNAs encoding chimaeric Ca2+ channels in which one or more of the four repeats of the skeletal muscle dihydropyridine receptor are replaced by the corresponding repeats derived from the cardiac dihydropyridine receptor. We report here that repeat I determines whether the chimaeric Ca2+ channel shows slow (skeletal muscle-like) 12 or rapid (cardiac-like) 13 activation.

THE skeletal muscle dihydropyridine (DHP) receptor is essential in excitation–contraction (EC) coupling1–4. The receptor is postulated to be the voltage sensor giving rise to the intramembrane current, termed charge movement5. We have now tested this hypothesis using myotubes from mice with the muscular dysgenesis mutation, which alters the skeletal muscle DHP receptor gene and prevents its expression3,4,6. Our results indicate that charge movement is deficient in dysgenic myotubes but is fully restored following injection of an expression plasmid carrying the rabbit skeletal muscle DHP receptor complementary DNA, strongly supporting the hypothesis that the DHP receptor is the voltage sensor for EC coupling in skeletal muscle. Additionally, our data obtained for normal and chimaeric DHP receptor constructs demonstrate that DHP receptors with widely differing abilities to function as calcium channels and to mediate EC coupling produce very similar charge movements

There are dihydropyridine (DHP)-sensitive calcium currents in both skeletal and cardiac muscle cells, although the properties of these currents are very different in the two cell types (for simplicity, we refer to currents in both tissues as L-type). The mechanisms of depolarization-contraction coupling also differ. As the predominant voltage-dependent calcium current of cardiac cells, the L-type current represents a major pathway for entry of extracellular calcium. This entry triggers the subsequent large release of calcium from the sarcoplasmic reticulum (SR). In contrast, depolarization of skeletal muscle releases calcium from the SR without the requirement for entry of extracellular calcium through L-type calcium channels. To investigate the molecular basis for these differences in calcium currents and in excitation-contraction (E-C) coupling, we expressed complementary DNAs for the DHP receptors from skeletal and cardiac muscle in dysgenic skeletal muscle. We compared the properties of the L-type channels produced and showed that expression of a cardiac calcium channel in skeletal muscle cells results in E-C coupling resembling that of cardiac muscle.

The Torpedo and calf acetylcholine receptors and hybrids composed of subunits from the two species have been produced in Xenopus oocytes by the use of the cloned complementary DNAs. Single-channel current measurements indicate that these receptors form channels of similar conductance but with different gating behaviour.

The sodium channel is a voltage-gated ionic channel essential for the generation of action potentials1–3. It has been reported that the sodium channels purified from the electric organ of Electrophorus electricus (electric eel)4,5 and from chick cardiac muscle6 consist of a single polypeptide of relative molecular mass (Mr) ∼260,000 (260K), whereas those purified from rat brain7 and skeletal muscle8 contain, in addition to the large polypeptide, two or three smaller polypeptides of Mr 37–45K. Recently, we have elucidated the primary structure of the Electrophorus sodium channel by cloning and sequencing the DNA complementary to its messenger RNA9. Despite the apparent homogeneity of the purified sodium channel preparations, several types of tetrodotoxin (or saxitoxin) binding sites or sodium currents have been observed in many excitable membranes10–19. The occurrence of distinguishable populations of sodium channels may be attributable to different states of the same channel protein or to distinct channel proteins. We have now isolated complementary DNA clones derived from two distinct rat brain mRNAs encoding sodium channel large polypeptides and present here the complete amino-acid sequences of the two polypeptides (designated sodium channels I and II), as deduced from the cDNA sequences. A partial DNA sequence complementary to a third homologous mRNA from rat brain has also been cloned.

Cloning and sequence analysis of cDNA for the Electrophorus electricus electroplax sodium channel indicate that this protein, consisting of 1,820 amino acid residues, exhibits four repeated homology units, which are presumably oriented in a pseudosymmetric fashion across the membrane. Each homology unit contains a unique segment with clustered positively charged residues, which may be involved in the gating structure, possibly in conjunction with negatively charged residues clustered elsewhere.

Cloning and sequence analysis of DNA complementary to porcine cerebral messenger RNA encoding the muscarinic acetylcholine receptor predict the complete amino-acid sequence of this protein. Expression of the complementary DNA produced functional muscarinic receptor in Xenopus oocytes. The muscarinic receptor is homologous with the beta-adrenergic receptor and rhodopsin in both amino-acid sequence and suggested transmembrane topography.

The nicotinic acetylcholine receptor (AChR) from fish electric organ is well characterized and is known to consist of five subunits present in a molar stoichiometry of alpha 2 beta gamma delta (reviewed in refs 1-3). The mammalian skeletal muscle AChR is thought to have a similar subunit structure. We have recently elucidated the primary structures of the alpha-, beta-, gamma- and delta-subunit precursors of the Torpedo californica AChR by cloning and sequencing cDNAs for these polypeptides; cDNA sequences for the gamma-subunit precursor of the T. californica AChR and the alpha-subunit precursor of the Torpedo marmorata AChR have also been reported by other groups. The four subunits exhibit conspicuous sequence homology and are similar in hydrophilicity profile and predicted secondary structure, thus being most probably oriented in a pseudosymmetric fashion across the membrane. The transmembrane topology of the subunit molecules and the locations of functionally important regions, such as the acetylcholine binding site and the trans-membrane segments which may be involved in the ionic channel, have been proposed. We have now cloned cDNA for the alpha-subunit precursor of the calf skeletal muscle AChR and a human genomic DNA segment containing the corresponding gene. Nucleotide sequence analysis of the cloned DNAs has revealed the primary structures of the calf and human AChR alpha-subunit precursors, which exhibit marked sequence homology with their Torpedo counterpart. The protein-coding sequence of the human AChR alpha-subunit precursor gene is divided by eight introns into nine exons, which seem to correspond to different structural and functional domains of the subunit precursor molecule.

The availability of cloned cDNAs encoding the four subunits of the Torpedo acetylcholine receptor, which can be expressed to make functional receptors in Xenopus oocytes, has made possible a detailed investigation of the functions of the different structural components of the receptor. The functional analysis of receptors with alpha-subunits altered at specific sites by site-directed mutagenesis of the cDNA has allowed the location of specific regions of the alpha-subunit molecule involved in acetylcholine binding and forming a transmembrane ionic channel.

The structure-function relationship of the nicotinic acetylcholine receptor (AChR) has been effectively studied by the combination of complementary DNA manipulation and single-channel current analysis. Previous work with chimaeras between the Torpedo californica and bovine AChR delta-subunits has shown that the region comprising the hydrophobic segment M2 and its vicinity contains an important determinant of the rate of ion transport through the AChR channel. It has also been suggested that this region is responsible for the reduction in channel conductance caused by divalent cations and that segment M2 contributes to the binding site of noncompetitive antagonists. To identify those amino acid residues that interact with permeating ions, we have introduced various point mutations into the Torpedo AChR subunit cDNAs to alter the net charge of the charged or glutamine residues around the proposed transmembrane segments. The single-channel conductance properties of these AChR mutants expressed in Xenopus laevis oocytes indicate that three clusters of negatively charged and glutamine residues neighbouring segment M2 of the alpha-, beta-, gamma- and delta-subunits, probably forming three anionic rings, are major determinants of the rate of ion transport.

The sequence of 5,037 amino acids composing the ryanodine receptor from rabbit skeletal muscle sarcoplasmic reticulum has been deduced by cloning and sequencing the complementary DNA. The predicted structure suggests that the calcium release channel activity resides in the C-terminal region of the receptor molecule, whereas the remaining portion constitutes the 'foot' structure spanning the junctional gap between the sarcoplasmic reticulum and the transverse tubule.

The cloned cDNAs encoding the four subunits of the Torpedo californica acetylcholine receptor, each carried by a simian virus 40 vector, direct the synthesis of the functional receptor in a combined expression system consisting of COS monkey cells and Xenopus oocytes. Our results suggest that all four subunits are required to elicit a normal nicotinic response to acetylcholine, whereas only the alpha-subunit is indispensable for alpha-bungarotoxin binding activity.