Normalization of current kinetics by interaction between the alpha 1 and beta subunits of the skeletal muscle dihydropyridine-sensitive Ca2+ channel.

Purification of skeletal muscle dihydropyridine binding sites has enabled protein complexes to be isolated from which Ca2+ currents have been reconstituted. Complementary DNAs encoding the five subunits of the dihydropyridine receptor, alpha 1, beta, gamma, alpha 2 and delta, have been cloned and it is now recognized that alpha 2 and delta are derived from a common precursor. The alpha 1 subunit can itself produce Ca2+ currents, as was demonstrated using mouse L cells lacking alpha 2 delta, beta and gamma (our unpublished results). In L cells, stable expression of skeletal muscle alpha 1 alone was sufficient to generate voltage-sensitive, high-threshold L-type Ca2+ channel currents which were dihydropyridine-sensitive and blocked by Cd2+, but the activation kinetics were about 100 times slower than expected for skeletal muscle Ca2+ channel currents. This could have been due to the cell type in which alpha 1 was being expressed or to the lack of a regulatory component particularly one of the subunits that copurifies with alpha 1. We show here that coexpression of skeletal muscle beta with skeletal muscle alpha 1 generates cell lines expressing Ca2+ channel currents with normal activation kinetics as evidence for the participation of the dihydropyridine-receptor beta subunits in the generation of skeletal muscle Ca2+ channel currents.     

Induction of calcium currents by the expression of the alpha 1-subunit of the dihydropyridine receptor from skeletal muscle

The dihydropyridine (DHP) receptor purified from skeletal muscle comprises five protein subunits (alpha 1, alpha 2, beta, gamma and delta) and produces Ca2+ currents that are blocked by DHPs. Cloning of the alpha 1- and alpha 2-subunits, the former affinity-labelled by DHP, has shown that the alpha 1-subunit is expressed in skeletal muscle alone, whereas the alpha 2- and delta- subunits are also expressed in other tissues. Although the transient expression of the alpha 1-subunit in myoblasts from dysgenic mice (but not in oocytes) has been demonstrated, the use of these expression systems to determine the function of the alpha 1- subunit is complicated by the presence of endogenous Ca2+ currents, which may reflect the constitutive expression of proteins similar to the alpha 2-, beta-, gamma- and/or delta-subunits. We therefore selected a cell line which has no Ca2+ currents or alpha 2- subunit, and probably no delta-subunit for stable transformation with complementary DNA of the alpha 1- subunit. The transformed cells express DHP-sensitive, voltage-gated Ca2+ channels, indicating that the minimum structure of these channels is at most an alpha 1 beta gamma complex and possibly an alpha 1- subunit alone.     

Primary structure and functional expression of the cardiac dihydropyridine-sensitive calcium channel

In cardiac muscle, where Ca2+ influx across the sarcolemma is essential for contraction, the dihydropyridine (DHP)-sensitive L-type calcium channel represents the major entry pathway of extracellular Ca2+. We have previously elucidated the primary structure of the rabbit skeletal muscle DHP receptor by cloning and sequencing the complementary DNA. An expression plasmid carrying this cDNA, microinjected into cultured skeletal muscle cells from mice with muscular dysgenesis, has been shown to restore both excitation-contraction coupling and slow calcium current missing from these cells, so that a dual role for the DHP receptor in skeletal muscle transverse tubules is suggested. We report here the complete amino-acid sequence of the rabbit cardiac DHP receptor, deduced from the cDNA sequence. We also show that messenger RNA derived from the cardiac DHP receptor cDNA is sufficient to direct the formation of a functional DHP-sensitive calcium channel in Xenopus oocytes. Furthermore, higher calcium-channel activity is observed when mRNA specific for the polypeptide of relative molecular mass approximately 140,000 (alpha 2-subunit) associated with skeletal muscle DHP receptor is co-injected.     

Primary structure of the receptor for calcium channel blockers from skeletal muscle

The complete amino-acid sequence of the receptor for dihydropyridine calcium channel blockers from rabbit skeletal muscle is predicted by cloning and sequence analysis of DNA complementary to its messenger RNA. Structural and sequence similarities to the voltage-dependent sodium channel suggest that in the transverse tubule membrane of skeletal muscle the dihydropyridine receptor may act both as voltage sensor in excitation-contraction coupling and as a calcium channel.     

Extracellular domains mediating epsilon subunit interactions of muscle acetylcholine receptor.

Ligand-gated ion channels, a major class of cell-surface proteins, have a pseudosymmetric structure with five highly homologous subunits arranged around a central ion pore. The correct assembly of each channel, whose subunit composition varies with cell type and stage of development, requires specific recognition between the subunits. Assembly of the pentameric form of the acetylcholine receptor from adult muscle (AChR; alpha 2 beta epsilon delta) proceeds by a stepwise pathway starting with the formation of the heterodimers, alpha epsilon and alpha delta. The heterodimers than associate with the beta subunit and with each other to form the complete receptor. We have now determined which parts of the subunits mediate the interactions during assembly of the adult form of the receptor from mouse muscle by using a chimaeric subunit in which the N-terminal and C-terminal extracellular domains are derived from the epsilon subunit with the remainder from the beta subunit. The epsilon and beta subunits were chosen because the epsilon subunit forms a heterodimer with the alpha subunit in the pathway for assembly of the receptor, whereas the beta subunit does not. The epsilon beta chimera can substitute for the epsilon but not the beta subunit in the oligomeric receptor, indicating that the alpha subunit specifically recognizes an extracellular domain of the epsilon subunit.     

Primary structure and functional expression from complementary DNA of a brain calcium channel.

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.     

Vasoregulation by the beta1 subunit of the calcium-activated potassium channel

Small arteries exhibit tone, a partially contracted state that is an important determinant of blood pressure. In arterial smooth muscle cells, intracellular calcium paradoxically controls both contraction and relaxation. The mechanisms by which calcium can differentially regulate diverse physiological responses within a single cell remain unresolved. Calcium-dependent relaxation is mediated by local calcium release from the sarcoplasmic reticulum. These 'calcium sparks' activate calcium-dependent potassium (BK) channels comprised of alpha and beta1 subunits. Here we show that targeted deletion of the gene for the beta1 subunit leads to a decrease in the calcium sensitivity of BK channels, a reduction in functional coupling of calcium sparks to BK channel activation, and increases in arterial tone and blood pressure. The beta1 subunit of the BK channel, by tuning the channel's calcium sensitivity, is a key molecular component in translating calcium signals to the central physiological function of vasoregulation.     

Pentameric structure and subunit stoichiometry of a neuronal nicotinic acetylcholine receptor

Neuronal nicotinic acetylcholine receptors are members of a gene family of ligand-gated transmitter receptors that includes muscle nicotinic receptors, GABAA receptors and glycine receptors. Several lines of evidence indicate that neuronal nicotinic receptors can be made up of only two subunits, an alpha (alpha) subunit which binds ligand, and a non-alpha (n alpha) or beta (beta) subunit. The stoichiometry of each subunit in the functional receptor has been difficult to assess, however. Estimates of the molecular weight of neuronal nicotonic receptor macromolecules suggest that these receptors contain at least four subunits but probably not more than five. We have examined the subunit stoichiometry of the chick neuronal alpha 4/n alpha 1 receptor by first using site-directed mutagenesis to create subunits that confer different single channel properties on the receptor. Co-injection with wild-type and mutant subunits led to the appearance of receptors with wild-type, mutant and hybrid conductances. From the number of hybrid conductances, we could deduce the number of each subunit in the functional receptor.     

Function of a truncated dihydropyridine receptor as both voltage sensor and calcium channel.

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.     

Importance of a novel GABAA receptor subunit for benzodiazepine pharmacology

Neurotransmission effected by GABA (gamma-aminobutyric acid) is predominantly mediated by a gated chloride channel intrinsic to the GABAA receptor. This heterooligomeric receptor exists in most inhibitory synapses in the vertebrate central nervous system (CNS) and can be regulated by clinically important compounds such as benzodiazepines and barbiturates. The primary structures of GABAA receptor alpha- and beta-subunits have been deduced from cloned complementary DNAs. Co-expression of these subunits in heterologous systems generates receptors which display much of the pharmacology of their neural counterparts, including potentiation by barbiturates. Conspicuously, however, they lack binding sites for, and consistent electrophysiological responses to, benzodiazepines. We now report the isolation of a cloned cDNA encoding a new GABAA receptor subunit, termed gamma 2, which shares approximately 40% sequence identity with alpha- and beta-subunits and whose messenger RNA is prominently localized in neuronal subpopulations throughout the CNS. Importantly, coexpression of the gamma 2 subunit with alpha 1 and beta 1 subunits produces GABAA receptors displaying high-affinity binding for central benzodiazepine receptor ligands.     

Dihydropyridine receptors in muscle are voltage-dependent but most are not functional calcium channels

1,4-Dihydropyridines are a new class of compounds believed to bind specifically and with high affinity to voltage-dependent calcium channels. They may be the first example of a ligand of use in the extraction and purification of the Ca channel. Although Ca channels and dihydropyridine receptors are found in many tissues, the richest and most convenient source is skeletal muscle. Functionally, 1,4-dihydropyridines such as nifedipine and nitrendipine block Ca channels; this effect is believed to form the basis for their clinical importance as Ca antagonists in relaxing vascular smooth muscle. But where currents through Ca channels can be measured directly, the block has required 100-1,000 times higher concentrations of dihydropyridine than necessary for the saturation of dihydropyridine binding sites. This discrepancy has remained unresolved because the study of pharmacological effects on Ca channels has required intact cells, while it has been difficult to investigate binding in other than cell-free preparations. Here we describe a method for measuring dihydropyridine binding to intact skeletal muscle and we compare our results with voltage-clamp measurements of Ca-channel block. We conclude that less than a few per cent of the binding sites in skeletal muscle represent functional Ca channels, contrary to general belief.     

Cerebellar GABAA receptor selective for a behavioural alcohol antagonist

Benzodiazepines are widely prescribed anxiolytics and anticonvulsants which bind with high affinity to sites on the GABAA receptor/Cl- channel complex and potentiate the effect of the neurotransmitter GABA (gamma-aminobutyric acid). The heterogeneity of benzodiazepine recognition sites in the central nervous system was revealed by studies showing different classes of GABAA receptor subunits (classes alpha, beta and gamma) and variant subunits in these classes, particularly in the alpha-class. Expression of recombinant subunits produces functional receptors; when certain alpha-variants are coexpressed with beta- and gamma-subunits the resulting receptors have pharmacological properties characteristic of GABAA-benzodiazepine type I or type II receptors. The alpha-variants are differentially expressed in the central nervous system and can be photoaffinity-labelled with benzodiazepines. Here we report a novel alpha-subunit (alpha 6) of cerebellar granule cells. We show that recombinant receptors composed of alpha 6, beta 2 and gamma 2 subunits bind with high affinity to the GABA agonist [3H]muscimol and the benzodiazepine [3H]Ro15-4513 but not the other benzodiazepines or beta-carboniles. The same distinctive pharmacology is observed with GABAA receptors from rat cerebellum immunoprecipitated by an antiserum specific for the alpha 6 subunit. We conclude that this alpha-subunit is part of a cerebellar receptor subtype, selective for Ro15-4513, an antagonist of alcohol-induced motor incoordination and ataxia.     

A functional correlate for the dihydropyridine binding site in rat brain

Calcium channels, controlling the influx of extracellular Ca2+ and hence neurotransmitter release, exist in the brain. However, drugs classed as calcium antagonists and which inhibit Ca2+ entry through voltage-activated Ca2+ channels in heart and smooth muscle, seem not to affect any aspect of neuronal function in the brain at pharmacologically relevant concentrations. Yet the dihydropyridine calcium antagonists (for example, nitrendipine) bind stereospecifically with high affinity to a recognition site on brain-cell membranes thought to represent the Ca2+ channel and consequently, the physiological relevance of these sites has been questioned. However, activation of voltage-dependent Ca2+ channels can increase cytoplasmic Ca2+ and neurotransmitter release in neuronal tissue. We show here that Bay K8644, a dihydropyridine Ca2+-channel activator, can augment K+-stimulated release of serotonin from rat frontal cortex slices and that these effects can be antagonized by low concentrations of calcium antagonists. As 3H-dihydropyridine binding to cortical membrane preparations resembles the binding in heart and smooth muscle where there are good functional correlates we conclude that the dihydropyridine binding sites in the brain represent functional Ca2+ channels that can be unmasked under certain circumstances.     

Cloning, sequencing and expression of cDNA for a novel subunit of acetylcholine receptor from calf muscle

The nicotinic acetylcholine receptor (AChR) from fish electric organ has a subunit structure of alpha 2 beta gamma delta, and this is thought to be also the case for the mammalian skeletal muscle AChR. By cloning and sequencing the complementary or genomic DNAs, we have previously elucidated the primary structures of all four subunits of the Torpedo californica electroplax and calf muscle AChR and of the alpha- and gamma-subunits of the human muscle AChR; the primary structures of the gamma-subunit of the T. californica AChR and the alpha-subunit of the Torpedo marmorata AChR have also been deduced elsewhere. We have now cloned DNA complementary to the calf muscle messenger RNA encoding a novel polypeptide (the epsilon-subunit) whose deduced amino-acid sequence has features characteristic of the AChR subunits and which shows higher sequence homology with the gamma-subunit than with the other subunits. cDNA expression studies indicate that the calf epsilon-subunit, as well as the calf gamma-subunit, can replace the Torpedo gamma-subunit to form the functional receptor in combination with the Torpedo alpha-, beta- and delta-subunits.     

Regions of the skeletal muscle dihydropyridine receptor critical for excitation-contraction coupling

It is thought that in skeletal muscle excitation-contraction (EC) coupling, the release of Ca2+ from the sarcoplasmic reticulum is controlled by the dihydropyridine (DHP) receptor in the transverse tubular membrane, where it serves as the voltage sensor. We have shown previously that injection of an expression plasmid carrying the skeletal muscle DHP receptor complementary DNA restores EC coupling and L-type calcium current that are missing in skeletal muscle myotubes from mutant mice with muscular dysgenesis. This restored coupling resembles normal skeletal muscle EC coupling, which does not require entry of extracellular Ca2+. By contrast, injection into dysgenic myotubes of an expression plasmid carrying the cardiac DHP receptor cDNA produces L-type calcium current and cardiac-type EC coupling, which does require entry of extracellular Ca2+. To identify the regions responsible for this important functional difference between the two structurally similar DHP receptors, we have expressed various chimaeric DHP receptor cDNAs in dysgenic myotubes. The results obtained indicate that the putative cytoplasmic region between repeats II and III of the skeletal muscle DHP receptor is an important determinant of skeletal-type EC coupling.     

Novel dihydropyridines with positive inotropic action through activation of Ca2+ channels

Transmembrane influx of extracellular calcium through specific calcium channels is now accepted to have an important role in the excitation-contraction coupling of cardiac and smooth muscle. The importance of such slow calcium channels has been underlined by the development of specific calcium channel blocking agents, the 'calcium antagonists', typified by verapamil, nifedipine and diltiazem. These drugs have been used to investigate the properties of slow calcium channels in a variety of tissues. We have found that small modifications to the nifedipine molecule produce other dihydropyridine derivatives (see Fig. 1) with effects diametrically opposite to those of the calcium antagonists: cardiac contractility is stimulated and smooth muscle is contracted. These effects are competitively antagonized by nifedipine. Apparently, nifedipine and the novel compounds bind to the same specific dihydropyridine binding sites in or near the calcium channel. In contrast to nifedipine, however, the new compounds promote--instead of inhibiting--the influx of Ca2+ ions. We report here the properties of BAY K 8644 (methyl 1,4-dihydro-2,6-dimethyl-3-nitro-4-(2-trifluoromethylphenyl)- pyridine-5-carboxylate), one of the most potent of these novel compounds.     

Involvement of dihydropyridine receptors in excitation-contraction coupling in skeletal muscle

The transduction of action potential to muscle contraction (E-C coupling) is an example of fast communication between plasma membrane events and the release of calcium from an internal store, which in muscle is the sarcoplasmic reticulum (SR). One theory is that the release channels of the SR are controlled by voltage-sensing molecules or complexes, located in the transverse tubular (T)-membrane, which produce, as membrane voltage varies, 'intramembrane charge movements', but nothing is known about the structure of such sensors. Receptors of the Ca-channel-blocking dihydropyridines present in many tissues, are most abundant in T-tubular muscle fractions from which they can be isolated as proteins. Fewer than 5% of muscle dihydropyridines are functional Ca channels; there is no known role for the remainder in skeletal muscle physiology. We report here that low concentrations of a dihydropyridine inhibit charge movements and SR calcium release in parallel. The effect has a dependence on membrane voltage analogous to that of specific binding of dihydropyridines. We propose specifically that the molecule that generates charge movement is the dihydropyridine receptor.     

Cardiac-type excitation-contraction coupling in dysgenic skeletal muscle injected with cardiac dihydropyridine receptor cDNA

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.     

Structural homology of Torpedo californica acetylcholine receptor subunits

The nicotinic acetylcholine receptor (AChR) from the electroplax of the ray Torpedo californica is composed of five subunits present in a molar stoichiometry of alpha 2 beta gamma delta (refs 1-3) and contains both the binding site for the neurotransmitter and the cation gating unit (reviewed in refs 4-6). We have recently elucidated the complete primary structures of the alpha-, beta- and delta-subunit precursors of the T. californica AChR by cloning and sequencing cDNAs for these polypeptides. Here, we report the whole primary structure of the gamma-subunit precursor of the AChR deduced from the nucleotide sequence of the cloned cDNA. Comparison of the amino acid sequences of the four subunits reveals marked homology among them. The close resemblance among the hydrophilicity profiles and predicted secondary structures of all the subunits suggests that these polypeptides are oriented in a pseudosymmetric fashion across the membrane. Each subunit contains four putative transmembrane segments that may be involved in the ionic channel. The transmembrane topology of the subunit molecules has also been inferred.     

Inactivation of the sarcoplasmic reticulum calcium channel by protein kinase.

The ryanodine receptor protein of skeletal muscle sarcoplasmic reticulum (SR) membranes is a calcium ion channel which allows movement of calcium from the SR lumen into the cytoplasm during muscle activation. Gating of this channel is modulated by a number of physiologically important substances including calcium. Interestingly, calcium has both activating and inactivating effects which are concentration- and tissue-specific. In skeletal muscle, calcium-dependent inactivation of calcium release occurs at concentrations reached physiologically, suggesting that calcium may modulate the release process by a negative feedback mechanism. To determine the cellular mechanism responsible for calcium-dependent inactivation, we have investigated the ability of protein phosphorylation to affect single channel gating behaviour using the patch clamp technique. Here we demonstrate that the ryanodine receptor protein/calcium release channel of skeletal muscle SR is inactivated under conditions permissive for protein phosphorylation. This inactivation is reversed by the application of phosphatase and prevented by a peptide inhibitor specific for calcium/calmodulin-dependent protein kinase II. The results provide evidence for an endogenous protein kinase which is closely associated with the ryanodine receptor protein and regulates channel gating.