Skeletal Dihydropyridine Receptor Research Articles

Nitric oxide activates or inhibits skeletal muscle ryanodine receptors depending on its concentration, membrane potential and ligand binding.

We show that rabbit skeletal RyR channels in lipid bilayers can be activated or inhibited by NO, in a manner that depends on donor concentration, membrane potential and the presence of channel agonists. 10 microm S-nitroso-N-acetyl-penicillamine (SNAP) increased RyR activity at -40 mV within 15 sec of addition to the cis chamber, with a 2-fold increase in frequency of channel opening (F(o)). 10 microm SNAP did not alter activity at +40 mV and did not further activate RyRs previously activated by 2 mm cis ATP at +40 or -40 mV. In contrast to the increase in F(o) with 10 microm SNAP, 1 mm SNAP caused a 2-fold reduction in F(o) but a 1.5-fold increase in mean open time (T(o)) at -40 mV in the absence of ATP. 1 mm SNAP or 0.5 mm sodium nitroprusside (SNP) induced approximately 3-fold reductions in F(o) and T(o) at +40 or -40 mV when channels were activated by 2 mm cis ATP or in channels activated by 6.5 microm peptide A at -40 mV (peptide A corresponds to part of the II-III loop of the skeletal dihydropyridine receptor). Both SNAP-induced activation and SNAP/SNP-induced inhibition were reversed by 2 mm dithiothreitol. The results suggest that S-Nitrosylation or oxidation of at least three classes of protein thiols by NO each produced characteristic changes in RyR activity. We propose that, in vivo, initial release of NO activates RyRs, but stronger release increases [NO] and inhibits RyR activity and contraction.

Role of calcium permeation in dihydropyridine receptor function. Insights into channel gating and excitation-contraction coupling.

The skeletal and cardiac muscle dihydropyridine receptors (DHPRs) differ with respect to their rates of channel activation and in the means by which they control Ca2+ release from the sarcoplasmic reticulum (Adams, B.A., and K.G. Beam. 1990. FASEB J. 4:2809-2816). We have examined the functional properties of skeletal (SkEIIIK) and cardiac (CEIIIK) DHPRs in which a highly conserved glutamate residue in the pore region of repeat III was mutated to a positively charged lysine residue. Using expression in dysgenic myotubes, we have characterized macroscopic ionic currents, intramembrane gating currents, and intracellular Ca2+ transients attributable to these two mutant DHPRs. CEIIIK supported very small inward Ca2+ currents at a few potentials (from -20 to +20 mV) and large outward cesium currents at potentials greater than +20 mV. SkEIIIK failed to support inward Ca2+ flux at any potential. However, large, slowly activating outward cesium currents were observed at all potentials greater than + 20 mV. The difference in skeletal and cardiac Ca2+ channel activation kinetics was conserved for outward currents through CEIIIK and SkEIIIK, even at very depolarized potentials (at +100 mV; SkEIIIK: tau(act) = 30.7 +/- 1.9 ms, n = 11; CEIIIK: tau(act) = 2.9 +/- 0.5 ms, n = 7). Expression of SkEIIIK in dysgenic myotubes restored both evoked contractions and depolarization-dependent intracellular Ca(2+) transients with parameters of voltage dependence (V(0.5) = 6.5 +/- 3.2 mV and k = 9.3 +/- 0.7 mV, n = 5) similar to those for the wild-type DHPR (Garcia, J., T. Tanabe, and K.G. Beam. 1994. J. Gen. Physiol. 103:125-147). However, CEIIIK-expressing myotubes never contracted and failed to exhibit depolarization-dependent intracellular Ca2+ transients at any potential. Thus, high Ca2+ permeation is required for cardiac-type excitation-contraction coupling reconstituted in dysgenic myotubes, but not skeletal-type. The strong rectification of the EIIIK channels made it possible to obtain measurements of gating currents upon repolarization to -50 mV (Qoff) following either brief (20 ms) or long (200 ms) depolarizing pulses to various test potentials. For SkEIIIK, and not CEIIK, Qoff was significantly (P < 0.001) larger after longer depolarizations to +60 mV (121.4 +/- 2.0%, n = 6). The increase in Qoff for long depolarizations exhibited a voltage dependence similar to that of channel activation. Thus, the increase in Q(off) may reflect a voltage sensor movement required for activation of L-type Ca2+ current and suggests that most DHPRs in skeletal muscle undergo this voltage-dependent transition.

The II-III Loop of the Skeletal Muscle Dihydropyridine Receptor Is Responsible for the Bi-directional Coupling with the Ryanodine Receptor

The dihydropyridine receptor (DHPR) in the skeletal muscle plasmalemma functions as both voltage-gated Ca(2+) channel and voltage sensor for excitation-contraction (EC) coupling. As voltage sensor, the DHPR regulates intracellular Ca(2+) release via the skeletal isoform of the ryanodine receptor (RyR-1). Interaction with RyR-1 also feeds back to increase the Ca(2+) current mediated by the DHPR. To identify regions of the DHPR important for receiving this signal from RyR-1, we expressed in dysgenic myotubes a chimera (SkLC) having skeletal (Sk) DHPR sequence except for a cardiac (C) II-III loop (L). Tagging with green fluorescent protein (GFP) enabled identification of expressing myotubes. Dysgenic myotubes expressing GFP-SkLC or SkLC lacked EC coupling and had very small Ca(2+) currents. Introducing a short skeletal segment (alpha(1S) residues 720-765) into the cardiac II-III loop (replacing alpha(1C) residues 851-896) of GFP-SkLC restored both EC coupling and Ca(2+) current densities like those of the wild type skeletal DHPR. This 46-amino acid stretch of skeletal sequence was recently shown to be capable of transferring strong, skeletal-type EC coupling to an otherwise cardiac DHPR (Nakai, J., Tanabe, T., Konno, T., Adams, B., and Beam, K.G. (1998) J. Biol. Chem. 273, 24983-24986). Thus, this segment of the skeletal II-III loop contains a motif required for both skeletal-type EC coupling and RyR-1-mediated enhancement of Ca(2+) current.

Activation and Inhibition of Skeletal RyR Channels by a Part of the Skeletal DHPR II-III Loop: Effects of DHPR Ser687 and FKBP12

Peptides, corresponding to sequences in the N-terminal region of the skeletal muscle dihydropyridine receptor (DHPR) II-III loop, have been tested on sarcoplasmic reticulum (SR) Ca2+ release and ryanodine receptor (RyR) activity. The peptides were: A1, Thr671-Leu690; A2, Thr671-Leu690 with Ser687 Ala substitution; NB, Gly689-Lys708 and A1S, scrambled A1 sequence. The relative rates of peptide-induced Ca2+ release from normal (FKBP12+) SR were A2>A1>A1S>NB. Removal of FKBP12 reduced the rate of A1-induced Ca2+ release by ∼30%. A1 and A2 (but not NB or A1S), in the cytoplasmic (cis) solution, either activated or inhibited single FKBP12+ RyRs. Maximum activation was seen at −40mV, with 10μM A1 or 50nM A2. The greatest A1-induced increase in mean current (sixfold) was seen with 100nM cis Ca2+. Inhibition by A1 was greatest at +40mV (or when permeant ions flowed from cytoplasm to SR lumen) with 100μM cis Ca2+, where channel activity was almost fully inhibited. A1 did not activate FKBP12-stripped RyRs, although peptide-induced inhibition remained. The results show that peptide A activation of RyRs does not require DHPR Ser687, but required FKBP12 binding to RyRs. Peptide A must interact with different sites to activate or inhibit RyRs, because current direction-, voltage-, cis [Ca2+]-, and FKBP12-dependence of activation and inhibition differ.

Conversion of an inactive cardiac dihydropyridine receptor II-III loop segment into forms that activate skeletal ryanodine receptors

A 25 amino acid segment (Glu 666–Pro 691) of the II-III loop of the α 1 subunit of the skeletal dihydropyridine receptor, but not the corresponding cardiac segment (Asp 788–Pro 814), activates skeletal ryanodine receptors. To identify the structural domains responsible for activation of skeletal ryanodine receptors, we systematically replaced amino acids of the cardiac II-III loop with their skeletal counterparts. A cluster of five basic residues of the skeletal II-III loop ( 681RKRRK 685) was indispensable for activation of skeletal ryanodine receptors. In the cardiac segment, a negatively charged residue (Glu 804) appears to diminish the electrostatic potential created by this basic cluster. In addition, Glu 800 in the group of negatively charged residues 798EEEEE 802 of the cardiac II-III loop may serve to prevent the binding of the activation domain.

Activation of Ryanodine Receptors by Imperatoxin A and a Peptide Segment of the II-III Loop of the Dihydropyridine Receptor

Excitation-contraction coupling in skeletal muscle is believed to be triggered by direct protein-protein interactions between the sarcolemmal dihydropyridine-sensitive Ca2+ channel and the Ca2+ release channel/ryanodine receptor (RyR) of sarcoplasmic reticulum. A 138-amino acid cytoplasmic loop between repeats II and III of the alpha1 subunit of the skeletal dihydropyridine receptor (the II-III loop) interacts with a region of the RyR to elicit Ca2+ release. In addition, small segments (10-20 amino acid residues) of the II-III loop retain the capacity to activate Ca2+ release. Imperatoxin A, a 33-amino acid peptide from the scorpion Pandinus imperator, binds directly to the RyR and displays structural and functional homology with an activating segment of the II-III loop (Glu666-Leu690). Mutations in a structural motif composed of a cluster of basic amino acids followed by Ser or Thr dramatically reduce or completely abolish the capacity of the peptides to activate RyRs. Thus, the Imperatoxin A-RyR interaction mimics critical molecular characteristics of the II-III loop-RyR interaction and may be a useful tool to elucidate the molecular mechanism that couples membrane depolarization to sarcoplasmic reticulum Ca2+ release in vivo.

Localization in the II-III Loop of the Dihydropyridine Receptor of a Sequence Critical for Excitation-Contraction Coupling

Skeletal and cardiac muscles express distinct isoforms of the dihydropyridine receptor (DHPR), a type of voltage-gated Ca2+ channel that is important for excitation-contraction (EC) coupling. However, entry of Ca2+ through the channel is not required for skeletal muscle-type EC coupling. Previous work (Tanabe, T., Beam, K. G., Adams, B. A., Niidome, T., and Numa, S. (1990) Nature 346, 567-569) revealed that the loop between repeats II and III (II-III loop) is an important determinant of skeletal-type EC coupling. In the present study we have further dissected the regions of the II-III loop critical for skeletal-type EC coupling by expression of cDNA constructs in dysgenic myotubes. Because Ser687 of the skeletal II-III loop has been reported to be rapidly phosphorylated in vitro, we substituted this serine with alanine, the corresponding cardiac residue. This alanine-substituted skeletal DHPR retained the ability to mediate skeletal-type EC coupling. Weak skeletal-type EC coupling was produced by a chimeric DHPR, which was entirely cardiac except for a small amount of skeletal sequence (residues 725-742) in the II-III loop. Skeletal-type coupling was stronger when both residues 725-742 and adjacent residues were skeletal (e.g. a chimera containing skeletal residues 711-765). However, residues 725-742 appeared to be critical because skeletal-type coupling was not produced either by a chimera with skeletal residues 711-732 or by one with skeletal residues 734-765.

Antisense oligonucleotides against 'cardiac' and 'skeletal' DHP-receptors reveal a dual role for the 'skeletal' isoform in EC coupling of skeletal muscle cells in primary culture.

Two dihydropyridine receptor mRNA isoforms (cardiac and skeletal) are expressed in rat skeletal muscle cells in primary culture. The progressive changes in excitation-contraction coupling mode from dual mode ('skeletal' and 'cardiac') to predominant 'skeletal' one during in vitro myogenesis are thought to be linked to the developmental changes in the relative expression of the two types of molecular entity previously observed in this preparation. In order to test this hypothesis, myotube cultures (5- to 7-day-old) were treated with antisense phosphorothioated oligodeoxynucleotides against cardiac or skeletal alpha1 subunit of L-type calcium channel. The oligodeoxynucleotide uptake by cells was checked by means of imaging of fluorescent oligodeoxynucleotide derivatives within the cells. Optimum concentration used (10 microM in the extracellular medium) and incubation time (70 hours) were empirically determined. Antisense directed against the cardiac type led to a 54% decrease in the averaged L-type calcium current peak density at -10 mV. The same type of experiment was performed with antisense against the skeletal isoform and led to a same order of inhibition (46%). This result clearly shows that the two isoforms can work as a calcium channel. Conversely, analysis of the shape of T-V (relative contractile amplitude versus membrane potential) curves shows that the treatment with 'skeletal' antisense depressed the contractile response in the medium membrane potential range whereas treatment with 'cardiac' antisense had no effect. This and other results taken together suggest that the skeletal isoform of dihydropyridine receptor is involved in both 'cardiac' and 'skeletal' types of EC coupling mechanisms at work in early stages of myotubes in vitro development. The type of coupling probably depends on the proximity of the skeletal dihydropyridine receptor and the ryanodine receptor.

Molecular Origin of the L-Type Ca 2+ Current of Skeletal Muscle Myotubes Selectively Deficient in Dihydropyridine Receptor β1a Subunit

The origin of I βnull , the Ca 2+ current of myotubes from mice lacking the skeletal dihydropyridine receptor (DHPR) β 1a subunit, was investigated. The density of I βnull was similar to that of I dys, the Ca 2+ current of myotubes from dysgenic mice lacking the skeletal DHPR α 1S subunit (−0.6 ± 0.1 and −0.7 ± 0.1 pA/pF, respectively). However, I βnull activated at significantly more positive potentials. The midpoints of the G Ca- V curves were 16.3 ± 1.1 mV and 11.7 ± 1.0 mV for I βnull and I dys, respectively. I βnull activated significantly more slowly than I dys. At +30 mV, the activation time constant for I βnull was 26 ± 3 ms, and that for I dys was 7 ± 1 ms. The unitary current of normal L-type and β 1-null Ca 2+ channels estimated from the mean variance relationship at +20 mV in 10 mM external Ca 2+ was 22 ± 4 fA and 43 ± 7 fA, respectively. Both values were significantly smaller than the single-channel current estimated for dysgenic Ca 2+ channels, which was 84 ± 9 fA under the same conditions. I βnull and I dys have different gating and permeation characteristics, suggesting that the bulk of the DHPR α 1 subunits underlying these currents are different. I βnull is suggested to originate primarily from Ca 2+ channels with a DHPR α 1S subunit. Dysgenic Ca 2+ channels may be a minor component of this current. The expression of DHPR α 1S in β 1-null myotubes and its absence in dysgenic myotubes was confirmed by immunofluorescence labeling of cells.

Two Regions of the Ryanodine Receptor Involved in Coupling withl-Type Ca2+ Channels

Ryanodine receptors (RyRs) are present in the endoplasmic reticulum of virtually every cell type and serve critical roles, including excitation-contraction (EC) coupling in muscle cells. In skeletal muscle the primary control of RyR-1 (the predominant skeletal RyR isoform) occurs via an interaction with plasmalemmal dihydropyridine receptors (DHPRs), which function as both voltage sensors for EC coupling and as L-type Ca2+ channels (Rios, E., and Brum, G. (1987) Nature 325, 717-720). In addition to "receiving" the EC coupling signal from the DHPR, RyR-1 also "transmits" a retrograde signal that enhances the Ca2+ channel activity of the DHPR (Nakai, J., Dirksen, R. T., Nguyen, H. T., Pessah, I. N., Beam, K. G., and Allen, P. D. (1996) Nature 380, 72-76). A similar kind of retrograde signaling (from RyRs to L-type Ca2+ channels) has also been reported in neurons (Chavis, P., Fagni, L., Lansman, J. B., and Bockaert, J. (1996) Nature 382, 719-722). To investigate the molecular mechanism of reciprocal signaling, we constructed cDNAs encoding chimeras of RyR-1 and RyR-2 (the predominant cardiac RyR isoform) and expressed them in dyspedic myotubes, which lack an endogenous RyR-1. We found that a chimera that contained residues 1,635-2,636 of RyR-1 both mediated skeletal-type EC coupling and enhanced Ca2+ channel function, whereas a chimera containing adjacent RyR-1 residues (2, 659-3,720) was only able to enhance Ca2+ channel function. These results demonstrate that two distinct regions are involved in the reciprocal interactions of RyR-1 with the skeletal DHPR.

Functional nonequality of the cardiac and skeletal ryanodine receptors.

Dihydropyridine receptors (DHPRs), which are voltage-gated Ca2+ channels, and ryanodine receptors (RyRs), which are intracellular Ca2+ release channels, are expressed in diverse cell types, including skeletal and cardiac muscle. In skeletal muscle, there appears to be reciprocal signaling between the skeletal isoforms of both the DHPR and the RyR (RyR-1), such that Ca2+ release activity of RyR-1 is controlled by the DHPR and Ca2+ channel activity of the DHPR is controlled by RyR-1. Dyspedic skeletal muscle cells, which do not express RyR-1, lack excitation-contraction coupling and have an approximately 30-fold reduction in L-type Ca2+ current density. Here we have examined the ability of the predominant cardiac and brain RyR isoform, RyR-2, to substitute for RyR-1 in interacting with the skeletal DHPR. When RyR-2 is expressed in dyspedic muscle cells, it gives rise to spontaneous intracellular Ca2+ oscillations and supports Ca2+ entry-induced Ca2+ release. However, unlike RyR-1, the expressed RyR-2 does not increase the Ca2+ channel activity of the DHPR, nor is the gating of RyR-2 controlled by the skeletal DHPR. Thus, the ability to participate in skeletal-type reciprocal signaling appears to be a unique feature of RyR-1.

Unitary behavior of skeletal, cardiac, and chimeric L-type Ca2+ channels expressed in dysgenic myotubes.

Skeletal and cardiac dihydropyridine receptors function both as voltage-dependent L-type calcium channels (L-channels) and as critical proteins that trigger calcium release from the sarcoplasmic reticulum in muscle. In spite of these similarities, skeletal L-channels exhibit a markedly slower activation rate than cardiac L-channels. We investigated the mechanisms underlying this difference by comparing the unitary behavior of L-channels in cell-attached patches of dysgenic myotubes expressing skeletal, cardiac, or chimeric dihydropyridine receptors. Our results demonstrate that ensemble averages activate rapidly for the purely cardiac dihydropyridine receptor and approximately five times more slowly for L-channels attributable to the purely skeletal dihydropyridine receptor or a chimeric dihydropyridine receptor in which only the first internal repeat and all of the putative intracellular loops are of skeletal origin. All of the constructs studied similarly exhibit a brief (2-ms) and a long (> or = 15-ms) open time in the presence of Bay K 8644, neither of which depend significantly on voltage. In the absence of Bay K 8644, the fraction of total open events is markedly shifted to the briefer open time without altering the rate of ensemble activation. Closed time analysis of L-channels with cardiac-like, rapid activation (recorded in the presence of dihydropyridine agonist) reveals both a brief (approximately 1-ms) closed time and a second, voltage-dependent, long-lasting closed time. The time until first opening after depolarization is three to six times faster for rapidly activating L-channels than for slowly activating L-channels and depends strongly on voltage for both types of channels. The results suggest that a voltage-dependent, closed-closed transition that is fast in cardiac L-channels and slow in skeletal L-channels can account for the difference in activation rate between these two channels.

Binding sites of monoclonal antibodies and dihydropyridine receptor alpha 1 subunit cytoplasmic II-III loop on skeletal muscle triadin fusion peptides.

Triadin binds to the dihydropyridine receptor (DHPr) and the junction foot protein (JFP) in Western blot protein overlay experiments. Fusion peptides were synthesized using an expression system, pGSTag, which includes a protein kinase A phosphorylation site. Expressed peptides are DHPr664-799 encoding rabbit skeletal DHPr alpha1 subunit amino acids 664-799, triadin 1 (1-49), triadin 2 (68-389), triadin 2' (110-389), triadin 2a (68-278), triadin 2a1 (67-163), triadin 2a2 (165-240), triadin 2b (242-389), triadin 2b1 (242-299), triadin 3 (370-706), triadin 3a (370-562), triadin 3b (551-706), triadin 3b1 (551-672), and triadin 3b2 (673-706) (the numbers in parentheses correspond to the amino acid sequence of triadin). The triadin monoclonal antibodies, GE4.90 and AE8.91, bind to intact triadic vesicles as well as to vesicle fragments prepared after treatment with Triton X-100, indicating that they have cytoplasmic epitopes. MAb AE8.91 binds to triadin 2, 2', 2a, and 2a1, while mAb GE4.90 binds to triadin 3, 3b, and 3b2 indicating that residues 110-163 and the C-terminal 34 amino acids contain cytoplasmic domains. Radiolabeled DHPr664-799 binds to triadin in intact vesicles under nonreducing and reducing conditions. It binds to triadin fusion peptides, triadin 2, 2a, 3, 3b, and 3b1, but no to triadin 1 or triadin 3b2. The binding to triadin 2a is the most prominent. Direct binding between DHPr-644-799 and JFP was not seen. These experimental findings indicate that triadin contains an extensive cytoplasmic domain that binds to the domain of DHPr which is considered critical for signal transmission during skeletal muscle excitation-contraction sampling.

Co-expression in CHO cells of two muscle proteins involved in excitation-contraction coupling

Ryanodine receptors and dihydropyridine receptors are located opposite each other at the junctions between sarcoplasmic reticulum and either the surface membrane or the transverse tubules in skeletal muscle. Ryanodine receptors are the calcium release channels of the sarcoplasmic reticulum and their cytoplasmic domains form the feet, connecting sarcoplasmic reticulum to transverse tubules. Dihydropyridine receptors are L-type calcium channels that act as the voltage sensors of excitation-contraction coupling: they sense surface membrane and transverse tubule depolarization and induce opening of the sarcoplasmic reticulum release channels. In skeletal muscle, ryanodine receptors are arranged in extensive arrays and dihydropyridine receptors are grouped into tetrads, which in turn are associated with the four subunits of ryanodine receptors. The disposition allows for a direct interaction between the two sets of molecules. CHO cells were stably transformed with plasmids for skeletal muscle ryanodine receptors and either the skeletal dihydropyridine receptor, or a skeletal-cardiac dihydropyridine receptor chimera (CSk3) which can functionally substitute for the skeletal dihydropyridine receptor, in addition to plasmids for the alpha 2, beta and gamma subunits. RNA blot hybridization gave positive results for all components. Immunoblots, ryanodine binding, electron microscopy and exposure to caffeine show that the expressed ryanodine receptors forms functional tetrameric channels, which are correctly inserted into the endoplasmic reticulum membrane, and form extensive arrays with the same spacings as in skeletal muscle. Since formation of arrays does not require coexpression of dihydropyridine receptors, we conclude that self-aggregation is an independent property of ryanodine receptors. All dihydropyridine receptor-expressing clones show high affinity binding for dihydropyridine and immunolabelling with antibodies against dihydropyridine receptor. The presence of calcium currents with fast kinetics and immunolabelling for dihydropyridine receptors in the surface membrane of CSk3 clones indicate that CSk3-dihydropyridine receptors are appropriately targeted to the cell's plasmalemma. The expressed skeletal-type dihydropyridine receptors, however, remain mostly located within perinuclear membranes. In cells coexpressing functional dihydropyridine receptors and ryanodine receptors, no junctions between feet-bearing endoplasmic reticulum elements and surface membrane are formed, and dihydropyridine receptors do not assemble into tetrads. A separation between dihydropyridine receptors and ryanodine receptors is not unique to CHO cells, but is found also in cardiac muscle, in muscles of invertebrates and, under certain conditions, in skeletal muscle. We suggest that failure to form junctions in co-transfected CHO cell may be due to lack of an essential protein necessary either for the initial docking of the endoplasmic reticulum to the surface membrane or for maintaining the interaction between dihydropyridine receptors and ryanodine receptors. We also conclude that formation of tetrads requires a close interaction between dihydropyridine receptors and ryanodine receptors.

Activation of the skeletal muscle calcium release channel by a cytoplasmic loop of the dihydropyridine receptor.

Expression studies with skeletal and cardiac muscle cDNAs have suggested that the putative cytoplasmic loop region of the dihydropyridine receptor (DHPR) alpha 1 subunit between transmembrane repeats II and III (DCL) is a major determinant of the type of excitation-contraction coupling (skeletal or cardiac) in rescued dysgenic muscle cells (Tanabe, T., Beam, K. G., Adams, B. A., Niidome, T., and Numa, S. (1990) Nature 346, 567-569). In this study, the possibility of a direct functional interaction with the sarcoplasmic reticulum ryanodine receptor/Ca2+ release channel has been tested by expressing the DCLs of the mammalian skeletal and cardiac muscle DHPR alpha 1 subunit in Escherichia coli. The purified peptides activated the skeletal muscle ryanodine receptor/Ca2+ release channel in single channel and [3H]ryanodine binding measurements, by increasing channel open probability and the affinity of [3H]ryanodine binding, respectively. The two peptides did not activate the cardiac muscle Ca2+ release channel. Other proteins (polylysine, serum albumin) also increased [3H]ryanodine binding and Ca2+ release channel activity, but their activation mechanisms were distinguishable from DCLs. These results show that the II-III cytoplasmic loop of the skeletal and cardiac DHPR alpha 1 subunit functionally interacts with the skeletal, but not cardiac, muscle Ca2+ release channel. Furthermore, our studies suggest that in addition to the DHPR, the sarcoplasmic reticulum Ca2+ release channel may determine the type of E-C coupling that exists in muscle.

The gene coding for the ?1 subunit of the skeletal dihydropyridine receptor (Cchl1a3=mdg) maps to mouse Chromosome 1 and human 1q32

Using both chromosomal in situ hybridization and molecular techniques, we report the genetic localization of the gene coding for the alpha 1 subunit of the skeletal slow Ca2+ current channel/DHP receptor gene (Cchl1a3) on human Chromosome (Chr) 1 (1q31-1q32 region) and on mouse Chr 1 (region (F-G)). On the basis of single-strand conformation polymorphism (SSCP-PCR) analysis in an interspecific backcross, we have determined that the Cchl1a3 = mdg (muscular dysgenesis) locus is very closely linked to the myogenin (Myog) locus.