Skeletal muscle cells express different isoforms of the calcium channel α2/δ subunit

We examined developmental changes in calcium channel alpha2/delta subunit mRNA in skeletal muscle and their possible influence on L-type calcium currents (ICa-L). Several isoforms of alpha2/delta-1 mRNA were found in myotubes and muscle fibers, and their relative levels changed with time in culture or age of the animal. Levels of alpha2/delta-1a were largest in older myotubes and was the only alpha2/delta-1 isoform present in adult muscle. Both myotubes and muscle fibers also expressed low levels of alpha2/delta-2 and alpha2/delta-3 mRNA at all ages. alpha2/delta-4 mRNA could not be detected in either myotubes or muscle fibers. Changes in amplitude and voltage-dependent inactivation of the ICa-L concurred with the shift in alpha2/delta-1 isoform message, suggesting that alternative splicing of this subunit might be important for modulation of ICa-L.     

The triad targeting signal of the skeletal muscle calcium channel is localized in the COOH terminus of the alpha(1S) subunit

The specific localization of L-type Ca(2+) channels in skeletal muscle triads is critical for their normal function in excitation-contraction (EC) coupling. Reconstitution of dysgenic myotubes with the skeletal muscle Ca(2+) channel alpha(1S) subunit restores Ca(2+) currents, EC coupling, and the normal localization of alpha(1S) in the triads. In contrast, expression of the neuronal alpha(1A) subunit gives rise to robust Ca(2+) currents but not to triad localization. To identify regions in the primary structure of alpha(1S) involved in the targeting of the Ca(2+) channel into the triads, chimeras of alpha(1S) and alpha(1A) were constructed, expressed in dysgenic myotubes, and their subcellular distribution was analyzed with double immunofluorescence labeling of the alpha(1S)/alpha(1A) chimeras and the ryanodine receptor. Whereas chimeras containing the COOH terminus of alpha(1A) were not incorporated into triads, chimeras containing the COOH terminus of alpha(1S) were correctly targeted. Mapping of the COOH terminus revealed a triad-targeting signal contained in the 55 amino-acid sequence (1607-1661) proximal to the putative clipping site of alpha(1S). Transferring this triad targeting signal to alpha(1A) was sufficient for targeting and clustering the neuronal isoform into skeletal muscle triads and caused a marked restoration of Ca(2+)-dependent EC coupling.     

Heterologous regulation of the cardiac Ca2+ channel alpha 1 subunit by skeletal muscle beta and gamma subunits. Implications for the structure of cardiac L-type Ca2+ channels.

High threshold L-type Ca2+ channels of skeletal muscle are thought to consist of a complex of alpha 1, alpha 2 delta, beta, and gamma subunits. Expression of the cloned alpha 1 subunit from skeletal and cardiac muscle has established that this protein is the dihydropyridine-sensitive ion-conducting subunit. However, the kinetics of the skeletal muscle alpha 1 alone expressed in mouse L-cells were abnormally slow and were accelerated to within the normal range by coexpression with the skeletal muscle beta subunit. The kinetics of cardiac muscle alpha 1 were also slowed but to a lesser extent and were not altered by coexpression with skeletal muscle alpha 2. We show here that coexpression of the skeletal muscle beta subunit with the cardiac alpha 1 subunit in Xenopus laevis oocytes produced: 1) an increase in the peak voltage-sensitive current, 2) a shift of the peak current-voltage relationship to more hyperpolarized potentials, and 3) an increase in the rate of activation. Coexpression of the skeletal muscle gamma subunit did not have a significant effect on currents elicited by alpha 1. However, when gamma was coexpressed with beta and alpha 1, both peak currents and rates of activation at more negative potentials were increased. These results indicate that rather than simply amplifying expression of alpha 1, heterologous skeletal muscle beta and gamma subunits can modulate the biophysical properties of cardiac alpha 1.     

Altered inactivation of Ca2+ current and Ca2+ release in mouse muscle fibers deficient in the DHP receptor gamma1 subunit

Functional impacts of the skeletal muscle-specific Ca2+ channel subunit gamma1 have previously been studied using coexpression with the cardiac alpha1C polypeptide in nonmuscle cells and primary-cultured myotubes of gamma1-deficient mice. Data from single adult muscle fibers of gamma-/- mice are not yet available. In the present study, we performed voltage clamp experiments on enzymatically isolated mature muscle fibers of the m. interosseus obtained from gamma+/+ and gamma-/- mice. We measured L-type Ca2+ inward currents and intracellular Ca2+ transients during 100-ms step depolarizations from a holding potential of -80 mV. Ratiometric Ca2+ transients were analyzed with a removal model fit approach to calculate the flux of Ca2+ from the sarcoplasmic reticulum. Ca2+ current density, Ca2+ release flux, and the voltage dependence of activation of both Ca2+ current and Ca2+ release were not significantly different. By varying the holding potential and recording Ca2+ current and Ca2+ release flux induced by 100-ms test depolarizations to +20 mV, we studied quasi-steady-state properties of slow voltage-dependent inactivation. For the Ca2+ current, these experiments showed a right-shifted voltage dependence of inactivation. Importantly, we could demonstrate that a very similar shift occurred also in the inactivation curve of Ca2+ release. Voltages of half maximal inactivation were altered by 16 (current) and 14 mV (release), respectively. Muscle fiber bundles, activated by elevated potassium concentration (120 mM), developed about threefold larger contracture force in gamma-/- compared with gamma+/+. This difference was independent of the presence of extracellular Ca2+ and likely results from the lower sensitivity to voltage-dependent inactivation of Ca2+ release. These results demonstrate a specific alteration of voltage-dependent inactivation of both Ca2+ entry and Ca2+ release by the gamma1 subunit of the dihydropyridine receptor in mature muscle fibers of the mouse.     

Involvement of the carboxy-terminus region of the dihydropyridine receptor beta1a subunit in excitation-contraction coupling of skeletal muscle

Skeletal muscle knockout cells lacking the beta subunit of the dihydropyridine receptor (DHPR) are devoid of slow L-type Ca(2+) current, charge movements, and excitation-contraction coupling, despite having a normal Ca(2+) storage capacity and Ca(2+) spark activity. In this study we identified a specific region of the missing beta1a subunit critical for the recovery of excitation-contraction. Experiments were performed in beta1-null myotubes expressing deletion mutants of the skeletal muscle-specific beta1a, the cardiac/brain-specific beta2a, or beta2a/beta1a chimeras. Immunostaining was used to determine that all beta constructs were expressed in these cells. We examined the Ca(2+) conductance, charge movements, and Ca(2+) transients measured by confocal fluo-3 fluorescence of transfected myotubes under whole-cell voltage-clamp. All constructs recovered an L-type Ca(2+) current with a density, voltage-dependence, and kinetics of activation similar to that recovered by full-length beta1a. In addition, all constructs except beta2a mutants recovered charge movements with a density similar to full-length beta1a. Thus, all beta constructs became integrated into a skeletal-type DHPR and, except for beta2a mutants, all restored functional DHPRs to the cell surface at a high density. The maximum amplitude of the Ca(2+) transient was not affected by separate deletions of the N-terminus of beta1a or the central linker region of beta1a connecting two highly conserved domains. Also, replacement of the N-terminus half of beta1a with that of beta2a had no effect. However, deletion of 35 residues of beta1a at the C-terminus produced a fivefold reduction in the maximum amplitude of the Ca(2+) transients. A similar observation was made by deletion of the C-terminus of a chimera in which the C-terminus half was from beta1a. The identified domain at the C-terminus of beta1a may be responsible for colocalization of DHPRs and ryanodine receptors (RyRs), or may be required for the signal that opens the RyRs during excitation-contraction coupling. This new role of DHPR beta in excitation-contraction coupling represents a cell-specific function that could not be predicted on the basis of functional expression studies in heterologous cells.     

Functional properties of a new voltage-dependent calcium channel alpha(2)delta auxiliary subunit gene (CACNA2D2)

We have positionally cloned and characterized a new calcium channel auxiliary subunit, alpha(2)delta-2 (CACNA2D2), which shares 56% amino acid identity with the known alpha(2)delta-1 subunit. The gene maps to the critical human tumor suppressor gene region in chromosome 3p21.3, showing very frequent allele loss and occasional homozygous deletions in lung, breast, and other cancers. The tissue distribution of alpha(2)delta-2 expression is different from alpha(2)delta-1, and alpha(2)delta-2 mRNA is most abundantly expressed in lung and testis and well expressed in brain, heart, and pancreas. In contrast, alpha(2)delta-1 is expressed predominantly in brain, heart, and skeletal muscle. When co-expressed (via cRNA injections) with alpha(1B) and beta(3) subunits in Xenopus oocytes, alpha(2)delta-2 increased peak size of the N-type Ca(2+) currents 9-fold, and when co-expressed with alpha(1C) or alpha(1G) subunits in Xenopus oocytes increased peak size of L-type channels 2-fold and T-type channels 1.8-fold, respectively. Anti-peptide antibodies detect the expression of a 129-kDa alpha(2)delta-2 polypeptide in some but not all lung tumor cells. We conclude that the alpha(2)delta-2 gene encodes a functional auxiliary subunit of voltage-gated Ca(2+) channels. Because of its chromosomal location and expression patterns, CACNA2D2 needs to be explored as a potential tumor suppressor gene linking Ca(2+) signaling and lung, breast, and other cancer pathogenesis. The homologous location on mouse chromosome 9 is also the site of the mouse neurologic mutant ducky (du), and thus, CACNA2D2 is also a candidate gene for this inherited idiopathic generalized epilepsy syndrome.     

Dissociation of charge movement from calcium release and calcium current in skeletal myotubes by gabapentin

The skeletal muscle L-type calcium channel or dihydropyridine receptor (DHPR) plays an integral role in excitation-contraction (E-C) coupling. Its activation initiates three sequential events: charge movement (Q(r)), calcium release, and calcium current (I(Ca,L)). This relationship suggests that changes in Q(r) might affect release and I(Ca,L). Here we studied the effect of gabapentin (GBP) on the three events generated by DHPRs in skeletal myotubes in culture. GBP specifically binds to the alpha(2)/delta(1) subunit of the brain and skeletal muscle DHPR. Myotubes were stimulated with a protocol that included a depolarizing prepulse to inactivate voltage-dependent proteins other than DHPRs. Gabapentin (50 microM) significantly increased Q(r) while decreasing the rate of rise of calcium transients. Gabapentin also reduced the maximum amplitude of the I(Ca,L) (as we previously reported) without modifying the kinetics of activation. Exposure of GBP-treated myotubes to 10 microM nifedipine prevented the increase of Q(r) promoted by this drug, indicating that the extra charge recorded originated from DHPRs. Our data suggest that GBP dissociates the functions of the DHPR from the initial voltage-sensing step and implicates a role for the alpha(2)/delta(1) subunit in E-C coupling.     

Determinants for calmodulin binding on voltage-dependent Ca2+ channels

Calmodulin, bound to the alpha(1) subunit of the cardiac L-type calcium channel, is required for calcium-dependent inactivation of this channel. Several laboratories have suggested that the site of interaction of calmodulin with the channel is an IQ-like motif in the carboxyl-terminal region of the alpha(1) subunit. Mutations in this IQ motif are linked to L-type Ca(2+) current (I(Ca)) facilitation and inactivation. IQ peptides from L, P/Q, N, and R channels all bind Ca(2+)calmodulin but not Ca(2+)-free calmodulin. Another peptide representing a carboxyl-terminal sequence found only in L-type channels (designated the CB domain) binds Ca(2+)calmodulin and enhances Ca(2+)-dependent I(Ca) facilitation in cardiac myocytes, suggesting the CB domain is functionally important. Calmodulin blocks the binding of an antibody specific for the CB sequence to the skeletal muscle L-type Ca(2+) channel, suggesting that this is a calmodulin binding site on the intact protein. The binding of the IQ and CB peptides to calmodulin appears to be competitive, signifying that the two sequences represent either independent or alternative binding sites for calmodulin rather than both sequences contributing to a single binding site.     

Functional interaction of CaV channel isoforms with ryanodine receptors studied in dysgenic myotubes

The L-type Ca(2+) channels Ca(V)1.1 (alpha(1S)) and Ca(V)1.2 (alpha(1C)) share properties of targeting but differ by their mode of coupling to ryanodine receptors in muscle cells. The brain isoform Ca(V)2.1 (alpha(1A)) lacks ryanodine receptor targeting. We studied these three isoforms in myotubes of the alpha(1S)-deficient skeletal muscle cell line GLT under voltage-clamp conditions and estimated the flux of Ca(2+) (Ca(2+) input flux) resulting from Ca(2+) entry and release. Surprisingly, amplitude and kinetics of the input flux were similar for alpha(1C) and alpha(1A) despite a previously reported strong difference in responsiveness to extracellular stimulation. The kinetic flux characteristics of alpha(1C) and alpha(1A) resembled those in alpha(1S)-expressing cells but the contribution of Ca(2+) entry was much larger. alpha(1C) but not alpha(1A)-expressing cells revealed a distinct transient flux component sensitive to sarcoplasmic reticulum depletion by 30 microM cyclopiazonic acid and 10 mM caffeine. This component likely results from synchronized Ca(2+)-induced Ca(2+) release that is absent in alpha(1A)-expressing myotubes. In cells expressing an alpha(1A)-derivative (alpha(1)Aas(1592-clip)) containing the putative targeting sequence of alpha(1S), a similar transient component was noticeable. Yet, it was considerably smaller than in alpha(1C), indicating that the local Ca(2+) entry produced by the chimera is less effective in triggering Ca(2+) release despite similar global Ca(2+) inward current density.     

The beta subunit controls the gating and dihydropyridine sensitivity of the skeletal muscle Ca2+ channel.

The skeletal muscle (SKM) L-type Ca2+ channel is composed of a central subunit designated alpha 1, which contains the pore and the dihydropyridine (DHP) binding domains and three associated subunits, alpha 2/delta, beta, and gamma, which influence the activity of the SKM alpha 1. Coexpression of SKM alpha 1 and SKM beta in stably transfected mouse L cells results in a dramatic increase in DHP binding accompanied by fast gated Ba2+ currents. We report here that this "SKM alpha 1 beta-related phenotype" can be converted upon intracellular trypsin treatment into a slowly inactivating, DHP sensitive "SKM alpha 1 phenotype." These observations indicate that current amplitude, fast inactivation, and DHP sensitivity are modulated by an interaction of SKM alpha 1 and SKM beta on the internal side of the membrane.     

Ca2+-dependent excitation-contraction coupling triggered by the heterologous cardiac/brain DHPR beta2a-subunit in skeletal myotubes

Molecular determinants essential for skeletal-type excitation-contraction (EC) coupling have been described in the cytosolic loops of the dihydropyridine receptor (DHPR) alpha1S pore subunit and in the carboxyl terminus of the skeletal-specific DHPR beta1a-subunit. It is unknown whether EC coupling domains present in the beta-subunit influence those present in the pore subunit or if they act independent of each other. To address this question, we investigated the EC coupling signal that is generated when the endogenous DHPR pore subunit alpha1S is paired with the heterologous heart/brain DHPR beta2a-subunit. Studies were conducted in primary cultured myotubes from beta1 knockout (KO), ryanodine receptor type 1 (RyR1) KO, ryanodine receptor type 3 (RyR3) KO, and double RyR1/RyR3 KO mice under voltage clamp with simultaneous monitoring of confocal fluo-4 fluorescence. The beta2a-mediated Ca2+ current recovered in beta1 KO myotubes lacking the endogenous DHPR beta1a-subunit verified formation of the alpha1S/beta1a pair. In myotube genotypes which express no or low-density L-type Ca2+ currents, namely beta1 KO and RyR1 KO, beta2a overexpression recovered a wild-type density of nifedipine-sensitive Ca2+ currents with a slow activation kinetics typical of skeletal myotubes. Concurrent with Ca2+ current recovery, there was a drastic reduction of voltage-dependent, skeletal-type EC coupling and emergence of Ca2+ transients triggered by the Ca2+ current. A comparison of beta2a overexpression in RyR3 KO, RyR1 KO, and double RyR1/RyR3 KO myotubes concluded that both RyR1 and RyR3 isoforms participated in Ca2+-dependent Ca2+ release triggered by the beta2a-subunit. In beta1 KO and RyR1 KO myotubes, the Ca2+-dependent EC coupling promoted by beta2a overexpression had the following characteristics: 1), L-type Ca2+ currents had a wild-type density; 2), Ca2+ transients activated much slower than controls overexpressing beta1a, and the rate of fluorescence increase was consistent with the activation kinetics of the Ca2+ current; 3), the voltage dependence of the Ca2+ transient was bell-shaped and the maximum was centered at approximately +30 mV, consistent with the voltage dependence of the Ca2+ current; and 4), Ca2+ currents and Ca2+ transients were fully blocked by nifedipine. The loss in voltage-dependent EC coupling promoted by beta2a was inferred by the drastic reduction in maximal Ca2+ fluorescence at large positive potentials (DeltaF/Fmax) in double dysgenic/beta1 KO myotubes overexpressing the pore mutant alpha1S (E1014K) and beta2a. The data indicate that beta2a, upon interaction with the skeletal pore subunit alpha1S, overrides critical EC coupling determinants present in alpha1S. We propose that the alpha1S/beta pair, and not the alpha1S-subunit alone, controls the EC coupling signal in skeletal muscle.     

Cardiac-type EC-coupling in dysgenic myotubes restored with Ca2+ channel subunit isoforms alpha1C and alpha1D does not correlate with current density

Ca(2+)-induced Ca(2+)-release (CICR)-the mechanism of cardiac excitation-contraction (EC) coupling-also contributes to skeletal muscle contraction; however, its properties are still poorly understood. CICR in skeletal muscle can be induced independently of direct, calcium-independent activation of sarcoplasmic reticulum Ca(2+) release, by reconstituting dysgenic myotubes with the cardiac Ca(2+) channel alpha(1C) (Ca(V)1.2) subunit. Ca(2+) influx through alpha(1C) provides the trigger for opening the sarcoplasmic reticulum Ca(2+) release channels. Here we show that also the Ca(2+) channel alpha(1D) isoform (Ca(V)1.3) can restore cardiac-type EC-coupling. GFP-alpha(1D) expressed in dysgenic myotubes is correctly targeted into the triad junctions and generates action potential-induced Ca(2+) transients with the same efficiency as GFP-alpha(1C) despite threefold smaller Ca(2+) currents. In contrast, GFP-alpha(1A), which generates large currents but is not targeted into triads, rarely restores action potential-induced Ca(2+) transients. Thus, cardiac-type EC-coupling in skeletal myotubes depends primarily on the correct targeting of the voltage-gated Ca(2+) channels and less on their current size. Combined patch-clamp/fluo-4 Ca(2+) recordings revealed that the induction of Ca(2+) transients and their maximal amplitudes are independent of the different current densities of GFP-alpha(1C) and GFP-alpha(1D). These properties of cardiac-type EC-coupling in dysgenic myotubes are consistent with a CICR mechanism under the control of local Ca(2+) gradients in the triad junctions.     

Sequence differences between alpha1C and alpha1S Ca2+ channel subunits reveal structural determinants of a guarded and modulated benzothiazepine receptor

The molecular basis of the Ca2+ channel block by (+)-cis-diltiazem was studied in class A/L-type chimeras and mutant alpha1C-a Ca2+ channels. Chimeras consisted of either rabbit heart (alpha1C-a) or carp skeletal muscle (alpha1S) sequence in transmembrane segments IIIS6, IVS6, and adjacent S5-S6 linkers. Only chimeras containing sequences from alpha1C-a were efficiently blocked by (+)-cis-diltiazem, whereas the phenylalkylamine (-)-gallopamil efficiently blocked both constructs. Carp skeletal muscle and rabbit heart Ca2+ channel alpha1 subunits differ with respect to two nonconserved amino acids in segments IVS6. Transfer of a single leucine (Leu1383, located at the extracellular mouth of the pore) from IVS6 alpha1C-a to IVS6 of alpha1S significantly increased the (+)-cis-diltiazem sensitivity of the corresponding mutant L1383I. An analysis of the role of the two heterologous amino acids in a L-type alpha1 subunit revealed that corresponding amino acids in position 1487 (outer channel mouth) determine recovery of resting Ca2+ channels from block by (+)-cis-diltiazem. The second heterologous amino acid in position 1504 of segment IVS6 (inner channel mouth) was identified as crucial inactivation determinant of L-type Ca2+ channels. This residue simultaneously modulates drug binding during membrane depolarization. Our study provides the first evidence for a guarded and modulated benzothiazepine receptor on L-type channels.     

Functional impact of the ryanodine receptor on the skeletal muscle L-type Ca(2+) channel

L-type Ca(2+) channel (L-channel) activity of the skeletal muscle dihydropyridine receptor is markedly enhanced by the skeletal muscle isoform of the ryanodine receptor (RyR1) (Nakai, J., R.T. Dirksen, H. T. Nguyen, I.N. Pessah, K.G. Beam, and P.D. Allen. 1996. Nature. 380:72-75.). However, the dependence of the biophysical and pharmacological properties of skeletal L-current on RyR1 has yet to be fully elucidated. Thus, we have evaluated the influence of RyR1 on the properties of macroscopic L-currents and intracellular charge movements in cultured skeletal myotubes derived from normal and "RyR1-knockout" (dyspedic) mice. Compared with normal myotubes, dyspedic myotubes exhibited a 40% reduction in the amount of maximal immobilization-resistant charge movement (Q(max), 7.5 +/- 0.8 and 4.5 +/- 0.4 nC/muF for normal and dyspedic myotubes, respectively) and an approximately fivefold reduction in the ratio of maximal L-channel conductance to charge movement (G(max)/Q(max)). Thus, RyR1 enhances both the expression level and Ca(2+) conducting activity of the skeletal L-channel. For both normal and dyspedic myotubes, the sum of two exponentials was required to fit L-current activation and resulted in extraction of the amplitudes (A(fast) and A(slow)) and time constants (tau(slow) and tau(fast)) for each component of the macroscopic current. In spite of a >10-fold in difference current density, L-currents in normal and dyspedic myotubes exhibited similar relative contributions of fast and slow components (at +40 mV; A(fast)/[A(fast) + A(slow)] approximately 0.25). However, both tau(fast) and tau(slow) were significantly (P < 0.02) faster for myotubes lacking the RyR1 protein (tau(fast), 8.5 +/- 1.2 and 4.4 +/- 0.5 ms; tau(slow), 79.5 +/- 10.5 and 34.6 +/- 3.7 ms at +40 mV for normal and dyspedic myotubes, respectively). In both normal and dyspedic myotubes, (-) Bay K 8644 (5 microM) caused a hyperpolarizing shift (approximately 10 mV) in the voltage dependence of channel activation and an 80% increase in peak L-current. However, the increase in peak L-current correlated with moderate increases in both A(slow) and A(fast) in normal myotubes, but a large increase in only A(fast) in dyspedic myotubes. Equimolar substitution of Ba(2+) for extracellular Ca(2+) increased both A(fast) and A(slow) in normal myotubes. The identical substitution in dyspedic myotubes failed to significantly alter the magnitude of either A(fast) or A(slow). These results demonstrate that RyR1 influences essential properties of skeletal L-channels (expression level, activation kinetics, modulation by dihydropyridine agonist, and divalent conductance) and supports the notion that RyR1 acts as an important allosteric modulator of the skeletal L-channel, analogous to that of a Ca(2+) channel accessory subunit.     

Molecular origin of the L-type Ca2+ current of skeletal muscle myotubes selectively deficient in dihydropyridine receptor beta1a subunit.

The origin of Ibetanull, the Ca2+ current of myotubes from mice lacking the skeletal dihydropyridine receptor (DHPR) beta1a subunit, was investigated. The density of Ibetanull was similar to that of Idys, the Ca2+ current of myotubes from dysgenic mice lacking the skeletal DHPR alpha1S subunit (-0.6 +/- 0.1 and -0.7 +/- 0.1 pA/pF, respectively). However, Ibetanull activated at significantly more positive potentials. The midpoints of the GCa-V curves were 16.3 +/- 1.1 mV and 11.7 +/- 1.0 mV for Ibetanull and Idys, respectively. Ibetanull activated significantly more slowly than Idys. At +30 mV, the activation time constant for Ibetanull was 26 +/- 3 ms, and that for Idys was 7 +/- 1 ms. The unitary current of normal L-type and beta1-null Ca2+ channels estimated from the mean variance relationship at +20 mV in 10 mM external Ca2+ was 22 +/- 4 fA and 43 +/- 7 fA, respectively. Both values were significantly smaller than the single-channel current estimated for dysgenic Ca2+ channels, which was 84 +/- 9 fA under the same conditions. Ibetanull and Idys have different gating and permeation characteristics, suggesting that the bulk of the DHPR alpha1 subunits underlying these currents are different. Ibetanull is suggested to originate primarily from Ca2+ channels with a DHPR alpha1S subunit. Dysgenic Ca2+ channels may be a minor component of this current. The expression of DHPR alpha1S in beta1-null myotubes and its absence in dysgenic myotubes was confirmed by immunofluorescence labeling of cells.     

Heterologous expression of BI Ca2+ channels in dysgenic skeletal muscle

We have examined the ability of BI (class A) Ca2+ channels, cloned from rabbit brain, to mediate excitation-contraction (E-C) coupling in skeletal muscle. Expression plasmids carrying cDNA encoding BI channels were microinjected into the nuclei of dysgenic mouse myotubes grown in primary culture. Ionic currents and intramembrane charge movements produced by the BI channels were recorded using the whole-cell patch- clamp technique. Injected myotubes expressed high densities of ionic BI Ca2+ channel current (average 31 pA/pF) but did not display spontaneous contractions, and only very rarely displayed evoked contractions. The expressed ionic current was pharmacologically distinguished from the endogenous L-type current of dysgenic skeletal muscle (Idys) by its insensitivity to the dihydropyridine antagonist (+)-PN 200-110. Peak BI Ca2+ currents activated with a time constant (tau a) of approximately 2 ms and inactivated with a time constant (tau h) of approximately 260 ms (20-23 degrees C). The time constant of inactivation (tau h) was not increased by substituting Ba2+ for Ca2+ as charge carrier, demonstrating that BI channels expressed in dysgenic myotubes do not undergo Ca(2+)-dependent inactivation. The average maximal Ca2+ conductance (Gmax) produced by the BI channels was quite large (approximately 534 S/F). In contrast, the average maximal charge movement (Qmax) produced in the same myotubes (approximately 2.7 nC/microF) was quite small, being barely larger than Qmax in control dysgenic myotubes (approximately 2.3 nC/microF). Thus, the ratio Gmax/Qmax for the BI channels was considerably higher than previously found for cardiac or skeletal muscle L-type Ca2+ channels expressed in the same system, indicating that neuronal BI Ca2+ channels exhibit a much higher open probability than these L-type Ca2+ channels.     

Biochemical and biophysical evidence for gamma 2 subunit association with neuronal voltage-activated Ca2+ channels.

A novel gene (Cacng2; gamma(2)) encoding a protein similar to the voltage-activated Ca(2+) channel gamma(1) subunit was identified as the defective gene in the epileptic and ataxic mouse, stargazer. In this study, we analyzed the association of this novel neuronal gamma(2) subunit with Ca(2+) channels of rabbit brain, and the function of the gamma(2) subunit in recombinant neuronal Ca(2+) channels expressed in Xenopus oocytes. Our results showed that the gamma(2) subunit and a closely related protein (called gamma(3)) co-sedimented and co-immunoprecipitated with neuronal Ca(2+) channel subunits in vivo. Electrophysiological analyses showed that gamma(2) co-expression caused a significant decrease in the current amplitude of both alpha(1B)(alpha(1)2.2)-class (36.8%) and alpha(1A)(alpha(1)2.1)-class (39.7%) Ca(2+) channels (alpha(1)beta(3)alpha(2)delta). Interestingly, the inhibitory effects of the gamma(2) subunit on current amplitude were dependent on the co-expression of the alpha(2)delta subunit. In addition, co-expression of gamma(2) or gamma(1) also significantly decelerates the activation kinetics of alpha(1B)-class Ca(2+) channels. Taken together, these results suggest that the gamma(2) subunit is an important constituent of the neuronal Ca(2+) channel complex and that it down-regulates neuronal Ca(2+) channel activity. Furthermore, the gamma(2) subunit likely contributes to the fine-tuning of neuronal Ca(2+) channels by counterbalancing the effects of the alpha(2)delta subunit.