Alpha 1S Subunit Research Articles

Short-Communication: Variable Expression of Clinical Symptoms and an Unexpected Finding of Vacuolar Myopathy Related to a Pathogenic Variant in the CACNA1S Gene in a Previous Case Report.

Several clinical phenotypes have been described related to the CACNA1S gene (calcium channel voltage-dependent L-type alpha-1S subunit), such as autosomal dominant hypokalemic periodic paralysis 1 and autosomal dominant malignant hyperthermia susceptibility and are associated with autosomal dominant and recessive congenital myopathy. Recently, an interesting case of a 58-year-old male patient was published describing an unusual clinical presentation of hypokalemic periodic paralysis where a late-onset limb-girdle myopathy had developed 41 years after paralysis occurred when the patient was 11 years old. Muscle biopsy results were consistent with myopathic changes and revealed the presence of vacuoles, without inflammatory reaction. Later, molecular analysis revealed a pathogenic variant c.3716G>A (p.Arg1239His) in exon 30 of the CACNA1S gene. This technical report provides an extension of the molecular findings and evaluates the clinical and histopathological relationship previously published regarding this case.

Genetic Evidence Supporting the Role of the Calcium Channel, CACNA1S, in Tooth Cusp and Root Patterning.

In this study, we report a unique dominantly inherited disorganized supernumerary cusp and single root phenotype presented by 11 affected individuals belonging to 5 north-eastern Thai families. Using whole exome sequencing (WES) we identified a common single missense mutation that segregates with the phenotype in exon 6 of CACNA1S (Cav1.1) (NM_000069.2: c.[865A > G];[=] p.[Ile289Val];[=]), the Calcium Channel, Voltage-Dependent, L Type, Alpha-1s Subunit, OMIM ∗ 114208), affecting a highly conserved amino-acid isoleucine residue within the pore forming subdomain of CACNA1S protein. This is a strong genetic evidence that a voltage-dependent calcium ion channel is likely to play a role in influencing tooth morphogenesis and patterning.

Possible Involvement of the CACNA1E Gene in Migraine: A Search for Single Nucleotide Polymorphism in Different Clinical Phenotypes.

To search for differences in prevalence of a CACNA1E variant between migraine without aura, various phenotypes of migraine with aura, and healthy controls. Familial hemiplegic migraine type 1 (FHM1) is associated with mutations in the CACNA1A gene coding for the alpha 1A (Cav 2.1) pore-forming subunit of P/Q voltage-dependent Ca2+ channels. These mutations are not found in the common forms of migraine with or without aura. The alpha 1E subunit (Cav 2.3) is the counterpart of Cav 2.1 in R-type Ca2+ channels, has different functional properties, and is encoded by the CACNA1E gene. First, we performed a total exon sequencing of the CACNA1E gene in three probands selected because they had no abnormalities in the three FHM genes. In a patient suffering from basilar-type migraine, we identified a single nucleotide polymorphism (SNP) in exon 20 of the CACNA1E gene (Asp859Glu - rs35737760; Minor Allele Frequency 0.2241) hitherto not studied in migraine. In a second step, we determined its occurrence in four groups by direct sequencing on blood genomic DNA: migraine patients without aura (N = 24), with typical aura (N = 55), complex neurological auras (N = 19; hemiplegic aura: N = 15; brain stem aura: N = 4), and healthy controls (N = 102). The Asp859Glu - rs35737760 SNP of the CACNA1E gene was present in 12.7% of control subjects and in 20.4% of the total migraine group. In the migraine group it was significantly over-represented in patients with complex neurological auras (42.1%), OR 4.98 (95% CI: 1.69-14.67, uncorrected P = .005, Bonferroni P = .030, 2-tailed Fisher's exact test). There was no significant difference between migraine with typical aura (10.9%) and controls. We identified a polymorphism in exon 20 of the CACNA1E gene (Asp859Glu - rs35737760) that is more prevalent in hemiplegic and brain stem aura migraine. This missense variant causes a change from aspartate to glutamate at position 859 of the Cav 2.3 protein and might modulate the function of R-type Ca2+ channels. It could thus be relevant for migraine with complex neurological aura, although this remains to be proven.

CNS Voltage-gated Calcium Channel Gene Variation And Prolonged Recovery Following Sport-related Concussion

Objectives:To examine the association between concussion duration and two calcium channel, voltage-dependent, R type, alpha 1E subunit (CACNA1E) single nucleotide polymorphisms (i.e., rs35737760 and rs704326). A secondary purpose was to examine the association between CACNA1E single nucleotide polymorphisms (SNPs) and three acute concussion severity scores (i.e., vestibule-ocular reflex test, balance error scoring scale, and Immediate Post-Concussion Assessment and Cognitive Testing).Methods:Forty athletes with a diagnosed concussion from a hospital concussion program completed a standardized initial evaluation. Concussion injury characteristics, acute signs and symptoms followed by an objective screening (i.e., vestibular ocular assessments, balance error scoring system test, and Immediate Post-Concussion Assessment and Cognitive Testing exam) were assessed. Enrolled participants provided salivary samples for isolation of DNA. Two exon SNPs rs35737760 and rs704326 within CACNA1E were genotyped.Results:There was a significant difference found between acute balance deficits and prolonged recovery group (X2 = 5.66, p = 0.017). There was an association found between the dominant model GG genotype (X2 = 5.41, p = 0.027) within the rs704326 SNP and prolonged recovery group. Significant differences were identified for the rs704326 SNP within the dominant model GG genotype (p = 0.030) for VOR scores by recovery. A significant difference was found between the rs704326 SNP codominant model AA (p = 0.042) and visual memory. There was an association between acute balance deficits and prolonged recovery (X2 = 5.66, p = 0.017) for the rs35737760 SNP. No significant associations between concussion severity and genotype for rs35737760 SNP.Conclusion:Athletes carrying the CACNA1E rs704326 homozygous genotype GG are at a greater risk of a prolonged recovery. Athletes that reported balance deficits at the time of injury were more likely to have prolonged recovery. These polymorphisms within CACNA1E could alter the CACNA1E protein and allow for an increase of calcium leading to deficits to the granule cells within the brain.

Mutation analysis of CACNA1S and SCN4A in patients with hypokalemic periodic paralysis.

Mutations in CACNA1S (calcium channel, voltage‑dependent, L type, alpha 1S subunit) and SCN4A (sodium channel, voltage‑gated, type IV, alpha subunit) are associated with hypokalemic periodic paralysis (HPP). The aim of the current study was to investigate CACNA1S and SCN4A mutations in patients with HPP. Mutations in CACNA1S and SCN4A were detected in three familial hypokalemic periodic paralysis (FHPP) pedigrees and in two thyrotoxic hypokalemic periodic paralysis (THPP) pedigrees using polymerase chain reaction, DNA sequencing and sequence alignment with GenBank data. A single base mutation from cytosine to guanine at site 1582 was identified in exon 11 of CACNA1S in one FHPP pedigree, resulting in an arginine to glycine (R528G) substitution. A single base mutation from thymine to cytosine at site 2012 was identified in exon 12 of SCN4A in one THPP pedigree, resulting in a phenylalanine to serine (F671S) substitution. No mutations in CACNA1S or SCN4A were identified in the remaining three pedigrees. The present study indicated that CACNA1S and SCN4A mutations are relatively rare in patients with HPP, and further studies are required to determine whether these mutation‑associated substitutions are representative of patients with HPP.

3D Structure of the Dihydropyridine Receptor of Skeletal Muscle.

Excitation contraction coupling, the rapid and massive Ca2+ release under control of an action potential that triggers muscle contraction, takes places at specialized regions of the cell called triad junctions. There, a highly ordered supramolecular complex between the dihydropyridine receptor (DHPR) and the ryanodine receptor (RyR1) mediates the quasi-instantaneous conversion from T-tubule depolarization into Ca2+ release from the sarcoplasmic reticulum (SR). The DHPR has several key modules required for EC coupling: the voltage sensors and II-III loop in the alpha1s subunit, and the beta subunit. To gain insight into their molecular organization, this review examines the most updated 3D structure of the DHPR as obtained by transmission electron microscopy and image reconstruction. Although structure determination of a heteromeric membrane protein such as the DHPR is challenging, novel technical advances in protein expression and 3D labeling facilitated this task. The 3D structure of the DHPR complex consists of a main body with five irregular corners around its perimeter encompassing the transmembrane alpha 1s subunit besides the intracellular beta subunit, an extended extracellular alpha 2 subunit, and a bulky intracellular II-III loop. The structural definition attained at 19 Å resolution enabled docking of the atomic coordinates of structural homologs of the alpha1s and beta subunits. These structural features, together with their relative location with respect to the RyR1, are discussed in the context of the functional data.

Association between genetic polymorphisms in Ca(v)2.3 (R-type) Ca2+ channels and fentanyl sensitivity in patients undergoing painful cosmetic surgery.

Individual differences in the sensitivity to fentanyl, a widely used opioid analgesic, lead to different proper doses of fentanyl, which can hamper effective pain treatment. Voltage-activated Ca2+ channels (VACCs) play a crucial role in the nervous system by controlling membrane excitability and calcium signaling. Cav2.3 (R-type) VACCs have been especially thought to play critical roles in pain pathways and the analgesic effects of opioids. However, unknown is whether single-nucleotide polymorphisms (SNPs) of the human CACNA1E (calcium channel, voltage-dependent, R type, alpha 1E subunit) gene that encodes Cav2.3 VACCs influence the analgesic effects of opioids. Thus, the present study examined associations between fentanyl sensitivity and SNPs in the human CACNA1E gene in 355 Japanese patients who underwent painful orofacial cosmetic surgery, including bone dissection. We first conducted linkage disequilibrium (LD) analyses of 223 SNPs in a region that contains the CACNA1E gene using genomic samples from 100 patients, and a total of 13 LD blocks with 42 Tag SNPs were observed within and around the CACNA1E gene region. In the preliminary study using the same 100 genomic samples, only the rs3845446 A/G SNP was significantly associated with perioperative fentanyl use among these 42 Tag SNPs. In a confirmatory study using the other 255 genomic samples, this SNP was also significantly associated with perioperative fentanyl use. Thus, we further analyzed associations between genotypes of this SNP and all of the clinical data using a total of 355 samples. The rs3845446 A/G SNP was associated with intraoperative fentanyl use, 24 h postoperative fentanyl requirements, and perioperative fentanyl use. Subjects who carried the minor G allele required significantly less fentanyl for pain control compared with subjects who did not carry this allele. Although further validation is needed, the present findings show the possibility of the involvement of CACNA1E gene polymorphisms in fentanyl sensitivity.

Novel missense mutations and unexpected multiple changes of RYR1 gene in 75 malignant hyperthermia families

Malignant hyperthermia (MH) is an autosomal dominant pharmacogenetic disorder of skeletal muscle characterized by disturbance of intracellular calcium homeostasis in the sarcoplasmic reticulum. Mutations of the ryanodine receptor 1 (RYR1) gene account for most cases, with some studies claiming up to 86% of mutations in this locus. However, RYR1 gene is large and variants are common even in the normal population. We examined 54 families with MH susceptibility and 21 diagnosed with equivocal MH. Thirty-five were selected for an anesthetic reaction, whereas the remainder for hyperCKemia. In these, we studied all 106 exons of the RYR1 gene. When no mutation was found, we also screened: sodium channel voltage-gated, type IV alpha subunit (SCN4A), calcium channel voltage-dependent, L type, alpha 1S subunit (CACNA1S), and L-type voltage-gated calcium channel alpha 2/delta-subunit (CACNL2A). Twenty-nine different RYR1 mutations were discovered in 40 families. Three other MH genes were tested in negative cases. Fourteen RYR1 amino acid changes were novel, of which 12 were located outside the mutational 'hot spots'. In two families, the known mutation p.R3903Q was also observed in malignant hyperthermia-nonsusceptible (MHN) individuals. Unexpectedly, four changes were also found in the same family and two in another. Our study confirms that MH is genetically heterogeneous and that a consistent number of cases are not due to RYR1 mutations. The discordance between in vitro contracture test status and the presence of a proven causative RYR1 mutation suggests that the penetrance may vary due to as yet unknown factors.

In Vitro Interactions Between the Beta1a Subunit of the Skeletal Muscle Dhpr and RyR1

The beta1a subunit of the skeletal muscle DHPR plays two important roles in excitation-contraction (EC) coupling in skeletal muscle. Beta1a was originally found to target the alpha1S subunit of the DHPR to sarcolemmal tetrads which oppose skeletal ryanodine receptor (RyR1) channels in the sarcoplasmic reticulum. It was later found to also aid in transmission of EC coupling between the alpha1S subunit and RyR1 (1,2,3), through its C-terminal tail residues (1,2). This presumably depends on an interaction between the C-terminal tail of beta1a and RyR1. The full beta1a subunit binds to RyR1 in affinity chromatography experiments (4), but direct binding of the C-tail to RyR1 has not been reported, nor have direct functional interactions between the proteins. We show here that a peptide corresponding to the native sequence of residues in the C-terminus of beta1a, but not a scrambled sequence, bind to RyR1. In addition the peptide and the full β1a subunit significantly increase both [3H]ryanodine binding and single RyR1 channel activity at concentrations of 0.1 to 1nM, with maximum 3- to 5-fold activation at a concentration of ∼10nM. The increase in RyR1 channel activity with both constructs is essentially irreversible within the lifetime of the bilayer, indicating tight binding. These results show that the C-terminal tail of the beta1a subunit is capable of directly binding to and activating RyR1 and hence suggest that beta1a may enhance EC coupling by virtue of its ability to activate RyR1.1. Beurg M et al. Biophys J1999 77, 2953-2967.2. Zhou W et al. Cell Calcium 2006 39, 227-236.3. Schredelseker J et al. J Biol Chem 2009 284, 1242-51.4. Cheng W et al. Proc Natl Acad Sci 2005 102, 19225-19230.

Increased CaVβ1a expression with aging contributes to skeletal muscle weakness

Ca2+ release from the sarcoplasmic reticulum (SR) into the cytosol is a crucial part of excitation-contraction (E-C) coupling. Excitation-contraction uncoupling, a deficit in Ca2+ release from the SR, is thought to be responsible for at least some of the loss in specific force observed in aging skeletal muscle. Excitation-contraction uncoupling may be caused by alterations in expression of the voltage-dependent calcium channel alpha1s (CaV1.1) and beta1a (CaVbeta1a) subunits, both of which are necessary for E-C coupling to occur. While previous studies have found CaV1.1 expression declines in old rodents, CaVbeta1a expression has not been previously examined in aging models. Western blot analysis shows a substantial increase of CaVbeta1a expression over the full lifespan of Friend Virus B (FVB) mice. To examine the specific effects of CaVbeta1a overexpression, a CaVbeta1a-YFP plasmid was electroporated in vivo into young animals. The resulting increase in expression of CaVbeta1a corresponded to decline of CaV1.1 over the same time period. YFP fluorescence, used as a measure of CaVbeta1a-YFP expression in individual fibers, also showed an inverse relationship with charge movement, measured using the whole-cell patch-clamp technique. Specific force was significantly reduced in young CaVbeta1a-YFP electroporated muscle fibers compared with sham-electroporated, age-matched controls. siRNA interference of CaVbeta1a in young muscles reduced charge movement, while charge movement in old was restored to young control levels. These studies imply CaVbeta1a serves as both a positive and negative regulator CaV1.1 expression, and that endogenous overexpression of CaVbeta1a during old age may play a role in the loss of specific force.

Presence of free haptoglobin alpha 1S-subunit in acute porcine reproductive and respiratory syndrome virus infection

The biochemical events triggered by viral infection are critical to the outcome of a host immune response. Porcine reproductive and respiratory syndrome virus (PRRSV) causes the most significant disease of swine worldwide. Onset of infection is insidious and subclinical. Clinical signs may not appear for days and antibodies cannot be detected for a week or more. To understand better the early pathophysiological response of swine to PRRSV infection and its inapparent onset, we examined serum samples in the first days of infection for evidence of early biochemical changes. Sera from pigs infected with various isolates of PRRSV were extracted to remove high molecular mass proteins, desalted and analysed by matrix assisted laser desorption/ionization-time of flight mass spectrometry (MS). Comparative analysis of low molecular mass serum protein profiles revealed that one protein, with an m/z value of 9244+/-2, appeared within 1 day of infection. The 9244+/-2 peak was identified as the alpha 1S (alpha1S)-subunit of porcine haptoglobin (Hp) by tandem MS sequencing and confirmed by immunoblotting with anti-porcine Hp antibody. Hp is an acute phase haem-binding protein consisting of alpha-beta heterodimers that is secreted from the liver in response to stresses, including infection. However, the presence of free alpha1S-subunit in response to infection is novel and may provide new insights into biochemical processing of Hp and its role in disease pathogenesis, including PRRS.

Participation of low-threshold Ca2+ spike in the Purkinje cells complex spike

In Purkinje cells from cerebellar slice cultures, low-threshold Ca spike (LTS) gives rise to complex bursts in the soma that resemble the complex spike induced by climbing fibers stimulation. We show that LTS is reduced by T-type and R-type Ca channel blockers (SNX-482, nickel, or mibefradil). We propose that LTS is generated by openings of T-type Ca channels (alpha-1G and/or alpha-1I subunits) and R-type Ca channels (alpha-1E subunit isoforms with a weak sensitivity to SNX-482 and to nickel). Using mibefradil we show that climbing fiber stimulation activates LTS, which contributes to the shape of the response. This Ca entry may be involved in Ca-dependent synaptic plasticity of the parallel fiber input induced by climbing fiber activation.

Absence of regulation of the T-type calcium current by Cav1.1, β1a and γ1 dihydropyridine receptor subunits in skeletal muscle cells

The subunit structure of low voltage activated T-type Ca2+ channels is still unknown. Co-expression of dihydropyridine receptor (DHPR) auxiliary subunits with T-type alpha1 subunits in heterologous systems has produced conflicting results. In developing foetal skeletal muscle fibres which abundantly express DHPR subunits, Cav3.2 (alpha1H) subunits are believed to underlie T-type calcium currents which disappear 2 to 3 weeks after birth. Therefore, a possible regulation of foetal skeletal muscle T-type Ca2+ channels by DHPR subunits was investigated in freshly isolated foetal skeletal muscle using knockout mice, which provide a powerful tool to address this question. The possible involvement of alpha1S (Cav1.1), beta1 and gamma1 DHPR subunits was tested using dysgenic (alpha1S-null), beta1a and gamma1 knockout mice. The results show that the absence of alpha1S, beta1 or gamma1 DHPR subunits does not significantly affect the electrophysiological properties of T-type Ca2+ currents in skeletal muscle, suggesting that (1) native Cav3.2 is not regulated by beta1 or gamma1 DHPR subunits; (2) T-type and L-type currents have distinct and not interchangeable roles.

Transmitter modulation of spike‐evoked calcium transients in arousal related neurons: muscarinic inhibition of SNX‐482‐sensitive calcium influx

Nitric oxide synthase (NOS)-containing cholinergic neurons in the laterodorsal tegmentum (LDT) influence behavioral and motivational states through their projections to the thalamus, ventral tegmental area and a brainstem 'rapid eye movement (REM)-induction' site. Action potential-evoked intracellular calcium transients dampen excitability and stimulate NO production in these neurons. In this study, we investigated the action of several arousal-related neurotransmitters and the role of specific calcium channels in these LDT Ca(2+)-transients by simultaneous whole-cell recording and calcium imaging in mouse (P14-P30) brain slices. Carbachol, noradrenaline and adenosine inhibited spike-evoked Ca(2+)-transients, while histamine, t-ACPD, a metabotropic glutamate receptor agonist, and orexin-A did not. Carbachol inhibition was blocked by atropine, was insensitive to blockade of G-protein-coupled inward rectifier (GIRK) channels and was not inhibited by nifedipine, omega-conotoxin GVIA or omega-agatoxin IVA, which block L-, N- and P/Q-type calcium channels, respectively. In contrast, SNX-482 (100 nm), a selective antagonist of R-type calcium channels containing the alpha1E (Cav2.3) subunit, attenuated carbachol inhibition of the somatic spike-evoked calcium transient. To our knowledge, this is the first demonstration of muscarinic inhibition of native SNX-482-sensitive R-channels. Our findings indicate that muscarinic modulation of these channels plays an important role in the feedback control of cholinergic LDT neurons and that inhibition of spike-evoked Ca(2+)-transients is a common action of neurotransmitters that also activate GIRK channels in these neurons. Because spike-evoked calcium influx dampens excitability, our findings suggest that these 'inhibitory' transmitters could boost firing rate and enhance responsiveness to excitatory inputs during states of high firing, such as waking and REM sleep.

Voltage-Dependent Calcium Channels in Young and Old Human Red Cells

Presence of subtypes of voltage-dependent Ca channels was investigated in young and old human red cells, employing immunological and flux-kinetics methods. Western blots showed specific reaction toward polyclonal rabbit antibodies raised against a highly conserved residue of alpha1 subunit of high-voltage activated Ca channels (pan alpha1) and against conserved residues of alpha1C and alpha1E subunits. No specific reaction was detected with antibodies against conserved residues of alpha1A, alpha1B, or alpha1D subunits. Only a single band (approx 260 kDa) was revealed on anti-pan alpha1 or anti-alpha1E blots, whereas two bands (200 and 230 kDa) were detected by anti-alpha1C exposure. Blots from old cells always showed diminished band intensity. Channel activity was assessed by studying the effect of voltage-dependent Ca channels blockers under conditions likely to alter the red cell membrane potential, through incubation in media of different composition. In a 150 mM NaCl + 5 mM KCl medium, blockers of L-, R-, and Q-type caused a 15-50% reduction of 45Ca influx into cells, which had the Ca pump inactivated by either exhaustive adenosine triphosphate depletion or presence of vanadate plus substrates. Additionally, some P/Qand N-type blockers also reduced Ca influx to various extents (25-60%). Old cells were generally insensitive to L-type but not to non-L-type blockers. Raising external K to about 70-80 mM reduced by 50-100% inhibition by L-type blockers. Incubation in a gluconate medium containing 150 mM Na+5 mM K practically abolished the action of L-type blockers, but only slightly reducing that by non-L-type. The results clearly demonstrate presence of L- and R-type Ca channels, apparently occurring in different functional states in young and old cells. Other non-L-type channels were also demonstrated only by pharmacological means. A possible physiological role for these channels is discussed.

Mapping Sites of Potential Proximity between the Dihydropyridine Receptor and RyR1 in Muscle Using a Cyan Fluorescent Protein-Yellow Fluorescent Protein Tandem as a Fluorescence Resonance Energy Transfer Probe

Excitation-contraction coupling in skeletal muscle involves conformational coupling between the dihydropyridine receptor (DHPR) and the type 1 ryanodine receptor (RyR1) at junctions between the plasma membrane and sarcoplasmic reticulum. In an attempt to find which regions of these proteins are in close proximity to one another, we have constructed a tandem of cyan and yellow fluorescent proteins (CFP and YFP, respectively) linked by a 23-residue spacer, and measured the fluorescence resonance energy transfer (FRET) of the tandem either in free solution or after attachment to sites of the alpha1S and beta1a subunits of the DHPR. For all of the sites examined, attachment of the CFP-YFP tandem did not impair function of the DHPR as a Ca2+ channel or voltage sensor for excitation-contraction coupling. The free tandem displayed a 27.5% FRET efficiency, which decreased significantly after attachment to the DHPR subunits. At several sites examined for both alpha1S (N-terminal, proximal II-III loop of a two fragment construct) and beta1a (C-terminal), the FRET efficiency was similar after expression in either dysgenic (alpha1S-null) or dyspedic (RyR1-null) myotubes. However, compared with dysgenic myotubes, the FRET efficiency in dyspedic myotubes increased from 9.9 to 16.7% for CFP-YFP attached to the N-terminal of beta1a, and from 9.5 to 16.8% for CFP-YFP at the C-terminal of alpha1S. Thus, the tandem reporter suggests that the C terminus of alpha1S and the N terminus of beta1a may be in close proximity to the ryanodine receptor.

Regulation of muscle Cav1.1 channels by long-term depolarization involves proteolysis of the alpha1s subunit.

The effects of long-term depolarization on frog skeletal muscle Cav1.1 channels were assessed. Voltage-clamp and Western-blot experiments revealed that long-term depolarization brings about a drastic reduction in the amplitude of currents flowing through Cav1.1 channels and in the levels of the alpha1s subunit, the main subunit of muscle L-type channels. The decline of both phenomena was prevented by the action of the protease inhibitors E64 (50 microM) and leupeptin (50 microM). In contrast, long-term depolarization had no effect on beta1, the auxiliary subunit of alpha1s. The levels of mRNAs coding the alpha1s and the beta1 subunits were measured by RNase protection assays. Neither the content of the alpha1s nor the beta1 subunit mRNAs were affected by long-term depolarization, indicating that the synthesis of Cav1.1 channels remained unaffected. Taken together, our experiments suggest that the reduction in the amplitude of membrane currents and in the alpha1s subunit levels is caused by increased degradation of this subunit by a Ca2+-dependent protease.

Functional equivalence of dihydropyridine receptor a1S and b1a subunits in triggering excitation-contraction coupling in skeletal muscle

Molecular understanding of the mechanism of excitation-contraction (EC) coupling in skeletal muscle has been made possible by cultured myotube models lacking specific dihydropyridine receptor (DHPR) subunits and ryanodine receptor type 1 (RyR1) isoforms. Transient expression of missing cDNAs in mutant myotubes leads to a rapid recovery, within days, of various Ca2+ current and EC coupling phenotypes. These myotube models have thus permitted structure-function analysis of EC coupling domains present in the DHPR controlling the opening of RyR1. The purpose of this brief review is to highlight advances made by this laboratory towards understanding the contribution of domains present in alpha1S and beta1a subunits of the skeletal DHPR to EC coupling signaling. Our main contention is that domains of the alpha1S II-III loop are necessary but not sufficient to recapitulate skeletal-type EC coupling. Rather, the structural unit that controls the EC coupling signal appears to be the alpha1S/beta1a pair.

Developmental induction of DHPR alpha 1s and RYR1 gene expression does not require neural or mechanical signals.

This study compares dihydropyridine receptor (DHPR) and ryanodine receptor (RyR1) gene expression in the diaphragm and hindlimb skeletal muscles of neonatal mice, and examines the contribution of neural and mechanical signals to their developmental induction in vivo. DHPR alpha 1s subunit and RyR1 protein are expressed concurrently, while their respective mRNAs are induced sequentially, with DHPR mRNA ahead of RyR1 mRNA. Both DHPR and RyR1 are more abundant in the diaphragm at birth, and become more abundant in the hindlimb at maturity. These patterns are consistent across different muscles and species. A critical period for DHPR alpha 1s and RyR1 gene expression in the hindlimb occurs between days 5 and 19 postnatal. Their mRNA expression during this period is unchanged by denervation or tenotomy, but DHPR protein decreases after tenotomy. These results demonstrate that both transcriptional and post-transcriptional mechanisms contribute to the muscle-specific and coordinated assembly of the functional DHPR-RyR1 complex, and that the developmental induction of DHPR and RyR1 gene transcription does not require neural or mechanical signals.

Alternative Splicing of the β4Subunit Has α1Subunit Subtype-Specific Effects on Ca2+Channel Gating

Ca2+ channel beta subunits are important molecular determinants of the kinetics and voltage dependence of Ca2+ channel gating. Through direct interactions with channel-forming alpha1 subunits, beta subunits enhance expression levels, accelerate activation, and have variable effects on inactivation. Four distinct beta subunit genes each encode five homologous sequence domains (D1-5), three of which (D1, D3, and D5) undergo alternative splicing. We have isolated from human spinal cord a novel alternatively spliced beta4 subunit containing a short form of domain D1 (beta4a) that is highly homologous to N termini of Xenopus and rat beta3 subunits. The purpose of this study was to compare the gating properties of various alpha1 subunit complexes containing beta4a with those of complexes containing a beta4 subunit with a longer form of domain D1, beta4b. Expression in Xenopus oocytes revealed that, relative to alpha1A and alpha1B complexes containing beta4b, the voltage dependence of activation and inactivation of complexes containing beta4a were shifted to more depolarized potentials. Moreover, alpha1A and alpha1B complexes containing beta4a inactivated at a faster rate. Interestingly, beta4 subunit alternative splicing did not influence the gating properties of alpha1C and alpha1E subunits. Experiments with beta4 deletion mutants revealed that both the N and C termini of the beta4 subunit play critical roles in setting voltage-dependent gating parameters and that their effects are alpha1 subunit specific. Our data are best explained by a model in which distinct modes of activation and inactivation result from beta-subunit splice variant-specific interactions with an alpha1 subunit gating structure.