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 Monoamine Neurons of the Rat Brain Preferentially Express a Splice Variant of α1B Subunit of the N-Type Calcium Channel

The N-type voltage-dependent calcium channels play a significant role in neurotransmitter release. The alpha1B subunit of the N-type calcium channel functions as the primary subunit that forms the pore and contains the structural motifs that mediate the pharmacological and gating properties of the channel. We report on an isoform of the alpha1B subunit that is preferentially expressed by the monoaminergic neurons of the rat brain. This isoform contains a 21-amino acid cassette in the synprint site present in the cytoplasmic loop between domains IIS6 and IIIS1. RT-PCR of micropunched tissue was used to show preferential expression of this isoform in regions of the brain containing monoaminergic neurons and to a lesser extent in the cerebellum. Double-label in situ hybridization was used to show expression of this isoform mRNA in dopaminergic neurons of the ventral mesencephalon. The expression of two distinct N-type calcium channels containing these alpha1B subunit isoforms by the monoaminergic neurons may provide for synapse-specific regulation of neurotransmitter release.     

The fastest contracting muscles of nonmammalian vertebrates express only one isoform of the ryanodine receptor.

The skeletal muscles of chickens, frogs, and fish have been reported to express two isoforms (alpha and beta) of the sarcoplasmic reticulum calcium release channel (ryanodine receptor or RYR), while mammals express only one. We have studied patterns of RYR isoform expression in skeletal muscles from a variety of fish, reptiles, and birds with immunological techniques. Immunoblot analysis with a monoclonal antibody that recognizes both nonmammalian RYR isoforms and a polyclonal antibody specific to the alpha isoform show two key results: (a) two reptilian orders share with mammals the pattern of expressing only the alpha (skeletal) RYR isoform in skeletal muscle; and (b) certain functionally specialized muscles of fish and birds express only the alpha RYR isoforms. While both isoforms are expressed in the body musculature of fish and birds, the alpha isoform is expressed alone in extraocular muscles and swimbladder muscles. The appearance of the alpha RYR isoform alone in the extraocular muscles and a fast-contracting sonic muscle in fish (toadfish swimbladder muscle) provides evidence that this isoform is selectively expressed when rapid contraction is required. The functional and phylogenetic implications of expression of the alpha isoform alone are discussed in the context of the mechanism and evolution of excitation-contraction coupling.     

Mice with cardiac-specific sequestration of the beta-subunit of the L-type calcium channel

The beta subunit of the L-type voltage-dependent calcium channel modifies the properties of the channel complex by both allosteric modulation of the alpha1 subunit function and by chaperoning the translocation of the alpha1 subunit to the plasma membrane. The goal of this study was to investigate the functional effect of changing the in vivo stoichiometry between the alpha1 and beta subunits by creating a dominant negative expression system in a transgenic mouse model. The high affinity beta subunit-binding domain of the alpha1 subunit was overexpressed in a cardiac-specific manner to act as a beta subunit trap. We found that the predominant beta isoform was located primarily in the membrane bound fraction of heart protein, whereas the beta1 and beta3 were mostly cytosolic. There was a significant diminution of the amount of beta2 in the membrane fraction of the transgenic animals, resulting in a decrease in contractility of the heart and a decrease in L-type calcium current density in the myocyte. However, there were no distinguishable differences in beta1 and beta3 protein expression levels in the membrane bound fraction between transgenic and non-transgenic animals. Since the beta1 and beta3 isoforms only make up a small portion of the total beta subunit in the heart, slight changes in this fraction are not detectable using Western analysis. In contrast, beta1 and beta3 in skeletal muscle and brain, the predominant isoforms in these tissues, respectively, are membrane bound.     

Evidence for the existence of a cardiac specific isoform of the alpha 1 subunit of the voltage dependent calcium channel

Biochemical, pharmacological and electrophysiological evidence implies the existence of tissue specific isoforms of the L-type VDCC. The alpha 1 and alpha 2 subunits of the skeletal muscle calcium channel have been previously cloned and their amino acid sequence deduced. Here we report the isolation and sequencing of a partial cDNA that encodes a heart specific isoform of the alpha 1 subunit. The amino acid sequence deduced from this part cDNA clone shows 64.7% similarity with the skeletal muscle alpha 1 subunit. Northern analysis reveals 2 hybridizing bands, 8.5 and 13 kb, in contrast to one 6.5 kb band in the skeletal muscle. Selective inhibition of mRNA expression in Xenopus oocytes by complementary oligodeoxy-nucleotides derived from the heart clone provides further evidence that the cDNA corresponds to an essential component of the VDCC. These data further support the existence of tissue-specific isoforms of the L-type VDCC.     

Stable and functional expression of the calcium channel alpha 1 subunit from smooth muscle in somatic cell lines.

Voltage-activated calcium channels are membrane spanning proteins that allow the controlled entry of Ca2+ into the cytoplasm of cells. The principal channel forming subunit of an L-type calcium channel is the alpha 1 subunit. Transfection of Chinese hamster ovary (CHO) cells with complementary DNA encoding the calcium channel alpha 1 subunit from smooth muscle led to the expression of functional calcium channels which bind calcium channel blockers and show the voltage-dependent activation and slow inactivation and unitary current conductance characteristic of calcium channels in smooth muscle. The currents mediated by these channels are sensitive towards dihydropyridine-type blockers and agonists indicating that the calcium channel blocker receptor sites were present in functional form. The smooth muscle alpha 1 subunit cDNA alone is sufficient for stable expression of functional calcium channels with the expected kinetic and pharmacological properties in mammalian somatic cells.     

Splice-variant changes of the CaV3.2 T-type calcium channel mediate voltage-dependent facilitation and associate with cardiac hypertrophy and development

Low voltage-activated T-type calcium (Ca) channels contribute to the normal development of the heart and are also implicated in pathophysiological states such as cardiac hypertrophy. Functionally distinct T-type Ca channel isoforms can be generated by alternative splicing from each of three different T-type genes (Ca_V3.1, Ca_V3.2, Ca_V3.3), although it remains to be described whether specific splice variants are associated with developmental states and pathological conditions. We aimed to identify and functionally characterize Ca_V3.2 T-type Ca channel alternatively spliced variants from newborn animals and to compare with adult normotensive and spontaneously hypertensive rats (SHR). DNA sequence analysis of full-length Ca_V3.2 cDNA generated from newborn heart tissue identified ten major regions of alternative splicing, the more common variants of which were analyzed by quantitative real-time PCR (qRT-PCR) and also subject to functional examination by whole-cell patch clamp. The main findings are that: (1) cardiac Ca_V3.2 T-type Ca channels are subject to considerable alternative splicing, (2) there is preferential expression of Ca_V3.2(−25) splice variant channels in newborn rat heart with a developmental shift in adult heart that results in approximately equal levels of expression of both (+25) and (−25) exon variants, (3) in the adult stage of hypertensive rats there is both an increase in overall Ca_V3.2 expression and a shift towards expression of Ca_V3.2(+25) containing channels as the predominant form and (4) alternative splicing confers a variant-specific voltage-dependent facilitation of Ca_V3.2 channels. We conclude that Ca_V3.2 alternative splicing generates significant T-type Ca channel structural and functional diversity with potential implications relevant to cardiac developmental and pathophysiological states.Key words: voltage-dependent facilitation, alternative splicing, T-type calcium channel, hypertension, cardiac hypertrophy     

Three differentially expressed Na,K-ATPase alpha subunit isoforms: structural and functional implications

We have characterized cDNAs coding for three Na,K-ATPase alpha subunit isoforms from the rat, a species resistant to ouabain. Northern blot and S1-nuclease mapping analyses revealed that these alpha subunit mRNAs are expressed in a tissue-specific and developmentally regulated fashion. The mRNA for the alpha 1 isoform, approximately equal to 4.5 kb long, is expressed in all fetal and adult rat tissues examined. The alpha 2 mRNA, also approximately equal to 4.5 kb long, is expressed predominantly in brain and fetal heart. The alpha 3 cDNA detected two mRNA species: a approximately equal to 4.5 kb mRNA present in most tissues and a approximately equal to 6 kb mRNA, found only in fetal brain, adult brain, heart, and skeletal muscle. The deduced amino acid sequences of these isoforms are highly conserved. However, significant differences in codon usage and patterns of genomic DNA hybridization indicate that the alpha subunits are encoded by a multigene family. Structural analysis of the alpha subunits from rat and other species predicts a polytopic protein with seven membrane-spanning regions. Isoform diversity of the alpha subunit may provide a biochemical basis for Na,K-ATPase functional diversity.     

Expression profiling of the gamma-subunit isoforms of AMP-activated protein kinase suggests a major role for gamma3 in white skeletal muscle

Expression patterns of the three isoforms of the regulatory gamma-subunit of AMP-activated protein kinase (AMPK) were determined in various tissues from adult humans, mice, and rats, as well as in human primary muscle cells. Real-time PCR-based quantification of mRNA showed similar expression patterns in the three species and a good correlation with protein expression in mice and rats. The gamma3-isoform appeared highly specific to skeletal muscle, whereas gamma1 and gamma2 showed broad tissue distributions. Moreover, the proportion of white, type IIb fibers in the mouse and rat muscle samples, as indicated by real-time PCR quantification of Atp1b2 mRNA, showed a strong positive correlation with the expression of gamma3. In samples of white skeletal muscle, gamma3 clearly appeared to be the most abundant gamma-isoform. Differentiation of human primary muscle cells from myoblasts into multinucleated myotubes was accompanied by upregulation of gamma3 mRNA expression, whereas levels of gamma1 and gamma2 remained largely unchanged. However, even in these cultured myotubes, gamma2 was the most highly expressed isoform, indicating a considerable difference compared with adult skeletal muscle. Immunoblot analysis of mouse gastrocnemius and quadriceps muscle extracts precipitated with a gamma3-specific antibody showed that gamma3 was exclusively associated with the alpha2- and beta2-subunit isoforms. The observation that the AMPKgamma3 isoform is expressed primarily in white skeletal muscle, in which it is the predominant gamma-isoform, strongly suggests that gamma3 has a key role in this tissue.     

Foot protein isoforms are expressed at different times during embryonic chick skeletal muscle development

We have investigated the time course of expression of the alpha and beta triad junctional foot proteins in embryonic chick pectoral muscle. The level of [3H]ryanodine binding in muscle homogenates is low until day E20 of embryonic development, then increases dramatically at the time of hatching reaching adult levels by day N7 posthatch. The alpha and beta foot protein isoforms increase in abundance concomitantly with [3H]ryanodine binding. Using foot protein isoform-specific antibodies, the alpha foot protein is detected in a majority of fibers in day E10 muscle, while the beta isoform is first observed at low levels in a few fibers in day E15 muscle. A high molecular weight polypeptide, distinct from the alpha and beta proteins, is recognized by antifoot protein antibodies. This polypeptide is observed in day E8 muscle and declines in abundance with continued development. It appears to exist as a monomer and does not bind [3H]ryanodine. In contrast, the alpha isoform present in day E10 muscle and the beta isoform in day E20 muscle are oligomeric and bind [3H]ryanodine suggesting that they may exist as functional calcium channels in differentiating muscle. Comparison of the intracellular distributions of the alpha foot protein, f-actin, the heavy chain of myosin and titin in day E10 muscle indicates that the alpha foot protein is expressed during myofibril assembly and Z line formation. The differential expression of the foot protein isoforms in developing muscle, and their continued expression in mature muscle, is consistent with these proteins making different functional contributions. In addition, the expression of the alpha isoform during the time of organization of a differentiated muscle morphology suggests that foot proteins may participate in events involved in muscle differentiation.     

Physiological differences between the alpha and beta ryanodine receptors of fish skeletal muscle.

Two isoforms of the sarcoplasmic reticulum Ca2+ release channel (ryanodine receptor or RYR) are expressed together in the skeletal muscles of most vertebrates. We have studied physiological properties of the two isoforms (alpha and beta) by comparing SR preparations from specialized fish muscles that express the alpha isoform alone to preparations from muscles containing both alpha and beta. Regulation of channel activity was assessed through [3H]ryanodine binding and reconstitution into planar lipid bilayers. Distinct differences were observed in the calcium-activation and -inactivation properties of the two isoforms. The fish alpha isoform, expressed alone in extraocular muscles, closely resembled the rabbit skeletal muscle RYR. Maximum [3H]ryanodine binding and maximum open probability (Po) of the alpha RYR were achieved from 1 to 10 microM free Ca2+. Millimolar Ca2+ reduced [3H]ryanodine binding and Po close to zero. The beta isoform more closely resembled the fish cardiac RYR in Ca2+ activation of [3H]ryanodine binding. The most prominent difference of the beta and cardiac isoforms from the alpha isoform was the lack of inactivation of [3H]ryanodine binding and Po by millimolar free Ca2+. Differences in activation of [3H]ryanodine binding by adenine nucleotides and inhibition by Mg2+ suggest that the beta and cardiac RYRs are not identical, however. [3H]ryanodine binding by the alpha RYR was selectively inhibited by 100 microM tetracaine, whereas cardiac and beta RYRs were much less affected. Tetracaine can thus be used to separate the properties of the alpha and beta RYRs in preparations in which both are present. The distinct physiological properties of the alpha and beta RYRs that are present together in most vertebrate muscles support models of EC coupling incorporating both directly coupled and Ca(2+)-coupled channels within a single triad junction.