Elsevier

Neuroscience Research

Volume 45, Issue 1, January 2003, Pages 129-137
Neuroscience Research

Human skeletal muscle calcium channel α1S is expressed in the basal ganglia: distinctive expression pattern among L-type Ca2+ channels

https://doi.org/10.1016/S0168-0102(02)00204-3Get rights and content

Abstract

Voltage-gated calcium channels (VGCCs) are essential molecules for neuronal function. VGCCs consist of five subunits, α1, α2, β, γ, and δ. Among the ten subtypes of the α1 subunit (α1A-I and S), expression of α1S was previously believed to be restricted to the skeletal muscle. We report here, however, that α1S is also expressed in human and rat central nervous system. First, we performed PCR screening for VGCC α1 subunits in human nervous system using degenerate primers, and identified α1S as well as all the eight α1 subunits with previously described expression. Intriguingly, α1S was selectively localized to the basal ganglia, particularly the caudate nucleus. In situ hybridization showed that α1S was expressed in medium-sized caudate neurons. Quantitative analysis using real time RT-PCR revealed a distinct pattern of α1S expression among L-type calcium channels. Furthermore, RT-PCR using laser-mediated manipulation of single cells suggested that human α1S was coexpressed with ryanodine receptors (RYRs) in GABAergic neurons. Our results suggest the potential relevance of α1S to dopaminergic signal transduction and calcium-induced calcium release in caudate neurons.

Introduction

Voltage-gated calcium channels (VGCCs) are essential molecules for excitatory cells, especially for neurons that mediate various important cellular functions. These channels initiate muscle contraction, modulate action potential waveforms, trigger release of neurotransmitters from nerve terminals or hormones from endocrine cells (Perney et al., 1986, Fossier et al., 1999), determine firing patterns and input–output characteristics of various neuronal cells (Llinas, 1988), regulate gene expression and cell cycle (Ramsdell, 1991, Bading et al., 1993, Hardingham et al., 1997), and mediate cell death (Lobner and Lipton, 1993, Porter et al., 1997). VGCCs are clinically important as well, and have been implicated in the pathogenesis of a number of diseases. For example, mutations of VGCCs cause hereditary neurological diseases such as hypokalemic periodic paralysis (Ptacek et al., 1994) and malignant hyperthermia (Monnier et al., 1997), and VGCC antibodies cause Lambert–Eaton myasthenic syndrome (Leys et al., 1989). VGCCs are also the targets for several therapeutic pharmaceuticals, including drugs for hypertension, arrhythmia, angina pectoris, and migraine (Abernethy and Schwartz, 1999).

VGCCs consist of five subunits, α1, α2, β, γ, and δ. Among the subunits, α1 is essential, as it can form a functional Ca2+ channel by itself, and it contains domains of the channel pore and the voltage sensor (Hofmann et al., 1994, Walker and De Waard, 1998). The structure of the α1 subunit consists of four homologous repeats, each with six putative transmembrane segments. The segments consist of highly conserved sequences among all the subtypes, whereas the cytoplasmic loop and the carboxyl terminal region are strikingly different among α1 subtypes.

To date, ten distinct genes encoding VGCC α1 subunit subtypes (α1A-I and S) have been identified. Nervous system expression has been reported for all subtypes except α1S (Snutch et al., 1990). VGCCs are classified, electrophysiologically and pharmacologically, into L-type (α1C, D, F, and S), P/Q-type (α1A), N-type (α1B), R-type (α1E), and T-type (α1G, H, and I). Among L-type calcium channels immunoprecipitated from rat cerebral cortex and the hippocampus, approximately 75% are α1C, and 20% are α1D (Hell et al., 1993). Based on channel currents, the remaining 5% of L-type channels are suspected to contain α1S and/or α1F, although skeletal muscle type α1S, and retinal type α1F, have not been identified in Northern blots from these brain regions (Ellis et al., 1988, Fisher et al., 1997, Strom et al., 1998). In this study, we screened human brain cDNA and found that α1S was also expressed in the central nervous system. We investigated the expression pattern of α1S in comparison with those of other L-type Ca2+ channels, α1C and α1D.

Section snippets

PCR screening

Total RNA from normal human whole brain was extracted and reverse transcribed, and nested PCR was performed using the degenerate primers as described previously (Jeong et al., 2000). We developed the degenerate primers using the consensus sequence of α1 subunit subtypes. The primer sets for α1A-F and S were Ca5, Ca3A, and Ca3B designated as set 1, and for α1G-I, Ca2F, Ca2FC, and Ca2R as set 2. The first PCR conditions were, 95 °C for 2 min, followed by 30 cycles consisting of 95 °C for 15 s, 56 °C

PCR screening and cloning using degenerate primers for VGCCs from human brain cDNA

In order to screen human whole brain cDNA by PCR, we designed two sets of degenerate primers, set 1 for α1A-F and α1S, and set 2 for α1G-I, so as to identify each subtype by its size and restriction enzyme patterns as described in the experimental methods. All eight of the α1 subunits with previously described expression (α1A-E and α1G-I) were obtained by this method. Retina was not included in the human cDNA, thus α1F was not obtained. Unexpectedly, we identified α1S, which was known as

Discussion

Our study revealed that human and rat α1S was also expressed in the nervous system, particularly in the basal ganglia. Three isoforms of α1S, a full-length isoform and two truncated isoforms, have been reported in the skeletal muscle. One truncated form, which was designated as a newborn form, has a deletion of 2047 bp from nucleotide 1431–3478 (Malouf et al., 1992), and contains the sequences for our RT-PCR primers and Northern blotting probes. The expression level of the newborn isoform in

Acknowledgements

This work was supported by grants-in-aid from SORST, Japan Science and Technology and grants-in-aid from Japan Ministry of Health, Labor, and Welfare. The authors thank R.P. Ruberu for technical advice.

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