Determinants of voltage-dependent inactivation affect Mibefradil block of calcium channels
Introduction
Voltage-gated calcium channels (VGCCs), are transmembrane proteins involved in the regulation of cellular excitability and Ca2+ homeostasis. VGCCs, classified as L-, N-, P-, Q-, R- and T-type based on their functional properties, are critical for many cellular functions including muscular contraction, neurotransmitter release and excitability. To date, ten Ca2+ channels genes have been reported and termed α1S for the first one identified in skeletal muscle and α1A through to α1I for the nine others isolated subsequently (Perez-Reyes et al., 1998). When transiently expressed from their cDNA in a host system together with their ancillary β and α2δ subunits, α1A exhibits the characteristics of P- and Q-type channels, α1B encodes an N-type channel, α1C α1D and α1S constitute three different types of L-type channels, and α1E seems to correlate to resistant R-type channels defined as VGCCs insensitive to all known Ca channel antagonists and toxins. The recently described α1F gene is highly homologous to α1D and probably corresponds to an L-type channel (Fisher et al., 1997). Finally, α1G α1H and α1I the latest genes identified, have been unambiguously characterized as members of the T-type Ca channel family (Perez-Reyes et al., 1998, Cribbs et al., 1998, Lee et al., 1999).
VGCCs are major targets for a variety of therapeutic agents such as dihydropyridines (DHPs), benzothiazepines (BTZs), and phenylalkylamines (PPAs) that are used in the treatment of hypertension, stroke, acute ischemia, migraine and epilepsy. These drugs are considered as L-type Ca2+ channel antagonists. In addition to the DHPs, BTZs and PAAs, a new molecule (Mibefradil, a tetralole derivative) has been described with a potent action on low-voltage activated T-type VGCCs (Mishra and Hermsmeyer, 1994). Mibefradil has proven to be effective in the treatment of cardiovascular diseases but the relationship between the clinical effect (hypotension with no increase in heart rate) and the inhibition of T-type channels has not clearly been established. In addition to the block of T-type Ca2+ channels, Mibefradil was also reported to block channels induced by α1A, α1B, α1C and α1E expression in Xenopus oocytes (Bezprozvanny and Tsien, 1995). To further compare Mibefradil blockade of different calcium channel subtypes, we studied its action on three major classes of cloned Ca2+ channels. Our results provide the first comparison of the effect of Mibefradil on recombinant α1A, α1C, α1E currents in nearly physiological conditions (transiently expressed in mammalian cells and currents recorded in physiological levels of external calcium). In addition, our results show that the voltage-dependent kinetics of inactivation of Ca2+ channels, determined by the nature of the associated β subunit isoform and/or alternative splicing of the α1 gene, is critical for the action of Mibefradil.
Section snippets
Transient expression of recombinant calcium channels
cDNAs encoding VGCCs α1, α2 and β subunits and reporter genes (CD8 or GFP) were inserted in vertebrate expression vectors. Rat brain α1A isoforms, α1E, β2a, and α2δ were cloned in pMT2 (Stea et al., 1994, Soong et al., 1993, Bourinet et al., 1999); rat brain α1C and β1b, in pCEP4 (Tomlinson et al., 1993); and CD8 (generously provided by Dr Brian Seed, Massachusetts General Hospital) and GFP (a kind gift from Dr Kye Chesnut, University of Florida) in CMV promoter driven vectors. Human embryonic
Recombinant Ca2+ currents in mammalian cells
Transfection of rat α1A, α1C, or α1E with α2-δ and β1b subunits produced a robust expression of Ca2+ channels as determined by the presence of a large Ca2+ current density. This allowed the comparison of these three major types of VGCCs with a physiological concentration of Ca2+ as the charge carrier. The α1A α1C and α1E channels were activated by depolarization greater than −40 to −30 mV, had a maximum amplitude around 0 mV, and reversed near +60 mV (Fig. 1). They all deactivated with
Discussion
The repertoire of therapeutic molecules acting on VGCCs (also called Ca2+ channel antagonists) is to date restricted to L type channels, and therefore preferentially targets cell types expressing these channels (mostly cardiovascular cells). In contrast a recently developed molecule, Mibefradil, has been reported to be more selective for T-type vs L-type Ca2+ channels in cardiovascular tissues (Mishra and Hermsmeyer, 1994, Benardeau and Ertel, 1998). Therefore T-type Ca2+ channel blockade
Concluding remarks
Altogether, our data are consistent with a voltage-dependent block which implies that Ca2+ channel diversity resulting from combinations of the different types of α1 subunits (and their respective variants) and the different β subunits may significantly affect pharmacological properties. Demonstration of a potent block by Mibefradil of various HVA calcium channels in the same range of concentration as that blocking T-type channels should contribute to our understanding of the drug effects, and
Acknowledgements
We are grateful to Ed Perez Reyes for providing the β2a cDNA and to Robert Koen for the Mibefradil. We thank Gerald Zamponi, Mike Seagar and Matteo Mangoni for careful reading of the manuscript. This work was supported by NATO grant CGR971546 (E.B.), by Ministerio de Educación y Cultura Español fellowship (C.J.), by AFM grant (J.N.), and by Produits Roche, Neuilly, France.
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