Abstract
We describe here several novel properties of the human alpha(1G) subunit that forms T-type calcium channels. The partial intron/exon structure of the corresponding gene CACNA1G was defined and several alpha(1G) isoforms were identified, especially two isoforms that exhibit a distinct III-IV loop: alpha(1G-a) and alpha(1G-b). Northern blot and dot blot analyses indicated that alpha(1G) mRNA is predominantly expressed in the brain, especially in thalamus, cerebellum, and substantia nigra. Additional experiments have also provided evidence that alpha(1G) mRNA is expressed at a higher level during fetal life in nonneuronal tissues (i.e. kidney, heart, and lung). Functional expression in HEK 293 cells of a full-length cDNA encoding the shortest alpha(1G) isoform identified to date, alpha(1G-b), resulted in transient, low threshold activated Ca(2+) currents with the expected permeability ratio (I(Sr) > I(Ca) >/= I(Ba)) and channel conductance ( approximately 7 pS). These properties, together with slowly deactivating tail currents, are typical of those of native T-type Ca(2+) channels. This alpha(1G)-related current was inhibited by mibefradil (IC(50) = 2 microM) and weakly blocked by Ni(2+) ions (IC(50) = 148 microM) and amiloride (IC(50) > 1 mM). We showed that steady state activation and inactivation properties of this current can generate a "window current" in the range of -65 to -55 mV. Using neuronal action potential waveforms, we show that alpha(1G) channels produce a massive and sustained Ca(2+) influx due to their slow deactivation properties. These latter properties would account for the specificity of Ca(2+) influx via T-type channels that occurs in the range of physiological resting membrane potentials, differing considerably from the behavior of other Ca(2+) channels.
Highlights
Transmembrane voltage-dependent calcium channels control Ca2ϩ ion entry from the extracellular space and thereby regulate various cellular processes such as muscle contraction, neuronal development and plasticity, secretion, and gene expression
Cloning of a Human ␣1G Subunit—Several cDNA clones covering from domain II to the C-terminal region of the human ␣1G isoform were isolated from a cerebellum cDNA library using a probe designed from the identified EST H06096 (Fig. 1A)
Our study provides the first evidence for alternatively spliced isoforms of the ␣1G subunit, revealing that the molecular diversity of T-type channels relies on the expression of three subunits encoded by distinct genes, ␣1G, ␣1H, and ␣1I [17]
Summary
Transmembrane voltage-dependent calcium channels control Ca2ϩ ion entry from the extracellular space and thereby regulate various cellular processes such as muscle contraction, neuronal development and plasticity, secretion, and gene expression. LVA calcium currents, mainly referred to as “T-type currents” due to their fast inactivation and small conductance, have been described in a wide variety of cell types [1]. Their physiological functions still remain obscure, they are thought to play a role in neuronal burst firing [2], pacemaker activity in heart [3, 4], aldosterone secretion [5], or fertilization [6]. The functional diversity of HVA calcium channels is primarily related to the existence of several ␣1 subunits (␣1A–F and ␣1S) encoded by distinct genes, many of which generate splice variants with specific properties, as described for the ␣1A isoforms that generate P/Q-type channels [14]. These recent data [15,16,17,18] provide clear evidence that
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