We have cloned and expressed a human ␣ 1I subunit that encodes a subtype of T-type calcium channels. The predicted protein is 95% homologous to its rat counterpart but has a distinct COOH-terminal region. Its mRNA is detected almost exclusively in the human brain, as well as in adrenal and thyroid glands. Calcium currents generated by the functional expression of human ␣ 1I and ␣ 1G subunits in HEK-293 cells were compared. The ␣ 1I current activated and inactivated ϳ10 mV more positively. Activation and inactivation kinetics were up to six times slower, while deactivation kinetics was faster and showed little voltage dependence. A slower recovery from inactivation, a lower sensitivity to Ni 2؉ ions (IC 50 ϳ180 M), and a larger channel conductance (ϳ11 picosiemens) were the other discriminative features of the ␣ 1I current. These data demonstrate that the ␣ 1I subunit encodes T-type Ca 2؉ channels functionally distinct from those generated by the human ␣ 1G or ␣ 1H subunits and point out that human and rat ␣ 1I subunits have species-specific properties not only in their primary sequence, but also in their expression profile and electrophysiological behavior.Voltage-dependent calcium channels control the rapid entry of Ca 2ϩ ions into a wide variety of cell types and are therefore involved in both electrical and cellular signaling. Electrophysiological studies have identified two major Ca 2ϩ channel types as high voltage-activated and low voltage-activated channels (1, 2) with this latter class being also identified as T-type Ca 2ϩ channels (3). T-type Ca 2ϩ channels were originally defined by their activation at low membrane potential, their fast time course, and their small single channel conductance (4, 5). These channels have been identified on a large variety of neurons, and it has become obvious that significant functional diversity exists in the gating behavior of T-type channels, particularly in inactivation kinetics, voltage dependence of steady-state inactivation, and pharmacology (6). The recent identification of several novel genes encoding a subset of homologous Ca 2ϩ channel ␣ 1 subunits, e.g. the ␣ 1G subunit (7-9), the ␣ 1H subunit (10,11), and the rat ␣ 1I subunit (12), has revealed that diversity of T-type voltage-dependent calcium channels is primarily related to the expression of distinct ␣ 1 subunits. Indeed, the expression of the various ␣ 1G and ␣ 1H subunits (7-12) produces Ca 2ϩ currents with the typical signature of T-type channels but with specific features, such as the block by Ni 2ϩ , which discriminates between ␣ 1G and ␣ 1H currents (13). By contrast, the biophysical properties of T-type channels generated by the ␣ 1I subunit markedly differ from those made of ␣ 1G and ␣ 1H subunits (12,14,15), and it was postulated that the ␣ 1I subunit is responsible for native "slow" T-type currents observed in rat thalamic neurons (16). To date the ␣ 1I subunit has only been cloned from rat (12) and whether the ␣ 1I subunit encodes an atypical T-type Ca 2ϩ channel certainly needs further investig...

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 ␣ 1S and ␤ 1a 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 Ca 2؉ 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 ␣ 1S (N-terminal, proximal II-III loop of a two fragment construct) and ␤ 1a (C-terminal), the FRET efficiency was similar after expression in either dysgenic (␣ 1S -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 ␤ 1a , and from 9.5 to 16.8% for CFP-YFP at the C-terminal of ␣ 1S . Thus, the tandem reporter suggests that the C terminus of ␣ 1S and the N terminus of ␤ 1a may be in close proximity to the ryanodine receptor.

T-type Ca(2+) channels have been suggested to play a role in cardiac automaticity, cell growth, and cardiovascular remodeling. Although three genes encoding for a T-type Ca(2+) channel have been identified, the nature of the isoform(s) supporting the cardiac T-type Ca(2+) current (I(Ca,T)) has not yet been determined. We describe the postnatal evolution of I(Ca,T) density in freshly dissociated rat atrial and ventricular myocytes and its functional properties at peak current density in young atrial myocytes. I(Ca,T) displays a classical low activation threshold, rapid inactivation kinetics, negative steady-state inactivation, slow deactivation, and the presence of a window current. Interestingly, I(Ca,T) is poorly sensitive to Ni(2+) and insensitive to R-type current toxin SNX-482. RT-PCR experiments and comparison of functional properties with recombinant Ca(2+) channel subtypes suggest that neonatal I(Ca,T) is related to the alpha(1G)-subunit. Atrial natriuretic factor (ANF) secretion was measured using peptide radioimmunoassays in atrial tissue. Pharmacological dissection of ANF secretion indicates an important contribution of I(Ca,T) to Ca(2+) signaling during the neonatal period.

Ca2+ channel antagonists of the dihydropyridine, benzothiazepine, and phenylalkylamine classes have selective effects on L-type versus T-type Ca2+ channels. In contrast, mibefradil was reported to be more selective for T-type channels. We used the whole-cell patch-clamp technique to investigate the effects of mibefradil on T-type and L-type Ca2+ currents (I(CaT) and I(CaL)) recorded at physiologic extracellular Ca2+ in different cardiac cell types. At a stimulation rate of 0.1 Hz, mibefradil blocked I(CaT) evoked from negative holding potentials (HPs) (-100 mV to -80 mV) with an IC50 of 0.1 microM in rat atrial cells. This concentration had no effect on I(CaL) in rat ventricular cells (IC50: approximately3 microM). However, block of I(CaL) was enhanced when the HP was depolarized to -50 mV (IC50: approximately 0.1 microM). Besides a resting block, mibefradil displayed voltage- and use-dependent effects on both I(CaT) and I(CaL). In addition, inhibition was enhanced by increasing the duration of the step-depolarizations. Similar effects were observed in human atrial and rabbit sinoatrial cells. In conclusion, mibefradil combines the voltage- and use-dependent effects of dihydropyridines and benzothiazepines on I(CaL). Inhibition of I(CaL), which has probably been underestimated before, may contribute to most of the cardiovascular effects of mibefradil.