MULTIPLE CALCIUM-CHANNEL SUBTYPES IN ISOLATED RAT CHROMAFFIN CELLS

By using the whole-cell configuration of the patch-clamp technique we have investigated the pharmacological properties of Ca2+ channels in short-term cultured rat chromaffin cells. In cells held at a membrane potential of -80 mV, using 10 mM Ba2+ as the charge carrier, only high-voltage-activated (HVA) Ca2+ channels were found. Ba2+ currents (I-Ba) showed variable sensitivity to dihydropyridine (DHP) Ca2+ channel agonists and antagonists. Furnidipine, a novel DHP antagonist, reversibly blocked the current amplitude by 22% and 48%, at 1 mu M and 10 mu M respectively, during short (15-50 ms) depolarizing pulses to 0 mV. The L-type Ca2+ channel agonist Bay K 8644 (1 mu M) caused a variable potentiation of HVA currents that could be better appreciated at low rather than at high depolarizing steps. Increase of I-Ba was accompanied by a 20-mV shift in the activation curves for Ca2+ channels towards more hyperpolarizing potentials. Application of the conus toxin omega-conotoxin GVIA (GVIA; 1 mu M) blocked 31% of I-Ba; blockade was irreversible upon removal of the toxin from the extracellular medium. omega-Agatoxin IVA (IVA; 100 nM) produced a 15% blockade of I-Ba. omega-Conotoxin MVIIC (MVIIC; 5 mu M) produced a 36% blockade of I-Ba; such blockade seems to be related to both GVIA-sensitive (N-type) and GVIA-resistant Ca2+ channels. The sequential addition of supramaximal concentrations of furnidipine (10 mu M), GVIA (1 mu M), IVA (100 nM) and MVIIC (3 mu M) produced partial inhibition of I,,, which were additive. Our data suggest that the whole cell I,, in rat chromaffin cells exhibits at least four components. About 50% of I-Ba is carried by L-type Ca2+ channels, 30% by N-type Ca2+ channels and 15% by P-type Ca2+ channels. These figures are close to those found in cat chromaffin cells. However, they differ considerably from those found in bovine chromaffin cells where P-like Ca2+ channels account for 45% of the current, N-type carry 35% and L-type Ca2+. channels are responsible for only 20-25% of the current. These drastic differences might have profound physiological implications for the relative contribution of each channel subtype to the regulation of catecholamine release in different animal species.