Characterization of Voltage-Dependent Ca²⁺ Channels in β-Cell Line

Although there is compelling pharmacological evidence based on Ca²⁺-channel antagonist studies suggesting that the voltage-dependent Ca²⁺ channels regulate insulin release, no direct comparison with Ca²⁺ currents exists. This is particularly important because of the recent demonstration in other cell types of one and possibly two Ca²⁺ channels that are insensitive to Ca²⁺-channel antagonists, the dihydropyridines and the phenylalkylamines. Using an SV40-transformed pancreatic β-cell line (HIT cells), we determined how voltage-dependent Ca²⁺ channels are involved in stimulus-secretion coupling. Ca²⁺ currents were measured with the tight-seal technique for wholecell recording. The cytosolic free-Ca²⁺ concentration ([Ca²⁺]₁) was followed with the fluorescent probe Fura 2, and the measurements were compared with insulin secretion stimulated by depolarizing the cells with K⁺. The Ca²⁺ current contained two components: arapidly decaying current activated at –50 to –40 mV that decayed with a time constant of 25 ms anda very slowly decaying component activated at –40 mV. Both components were sensitive to the Ca²⁺-channel antagonist nimodipine. There is excellent agreement in the concentration of nimodipine that inhibited Ca²⁺ current and the increase in [Ca²⁺₁], in response to K⁺ depolarization (IC₅₀ of 15 and 6 nM, respectively). Nimodipine inhibited insulin release over a similar dose-response range with an IC₅₀of 1.5 × 10 9 M. These studies indicate that the increase in [Ca²⁺₁], in response to β-cell depolarization can be accounted for by the influx of this ion through a single class of dihydropyridine-sensitive Ca²⁺ channels in the cell membrane.