High expression of the R-Type voltage-gated Ca2+ channel and its involvement in Ca2+-dependent gonadotropin-releasing hormone release in GT1-7 cells

The GT1 cell has been widely used as a model cell to study cellular functions of GnRH neurons. Despite the importance of Ca2+ channels, little is known except for L- and T-type Ca2+ channels in GT1 cells. Therefore, we studied the diversity of voltage-gated Ca2+ channels in GT1 - 7 cells with perforated-patch clamp and RT-PCR. An R-type Ca2+ channel blocker, SNX-482, inhibited the Ca2+ currents by 75.6% in all cells examined (n = 9). A T-type Ca2+ channel blocker, Ni2+, inhibited the Ca2+ currents by 12.6% in all cells examined ( n = 9). An L- type Ca2+ channel blocker, nimodipine, inhibited the Ca2+ currents by 17.9% in five of 11 cells examined. When using Ba2+ as a charge carrier, another dihydropyridine antagonist, nifedipine, clearly inhibited the currents by 12.1% in all cells examined ( n = 16). An N- type Ca2+ channel blocker, omega-conotoxin-GVIA, inhibited the Ca2+ currents by 13.8% in three of 20 cells examined. A P/Q type Ca2+ channel blocker, omega-agatoxin-IVA, had no effect on the currents ( n = 9). RT-PCR revealed that GT1-7 cells expressed the alpha(1B), alpha(1D), alpha(1E), and alpha(1H) subunit mRNA. Furthermore, SNX-482 and nifedipine inhibited the high K+-induced increase in the intracellular Ca2+ concentration and GnRH release. omega-Conotoxin-GVIA and omega-agatoxin-IVA had no effect. These results suggest that GT1-7 cells express R-, L-, N-, and T-type voltage-gated Ca2+ channels; the R- type was a major current component, and the L-, N-, and T-types were minor ones. The R- and L- type Ca2+ channels play a critical role in the regulation of Ca2+-dependent GnRH release.