Spergel D J, Catt K J, Rojas E
Endocrinology and Reproduction Research Branch, National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, MD 20892, USA.
Neuroendocrinology. 1996 Feb;63(2):101-11. doi: 10.1159/000126946.
The expression and function of large-conductance Ca2+ -activated K+ (BK) channels in the GT1-7 line of immortalized gonadotropin-releasing hormone (GnRH) neurons was investigated. Ionic currents were recorded using the patch-clamp technique, cytoplasmic free Ca2+ concentration ([Ca2+]i) was monitored using the fluorescent indicator, fura-2, and GnRH secretion was measured by radioimmunoassay. In cell-attached and inside-out patch-clamp recordings, K+ channels with a single-channel conductance of approximately 200 pS were detected. Depolarizing the patch increased the unitary current size and the open probability. In perforated-patch recordings, depolarizing pulses (50 ms) to potentials of -10 to +60 mV from a holding potential of -90 mV elicited outward current with early transient and sustained components. The transient current peaked 2-10 ms after the beginning of each pulse and increased in a voltage-dependent manner. This current was: (1) unaffected by the small-conductance Ca2+ -activated K+ channel blocker, apamin (100 nM); (2) reduced by the BK channel blocker, charybdotoxin (5 nM); (3) abolished by the Ca2+ channel blocker, CdCl2 (25 mu M), and (4) prolonged by the endoplasmic reticulum Ca2+ -ATPase inhibitor, thapsigargin (1 mu M). Based on the single-channel and whole-cell conductances, the number of channels per patch, the patch area, and the surface area of the cell, each GT1-7 cell contains 30-60 BK channels. The functional role of BK channels in GT1-7 cells was evaluated by measuring the effect of charybdotoxin (100 nM) on basal [Ca2+]i and GnRH secretion, as well as on the [Ca2+]i and GnRH secretory responses to gamma-aminobutyric acid (GABA, 100 mu M), an excitatory neurotransmitter in this system. Charybdotoxin had no effect on basal [Ca2+]i or GnRH secretion, or on the GABA-evoked [Ca2+]i and GnRH secretory responses. These results indicate that GT1-7 cells express BK channels; however, the physiological role of BK channels in GT1-7 cells remains elusive.
研究了大电导钙激活钾（BK）通道在永生化促性腺激素释放激素（GnRH）神经元GT1-7系中的表达及功能。采用膜片钳技术记录离子电流，使用荧光指示剂fura-2监测细胞质游离钙浓度（[Ca2+]i），并通过放射免疫分析法测定GnRH分泌。在细胞贴附式和内面向外式膜片钳记录中，检测到单通道电导约为200 pS的钾通道。使膜片去极化可增加单位电流大小和开放概率。在穿孔膜片记录中，从-90 mV的钳制电位向-10至+60 mV的电位施加50 ms的去极化脉冲，可引发具有早期瞬态和持续成分的外向电流。瞬态电流在每个脉冲开始后2 - 10 ms达到峰值，并呈电压依赖性增加。该电流：（1）不受小电导钙激活钾通道阻断剂蜂毒素（100 nM）的影响；（2）被BK通道阻断剂蝎毒素（5 nM）降低；（3）被钙通道阻断剂氯化镉（25 μM）消除；（4）被内质网钙ATP酶抑制剂毒胡萝卜素（1 μM）延长。根据单通道和全细胞电导、每个膜片的通道数量、膜片面积和细胞表面积，每个GT1-7细胞含有30 - 60个BK通道。通过测量蝎毒素（100 nM）对基础[Ca2+]i和GnRH分泌的影响，以及对γ-氨基丁酸（GABA，100 μM，该系统中的一种兴奋性神经递质）引起的[Ca2+]i和GnRH分泌反应的影响，评估了BK通道在GT1-7细胞中的功能作用。蝎毒素对基础[Ca2+]i或GnRH分泌，或对GABA诱发的[Ca2+]i和GnRH分泌反应均无影响。这些结果表明GT1-7细胞表达BK通道；然而，BK通道在GT1-7细胞中的生理作用仍不清楚。
