Varanda W A, de Carvalho A C
Department of Physiology, Faculty of Medicine of Ribeirão Preto, Universidade de São Paulo, Brazil.
Am J Physiol. 1994 Aug;267(2 Pt 1):C563-9. doi: 10.1152/ajpcell.1994.267.2.C563.
In this paper we describe the basic features of gap junctions in pairs of Leydig cells mechanically dissociated from mouse testes, studied with the double whole cell patch-clamp technique. These cells are extensively coupled with regard to dye injection and electrophysiological measurements. The mean junctional conductance (gj) measured in 61 pairs of cells was 10.6 +/- 1.5 (SE) nS. In most pairs gj was voltage dependent when transjunctional voltage exceeded +/- 50 mV. On imposition of a voltage gradient across the junction the transjunctional current decayed exponentially to a lower level, with a time constant of 3.3 s at 50 mV and 430 ms at 100 mV. As in other systems, octanol (600 microM final concentration) uncoupled the cells within approximately 2 min. In a few cell pairs, gj was low enough to permit recording of single channel currents without the use of uncoupling agents. Single channel conductance fluctuations measured using pipettes containing potassium aspartate were distributed mainly around three peaks, at 21, 39, and 60 pS, suggesting the presence of channels formed by connexin 43. Western blots of Percoll gradient purified Leydig cells using specific antibodies indicate that connexin 43 is indeed expressed in these cells, whereas connexin 26 and connexin 32 are not.
在本文中,我们描述了用双全细胞膜片钳技术研究的、从雄性小鼠睾丸中机械分离出来的成对睾丸间质细胞间缝隙连接的基本特征。在染料注入和电生理测量方面,这些细胞存在广泛的耦合。在61对细胞中测得的平均连接电导(gj)为10.6±1.5(SE)nS。在大多数细胞对中,当跨连接电压超过±50 mV时,gj呈电压依赖性。在连接处施加电压梯度时,跨连接电流呈指数衰减至较低水平,在50 mV时时间常数为3.3 s,在100 mV时为430 ms。与其他系统一样,辛醇(终浓度600μM)在约2分钟内使细胞解偶联。在少数细胞对中,gj足够低,无需使用解偶联剂就能记录单通道电流。使用含天冬氨酸钾的移液管测量的单通道电导波动主要分布在三个峰值附近,分别为21、39和60 pS,这表明存在由连接蛋白43形成的通道。使用特异性抗体对经Percoll梯度纯化的睾丸间质细胞进行的蛋白质免疫印迹分析表明,连接蛋白43确实在这些细胞中表达,而连接蛋白26和连接蛋白32则未表达。