Rispoli G, Martini M, Rossi M L, Rubbini G, Fesce R
INFM, Dipartimento di Biologia, Università di Ferrara, Italy.
Neuroreport. 2000 Aug 21;11(12):2769-74. doi: 10.1097/00001756-200008210-00032.
Hair cells in the frog semicircular canal, studied by the whole-cell patch-clamp technique, display three distinct Ca2+ currents: two non-inactivating components (L type and R type, the latter termed R2 in the following) and a second R type current (termed R1), which runs down first and inactivates in a Ca2+-dependent fashion. Since intracellular EGTA, up to 5 mM, did not display major effects on such inactivation, we used increasing amounts of BAPTA in the patch pipette, to control [Ca2+]i more efficiently and investigate whether modifications in [Ca2+]i at the cytoplasmic side of the channel affect the inactivation of the RI component and in general the gating of all channel types. The results here reported show that (1) K+ currents heavily contaminate recordings obtained using high concentrations of BAPTA in its commercially available K+ salt form; (2) BAPTA Cs+ salt can be satisfactorily employed to obtain reliable recordings; (3) the kinetics of channel gating and R1-channel inactivation are indeed markedly affected by effectively buffering [Ca2+]i.
采用全细胞膜片钳技术研究发现，青蛙半规管中的毛细胞呈现出三种不同的Ca2+电流：两种非失活成分（L型和R型，后者在下文称为R2）以及第二种R型电流（称为R1），R1电流首先衰减并以Ca2+依赖的方式失活。由于高达5 mM的细胞内EGTA对这种失活未显示出主要影响，我们在膜片电极中使用了越来越多的BAPTA，以更有效地控制[Ca2+]i，并研究通道胞质侧[Ca2+]i的变化是否会影响RI成分的失活以及一般情况下所有通道类型的门控。此处报道的结果表明：（1）使用市售K+盐形式的高浓度BAPTA获得的记录会受到K+电流的严重污染；（2）BAPTA Cs+盐可令人满意地用于获得可靠的记录；（3）通过有效缓冲[Ca2+]i，通道门控动力学和R1通道失活确实会受到显著影响。
