Shen Z, Marcus D C
Biophysics Laboratory, Boys Town National Research Hospital, Omaha, NE 68131, USA.
Hear Res. 1998 Sep;123(1-2):157-67. doi: 10.1016/s0378-5955(98)00110-5.
The IsK/KvLQT1 K+ channel in the apical membrane of strial marginal cells and vestibular dark cells is an essential ion transport pathway for the secretion of K+ into the endolymph of the inner ear. Study of this control point has been impeded by rundown of channel activity upon excision into commonly used cytosolic solutions. This paper describes conditions under which patches of apical membrane of strial marginal cells and vestibular dark cells from gerbil containing this channel can be excised, retaining its characteristic voltage dependence, kinetic properties, ion permeability sequence and pharmacological sensitivity, similar to those found during on-cell and perforated-patch whole cell recordings (Shen et al., Audit. Neurosci. 3 (1997) 215-230). Those excised-patch conditions include removal of Mg2+ from the cytosolic solution and use of a K+-rich pipette electrolyte. The inhibition of channel activity by Mg2+ was found to be a general feature of divalent cations; the channel was also inhibited by Ca2+, Ba2+ and Sr2+. The concentrations causing 50% inhibition of IsK/KvLQT1 channel current were 7 x 10(-5) M, 6 x 10(-6) M, 3 x 10(-4) M and 7 x 10(-5) M, respectively. It was also found that a chemical cross-linking agent, 3,3'-dithio-bis(sulfosuccinimidyl propionate) (DTSSP), which was previously shown to persistently activate IsK/KvLQTI channels expressed in Xenopus oocytes, maintained in excised patches channel activity which retained voltage dependence and pharmacological sensitivity. These data demonstrate that (1) the channel complex is inhibited by Ca2+, Mg2+ and other divalent cations, (2) the activation by Ca2+ observed previously in whole-cell preparations was due to action via other cellular pathways. These findings must be taken into account when considering the action of receptors which alter the cytosolic Ca2+ activity.
血管纹边缘细胞和前庭暗细胞顶端膜中的IsK/KvLQT1钾通道是将钾离子分泌到内耳内淋巴中的重要离子转运途径。由于切除后通道活性在常用的胞质溶液中逐渐降低，对这一控制点的研究受到了阻碍。本文描述了从沙鼠中分离出含有该通道的血管纹边缘细胞和前庭暗细胞顶端膜片的条件，这些膜片保留了其特征性的电压依赖性、动力学特性、离子通透性序列和药理学敏感性，类似于在细胞膜上和穿孔膜片全细胞记录中发现的情况（Shen等人，《听觉神经科学》3（1997）215 - 230）。这些切除膜片的条件包括从胞质溶液中去除镁离子以及使用富含钾离子的移液管电解质。发现镁离子对通道活性的抑制是二价阳离子的普遍特性；该通道也受到钙离子、钡离子和锶离子的抑制。导致IsK/KvLQT1通道电流50%抑制的浓度分别为7×10⁻⁵ M、6×10⁻⁶ M、3×10⁻⁴ M和7×10⁻⁵ M。还发现一种化学交联剂3,3'-二硫代双（磺基琥珀酰亚胺丙酸酯）（DTSSP），先前已证明其能持续激活非洲爪蟾卵母细胞中表达的IsK/KvLQTI通道，在切除的膜片中能维持通道活性，且保留电压依赖性和药理学敏感性。这些数据表明：（1）通道复合物受到钙离子、镁离子和其他二价阳离子的抑制；（2）先前在全细胞制剂中观察到的钙离子激活是通过其他细胞途径起作用的。在考虑改变胞质钙离子活性的受体作用时，必须考虑这些发现。
