Sunose H, Liu J, Shen Z, Marcus D C
Biophysics Laboratory, Boys Town National Research Hospital, Omaha, NE 68131, USA.
J Membr Biol. 1997 Mar 1;156(1):25-35. doi: 10.1007/s002329900184.
Adenosine 3',5'-cyclic monophosphate (cAMP) is known to stimulate exogenous IsK channel current in the Xenopus oocyte expression system. The present study was performed to determine whether elevation of cytosolic cAMP in a native mammalian epithelium known to secrete K+ through endogenously expressed IsK channels would stimulate K+ secretion through these channels. The equivalent short circuit current (Isc) across vestibular dark cell epithelium in gerbil was measured in a micro-Ussing chamber and the apical membrane current (IsK) and conductance (gIsK) of IsK channels was recorded with both the on-cell macro-patch and nystatin-perforated whole-cell patch-clamp techniques. It has previously been shown that Isc can be accounted for by transepithelial K+ secretion and that the apical IsK channels constitute a significant pathway for K+ secretion. The identification of the voltage-dependent whole-cell currents in vestibular dark cells was strengthened by the finding that a potent blocker of IsK channels, chromanol 293B, strongly reduced IIsK from 646 +/- 200 to 154 +/- 22 pA (71%) and gIsK from 7.5 +/- 2.6 to 2.8 +/- 0.4 nS (53%). Cytoplasmic cAMP was elevated by applying dibutyryl cyclic AMP (dbcAMP), or the phosphodiesterase inhibitors 3-isobutyl-1-methylxanthine (IBMX) and Ro-20-1724. dbcAMP (1 mM) increased Isc and IIsK from 410 +/- 38 to 534 +/- 40 microA/cm2 and from 4.3 +/- 0.8 to 11.4 +/- 2.2 pA, respectively. IBMX (1 mM) caused transient increases of Isc from 415 +/- 30 to 469 +/- 38 microA/cm2 and Ro-20-1724 (0.1 mM) from 565 +/- 43 to 773 +/- 58 microA/cm2. IBMX increased IIsK from 5.5 +/- 1.5 to 16.9 +/- 5.8 pA in on-cell experiments and from 191 +/- 31 to 426 +/- 53 pA in whole-cell experiments. The leak conductance due to all non-IsK channel sources did not change during dbcAMP and IBMX while 293B in the presence of dbcAMP reduced IIsK by 84% and gIsK by 62%, similar to unstimulated conditions. These results demonstrate that the cAMP pathway is constitutively active in vestibular dark cells and that the cAMP pathway stimulates transepithelial K+ secretion by increasing IsK channel current rather than by altering another transport pathway.
已知3',5'-环磷酸腺苷（cAMP）可刺激非洲爪蟾卵母细胞表达系统中的外源性IsK通道电流。本研究旨在确定在已知通过内源性表达的IsK通道分泌钾离子的天然哺乳动物上皮细胞中，胞质cAMP升高是否会通过这些通道刺激钾离子分泌。在微型尤斯灌流室中测量沙鼠前庭暗细胞上皮的等效短路电流（Isc），并使用细胞膜片钳和制霉菌素穿孔全细胞膜片钳技术记录IsK通道的顶膜电流（IsK）和电导（gIsK）。此前已表明，Isc可由跨上皮钾离子分泌来解释，且顶膜IsK通道构成钾离子分泌的重要途径。发现IsK通道的强效阻滞剂色满醇293B可使IIsK从646±200 pA大幅降至154±22 pA（71%），gIsK从7.5±2.6 nS降至2.8±0.4 nS（53%），这一结果强化了前庭暗细胞中电压依赖性全细胞电流的鉴定。通过应用二丁酰环磷腺苷（dbcAMP）或磷酸二酯酶抑制剂3-异丁基-1-甲基黄嘌呤（IBMX）和Ro-20-1724来升高细胞质cAMP。dbcAMP（1 mM）使Isc和IIsK分别从410±38 μA/cm²增加到534±40 μA/cm²以及从4.3±0.8 pA增加到11.4±2.2 pA。IBMX（1 mM）使Isc从415±30 μA/cm²短暂增加到469±38 μA/cm²，Ro-20-1724（0.1 mM）使Isc从565±43 μA/cm²增加到773±58 μA/cm²。在细胞膜片钳实验中，IBMX使IIsK从5.5±1.5 pA增加到16.9±5.8 pA，在全细胞膜片钳实验中从191±31 pA增加到426±53 pA。在dbcAMP和IBMX作用期间，所有非IsK通道来源的漏电导未发生变化，而在dbcAMP存在的情况下，293B使IIsK降低84%，gIsK降低62%，与未刺激条件下相似。这些结果表明，cAMP途径在前庭暗细胞中持续活跃，且cAMP途径通过增加IsK通道电流而非改变其他转运途径来刺激跨上皮钾离子分泌。
