Wangemann P, Liu J, Shen Z, Shipley A, Marcus D C
Cell Physiology Laboratory, Boystown National Research Hospital, Omaha, NE, USA.
J Membr Biol. 1995 Oct;147(3):263-73. doi: 10.1007/BF00234524.
Volume regulation of vestibular dark cells from the gerbilline inner ear in response to a hypo-osmotic challenge depends on the presence of cytosolic K+ and Cl-. The present study addresses the questions: (i) whether and by what mechanism K+ is released during volume regulation, (ii) whether the osmolarity of the basolateral medium has an effect on the steady-state rate of transepithelial K+ transport and (iii) whether there is cross-talk between the basolateral membrane responsible for K+ uptake and the apical membrane responsible for K+ release. K+ secretion (JK+,probe) and current density (Isc,probe) were measured with vibrating probes in the vicinity of the apical membrane and the transepithelial potential (Vt) and resistance (Rt) were measured in a micro-Ussing chamber. The equivalent short-circuit current (Isc) was calculated. The current (IIsK), conductance (gIsK) and inactivation time constant (tau IsK) of the IsK channel and the apparent reversal potential of the apical membrane (Vr) were obtained with the cell-attached macropatch technique. Vr was corrected (Vrc) for the membrane voltage (Vm) measured separately with microelectrodes. A hypo-osmotic challenge (294 to 154 mosM by removal of 150 mM mannitol) on the basolateral side of the epithelium increased JK+,probe and Isc,probe by a factor of 2.7 and 1.6. When this hypo-osmotic challenge was applied to both sides of the epithelium Vt and Isc increased from 5 to 14 mV and from 189 to 824 microA/cm2 whereas Rt decreased from 27 to 19 omega-cm2. With 3.6 mM K+ in the pipette IIsK was outwardly directed, tau IsK was 267 msec and the hypo-osmotic challenge caused IIsK and gIsK to increase from 14 to 37 pA and from 292 to 732 pS. Vrc hyperpolarized from -44 to -76 mV. With 150 mM K+ in the pipette IIsK was inwardly directed, tau IsK was 208 msec and the hypo-osmotic challenge caused IIsK and gIsK to increase in magnitude from 0 to -21 pA and from 107 to 1101 pS. Vrc remained unchanged (-2 vs. 1 mV). These data demonstrate that a hypo-osmotic challenge stimulates transepithelial K+ secretion and activates the apical IsK channel. The hypo-osmotically-induced increase in K+ secretion exceeded the estimated amount of K+ release necessary for the maintenance of constant cell volume, suggesting that the rate of basolateral K+ uptake was upregulated in the presence of the hypo-osmotic challenge and that cross-talk exists between the apical membrane and the basolateral membrane.
沙鼠内耳前庭暗细胞对低渗刺激的体积调节取决于胞质K⁺和Cl⁻的存在。本研究探讨以下问题：(i) 在体积调节过程中K⁺是否以及通过何种机制释放；(ii) 基底外侧介质的渗透压是否对跨上皮K⁺转运的稳态速率有影响；(iii) 负责K⁺摄取的基底外侧膜与负责K⁺释放的顶端膜之间是否存在相互作用。用振动探针在顶端膜附近测量K⁺分泌(JK⁺,探针)和电流密度(Isc,探针)，并在微型尤斯灌流室中测量跨上皮电位(Vt)和电阻(Rt)。计算等效短路电流(Isc)。用细胞贴附式大膜片钳技术获得IsK通道的电流(IIsK)、电导(gIsK)和失活时间常数(tau IsK)以及顶端膜的表观反转电位(Vr)。Vr用微电极单独测量的膜电压(Vm)进行校正(Vrc)。上皮基底外侧施加低渗刺激(通过去除150 mM甘露醇使渗透压从294降至154 mosM)使JK⁺,探针和Isc,探针增加2.7倍和1.6倍。当对上皮两侧施加这种低渗刺激时，Vt和Isc从5 mV增加到14 mV，从189 μA/cm²增加到824 μA/cm²，而Rt从27 Ω·cm²降至19 Ω·cm²。移液管中含3.6 mM K⁺时，IIsK向外，tau IsK为267毫秒，低渗刺激使IIsK和gIsK从14 pA增加到37 pA，从292 pS增加到732 pS。Vrc从 -44 mV超极化到 -76 mV。移液管中含150 mM K⁺时，IIsK向内，tau IsK为208毫秒，低渗刺激使IIsK和gIsK的幅度从0增加到 -21 pA，从107 pS增加到1101 pS。Vrc保持不变(-2 mV对1 mV)。这些数据表明，低渗刺激刺激跨上皮K⁺分泌并激活顶端IsK通道。低渗诱导的K⁺分泌增加超过维持细胞体积恒定所需的K⁺释放估计量，表明在低渗刺激存在下基底外侧K⁺摄取速率上调，并且顶端膜和基底外侧膜之间存在相互作用。
