Whole-cell patch-clamp recordings were used to determine the variation in the P2X receptor conductance, activated by extracellular ATP, in outer hair cells (OHCs) isolated from each of the four turns of the guinea-pig cochlea. 2. In standard solution (containing 1.5 mM Ca2+) slope conductances were determined in OHCs of known origin from current-voltage relationships obtained from voltage ramps applied between -100 and +50 mV. Membrane conductance throughout this voltage range was greatest in OHCs originating from the basal (high frequency encoding) region of the cochlea. This gradient in OHC conductance from apex to base of the cochlea can be attributed to variation in expression of both a negatively activated K+ conductance and a TEA-sensitive outwardly rectifying K+ conductance. OHC slope conductance measured about a membrane potential of -75 mV increased from a mean of 33.5 nS in the apical region (turn 4) to 96.8 nS in the basal region (turn 1) of the cochlea. 3. Removal of external Ca2+ reduced OHC conductance by an average of 25%, reflecting a Ca2+ dependence of the background conductances in these cells. In zero external Ca2+ the mean slope conductance measured at -75 mV in the apical turn was 25.0 nS compared with 73.8 nS in the basal turn. 4. In Ca(2+)-free solution both 2 mM and 4 microM ATP produced inward currents that were progressively larger in OHCs originating from more basal regions of the cochlea. The steady-state inward current elicited by 2 mM extracellular ATP increased from -1.44 to -3.26 nA for turns 4 and 1, respectively. 5. The P2X receptor conductance was determined between -100 and +50 mV by comparing voltage ramps in the presence and absence of extracellular ATP in Ca(2+)-free solution. The conductance was inwardly rectifying with a reversal potential close to 0 mV. Measured close to the resting membrane potential of the cells (-75 mV), 2 mM ATP elicited an average 300% increase in conductance in parallel with the systematic increase in background conductance which occurs in OHCs originating from the more basal regions of the cochlea. The conductance at -75 mV activated by 2 mM ATP increased from a mean of 59.6 nS in turn 4 OHCs to a mean of 166.2 nS in turn 1 OHCs. The conductance activated by 4 microM ATP was also greater in the basal turn OHCs (45.3 nS) than in the apical region OHCs (5.9 nS). 6. The number of ATP-gated ion channels on individual OHCs, presumed to be localized to the stereocilia, increases from approximately 6000 in turn 4 cells to 16,500 in turn 1 cells, based on estimates of unitary conductance and average maximum ATP-activated OHC conductance (2 mM ATP).
摘要
采用全细胞膜片钳记录技术，测定豚鼠耳蜗四个不同蜗旋中分离出的外毛细胞（OHC）中，由细胞外ATP激活的P2X受体电导的变化。2. 在标准溶液（含1.5 mM Ca2+）中，根据在-100至+50 mV之间施加电压斜坡得到的电流-电压关系，测定已知来源的OHC的斜率电导。在整个该电压范围内，耳蜗基底（高频编码）区域来源的OHC的膜电导最大。耳蜗OHC电导从蜗顶到蜗底的这种梯度变化，可归因于负激活K+电导和TEA敏感外向整流K+电导表达的差异。在约-75 mV膜电位下测得的OHC斜率电导，从耳蜗顶区（第4蜗旋）的平均33.5 nS增加到基底区（第1蜗旋）的96.8 nS。3. 去除细胞外Ca2+使OHC电导平均降低25%，这反映了这些细胞中背景电导对Ca2+的依赖性。在零细胞外Ca2+条件下，顶蜗旋在-75 mV测得的平均斜率电导为25.0 nS，而底蜗旋为73.8 nS。4. 在无Ca2+溶液中，2 mM和4 μM ATP均产生内向电流，且在耳蜗更基底区域来源的OHC中，内向电流逐渐增大。2 mM细胞外ATP引发的稳态内向电流，第4蜗旋和第1蜗旋分别从-1.44 nA增加到-3.26 nA。5. 通过比较无细胞外ATP和有细胞外ATP时在无Ca2+溶液中的电压斜坡，在-100至+50 mV之间测定P2X受体电导。该电导呈内向整流，反转电位接近0 mV。在接近细胞静息膜电位（-75 mV）处测量，2 mM ATP使电导平均增加300%，同时耳蜗更基底区域来源的OHC中背景电导也系统性增加。2 mM ATP在-75 mV激活的电导，第4蜗旋OHC平均为59.6 nS，第1蜗旋OHC平均为166.2 nS。4 μM ATP激活的电导在底蜗旋OHC（45.3 nS）中也大于顶区OHC（5.9 nS）。6. 根据单通道电导和平均最大ATP激活的OHC电导（2 mM ATP）估计，单个OHC上假定定位于静纤毛的ATP门控离子通道数量，从第4蜗旋细胞的约6000个增加到第1蜗旋细胞的16500个。
