Aiello E A, Malcolm A T, Walsh M P, Cole W C
Centro de Investigaciones Cardiovasculares, Facultad de Ciencias Médicas, Universidad Nacional de La Plata, La Plata, Argentina.
Am J Physiol. 1998 Aug;275(2):H448-59. doi: 10.1152/ajpheart.1998.275.2.H448.
Macroscopic 4-aminopyridine (4-AP)-sensitive, delayed rectifier K+ current of vascular smooth muscle cells is increased during beta-adrenoceptor activation with isoproterenol via a signal transduction pathway involving adenylyl cyclase and cAMP-dependent protein kinase (PKA) (Aiello, E. A., M. P. Walsh, and W. C. Cole. Am. J. Physiol. 268 (Heart Circ. Physiol. 37): H926-H934, 1995.). In this study, we identified the single delayed rectifier K+ (KDR) channel(s) of rabbit portal vein myocytes affected by treatment with isoproterenol or the catalytic subunit of PKA. 4-AP-sensitive KDR channels of 15.3 +/- 0.6 pS (n = 5) and 14.8 +/- 0.6 pS (n = 5) conductance, respectively, were observed in inside-out (I-O) and cell-attached (C-A) membrane patches in symmetrical KCl recording conditions. The kinetics of activation (time constant of 10.7 +/- 3. 02 ms) and inactivation (fast and slow time constants of 0.3 and 2.5 s, respectively) of ensemble currents produced by these channels mimicked those reported for inactivating, 4-AP-sensitive whole cell KDR current of vascular myocytes. Under control conditions, the open probability (NPo) of KDR channels of C-A membrane patches at -40 mV was 0.014 +/- 0.005 (n = 8). Treatment with 1 microM isoproterenol caused a significant, approximately threefold increase in NPo to 0. 041 +/- 0.02 (P < 0.05). KDR channels of I-O patches exhibited rundown after approximately 5 min, which was not affected by ATP (5 mM) in the bath solution. Treatment with the purified catalytic subunit of PKA (50 nM; 5 mM ATP) restored KDR channel activity and caused NPo to increase from 0.011 +/- 0.003 to 0.138 +/- 0.03 (P < 0. 05; n = 11). These data indicate that small-conductance, 15-pS KDR channels are responsible for inactivating the macroscopic delayed rectifier K+ current of rabbit portal vein myocytes and that the activity of these channels is enhanced by a signal transduction mechanism involving beta-adrenoceptors and phosphorylation by PKA at a membrane potential consistent with that observed in the myocytes in situ.
在通过异丙肾上腺素激活β-肾上腺素能受体期间，血管平滑肌细胞的宏观4-氨基吡啶（4-AP）敏感的延迟整流钾电流通过涉及腺苷酸环化酶和cAMP依赖性蛋白激酶（PKA）的信号转导途径增加（艾ello，E.A.，M.P.沃尔什和W.C.科尔。《美国生理学杂志》268（心脏循环生理学37）：H926 - H934，1995年）。在本研究中，我们鉴定了受异丙肾上腺素或PKA催化亚基处理影响的兔门静脉肌细胞的单个延迟整流钾（KDR）通道。在对称KCl记录条件下，在内外翻（I - O）和细胞贴附（C - A）膜片钳中分别观察到电导为15.3±0.6 pS（n = 5）和14.8±0.6 pS（n = 5）的4 - AP敏感KDR通道。这些通道产生的整体电流的激活动力学（时间常数为10.7±3.02毫秒）和失活动力学（快速和慢速时间常数分别为0.3和2.5秒）模仿了报道的血管肌细胞中失活的、4 - AP敏感的全细胞KDR电流的动力学。在对照条件下，C - A膜片钳中KDR通道在 - 40 mV时的开放概率（NPo）为0.014±0.005（n = 8）。用1μM异丙肾上腺素处理导致NPo显著增加约三倍，达到0.041±0.02（P < 0.05）。I - O膜片的KDR通道在大约5分钟后表现出电流衰减，这不受浴液中ATP（5 mM）的影响。用纯化的PKA催化亚基（50 nM；5 mM ATP）处理恢复了KDR通道活性，并使NPo从0.011±0.003增加到0.138±0.03（P < 0.05；n = 11）。这些数据表明，小电导的15 - pS KDR通道负责使兔门静脉肌细胞的宏观延迟整流钾电流失活，并且这些通道的活性通过涉及β - 肾上腺素能受体和PKA磷酸化的信号转导机制在与原位肌细胞中观察到的膜电位一致时增强。
