Bender Kirsten, Nasrollahzadeh Parastoo, Timpert Mathias, Liu Bing, Pott Lutz, Kienitz Marie-Cécile
Institute of Physiology, Ruhr-University Bochum, D-44780 Bochum, Germany.
J Physiol. 2008 Apr 15;586(8):2049-60. doi: 10.1113/jphysiol.2007.148346. Epub 2008 Feb 14.
The effect of beta-adrenergic stimulation on endogenous G-protein-activated K(+) (GIRK) current has been investigated in atrial myocytes from hearts of adult rats. Beta-adrenergic stimulation (10 microm isoprenaline, Iso) had no effect on activation kinetics, peak current or steady-state current but resulted in slowing of deactivation upon washout of acetylcholine (ACh), the time constant (tau(d)) being increased by a factor of about 2.5. The effect of Iso could be mimicked by inclusion of cAMP (500 microm) in the filling solution of the patch clamp pipette. The Iso-induced increase in tau(d) was blocked by the selective beta(1) receptor antagonist CGP-20112A (2 microm) and by the PKA inhibitor H9 (100 microm included in the pipette solution). A candidate for mediating these effects is RGS10, one of the regulators of G-protein signalling (RGS) species expressed in cardiac myocytes. Overexpression of RGS10 by adenoviral gene transfer resulted in a reduction in tau(d) of 60%. Sensitivity of tau(d) to Iso remained in cells overexpressing RGS10. Overexpression of RGS4 caused a comparable reduction in tau(d), which became insensitive to Iso. Expression of an RGS10 carrying a mutation (RGS10-S168A), which deletes a PKA phosphorylation site, caused a decrease in tau(d) comparable to overexpression of wild-type RGS10. Sensitivity of tau(d) to Iso was lost in RGS10-S168A-expressing myocytes. Silencing of RGS10 by means of adenovirus-mediated transcription of a short hairpin RNA did not affect basal tau(d) but removed sensitivity to Iso. These data suggest that endogenous RGS10 has GTPase-activating protein (GAP) activity on the G-protein species that mediates activation of atrial GIRK channels. Moreover, RGS10, via PKA-dependent phosphorylation, enables a crosstalk between beta-adrenergic and muscarinic cholinergic signalling.
已在成年大鼠心脏的心房肌细胞中研究了β-肾上腺素能刺激对内源性G蛋白激活的钾离子（GIRK）电流的影响。β-肾上腺素能刺激（10微摩尔异丙肾上腺素，Iso）对激活动力学、峰值电流或稳态电流没有影响，但在洗脱乙酰胆碱（ACh）后导致失活减慢，时间常数（τd）增加约2.5倍。在膜片钳移液管的灌流液中加入cAMP（500微摩尔）可模拟Iso的作用。Iso诱导的τd增加被选择性β1受体拮抗剂CGP-20112A（2微摩尔）和PKA抑制剂H9（移液管溶液中含100微摩尔）阻断。介导这些效应的一个候选分子是RGS10，它是心肌细胞中表达的G蛋白信号调节因子（RGS）之一。通过腺病毒基因转移过表达RGS10导致τd降低60%。在过表达RGS10的细胞中，τd对Iso的敏感性仍然存在。过表达RGS4导致τd有类似程度的降低，且对Iso不敏感。携带缺失PKA磷酸化位点突变（RGS10-S168A）的RGS10的表达导致τd降低，与野生型RGS10过表达相当。在表达RGS10-S168A的心肌细胞中，τd对Iso的敏感性丧失。通过腺病毒介导的短发夹RNA转录使RGS10沉默不影响基础τd，但消除了对Iso的敏感性。这些数据表明，内源性RGS10对介导心房GIRK通道激活的G蛋白具有GTP酶激活蛋白（GAP）活性。此外，RGS10通过PKA依赖性磷酸化，实现了β-肾上腺素能和毒蕈碱胆碱能信号之间的串扰。
