Abstract：
Signaling of G protein-activated inwardly rectifying K+ (GIRK) channels is an important mechanism of the parasympathetic regulation of the heart rate and cardiac excitability. GIRK channels are inhibited during stimulation of Gq-coupled receptors (GqPCRs) by depletion of phosphatidyl-4,5-bisphosphate (PIP2) and/or channel phosphorylation by protein kinase C (PKC). The GqPCR-dependent modulation of GIRK currents in terms of specific PKC isoform activation was analyzed in voltage-clamp experiments in rat atrial myocytes and in CHO or HEK 293 cells. By using specific PKC inhibitors, we identified the receptor-activated PKC isoforms that contribute to phenylephrine- and angiotensin-induced GIRK channel inhibition. We demonstrate that the cPKC isoform PKCα significantly contributes to GIRK inhibition during stimulation of wildtype α1B-adrenergic receptors (α1B-ARs). Deletion of the α1B-AR serine residues S396 and S400 results in a preferential regulation of GIRK activity by PKCβ. As a novel finding, we report that the AT1-receptor-induced GIRK inhibition depends on the activation of the nPKC isoform PKCε whereas PKCα and PKCβ do not mainly participate in the angiotensin-mediated GIRK reduction. Expression of the dominant negative (DN) PKCε prolonged the onset of GIRK inhibition and significantly reduced AT1-R desensitization, indicating that PKCε regulates both GIRK channel activity and the strength of the receptor signal via a negative feedback mechanism. The serine residue S418 represents an important phosphorylation site for PKCε in the GIRK4 subunit. To analyze the functional impact of this PKC phosphorylation site for receptor-specific GIRK channel modulation, we monitored the activity of a phosphorylation-deficient (GIRK4 (S418A)) GIRK4 channel mutant during stimulation of α1B-ARs or AT1-receptors. Mutation of S418 did not impede α1B-AR-mediated GIRK inhibition, suggesting that S418 within the GIRK4 subunit is not subject to PKCα-induced phosphorylation. Furthermore, activation of angiotensin receptors induced pronounced GIRK4 (S418A) channel inhibition, excluding that this phosphorylation site contributes to the AT1-R-induced GIRK reduction. Instead, phosphorylation of S418 has a facilitative effect on GIRK activity that was abolished in the GIRK4 (S418A) mutant. To summarize, the present study shows that the receptor-dependent regulation of atrial GIRK channels is attributed to the GqPCR-specific activation of different PKC isoforms. Receptor-specific activated PKC isoforms target distinct phosphorylation sites within the GIRK4 subunit, resulting in differential regulation of GIRK channel activity with either facilitative or inhibitory effects on GIRK currents.
摘要：
G蛋白激活的向内整流K（GIRK）通道的信号传导是心率和心脏兴奋性的副交感神经调节的重要机制。在刺激Gq偶联受体（GqPCR）期间，通过消耗磷脂酰-4,5-二磷酸（PIP2）和/或蛋白激酶C（PKC）的通道磷酸化来抑制GIRK通道。在大鼠心房肌细胞和CHO或HEK293细胞中的电压钳实验中，分析了GqPCR依赖性GIRK电流在特定PKC同种型激活方面的调节。通过使用特定的PKC抑制剂，我们鉴定了受体激活的PKC亚型,这些亚型有助于去氧肾上腺素和血管紧张素诱导的GIRK通道抑制.我们证明，在刺激野生型α1B-肾上腺素能受体（α1B-ARs）期间，cPKC同工型PKCα显着促进GIRK抑制。α1B-AR丝氨酸残基S396和S400的缺失导致PKCβ对GIRK活性的优先调节。作为一个新颖的发现，我们报道了AT1受体诱导的GIRK抑制依赖于nPKC同工型PKCε的激活,而PKCα和PKCβ并不主要参与血管紧张素介导的GIRK减少.显性阴性（DN）PKCε的表达延长了GIRK抑制的发作并显着降低了AT1-R脱敏，表明PKCε通过负反馈机制调节GIRK通道活性和受体信号的强度。丝氨酸残基S418代表GIRK4亚基中PKCε的重要磷酸化位点。为了分析PKC磷酸化位点对受体特异性GIRK通道调节的功能影响，我们监测了α1B-ARs或AT1受体刺激过程中磷酸化缺陷型(GIRK4(S418A))GIRK4通道突变体的活性.S418的突变不妨碍α1B-AR介导的GIRK抑制,表明GIRK4亚基内的S418不受PKCα诱导的磷酸化。此外，血管紧张素受体的激活诱导明显的GIRK4（S418A）通道抑制，排除该磷酸化位点有助于AT1-R诱导的GIRK减少。相反，S418的磷酸化对GIRK活性具有促进作用，该作用在GIRK4（S418A）突变体中被消除。总结一下,本研究表明,心房GIRK通道的受体依赖性调节归因于不同PKC亚型的GqPCR特异性激活.受体特异性激活的PKC亚型靶向GIRK4亚基内不同的磷酸化位点，导致GIRK通道活性的差异调节，对GIRK电流具有促进作用或抑制作用。
