Takezawa Ryuichi, Schmitz Carsten, Demeuse Philippe, Scharenberg Andrew M, Penner Reinhold, Fleig Andrea
Laboratory of Cell and Molecular Signaling, Center for Biomedical Research, The Queen's Medical Center and John A. Burns School of Medicine, University of Hawaii, Honolulu, HI 96813, USA.
Proc Natl Acad Sci U S A. 2004 Apr 20;101(16):6009-14. doi: 10.1073/pnas.0307565101. Epub 2004 Apr 6.
TRPM7 is a ubiquitously expressed and constitutively active divalent cation-selective ion channel, whose basal activity is regulated by intracellular levels of Mg(2+) and Mg.ATP. We have investigated receptor-mediated mechanisms that may actively regulate TRPM7 activity. We here report that TRPM7 currents are suppressed by intracellular GTPgammaS, suggesting the involvement of heterotrimeric G proteins. TRPM7 currents are also inhibited by stimulating endogenous muscarinic receptors, which is mediated by G(i) because the inhibitory effect is blunted by pertussis toxin. Conversely, stimulation of endogenous G(s)-coupled beta-adrenergic receptors potentiates TRPM7 currents, whereas G(q)-coupled thrombin receptors have little effect. Consistent with the involvement of G(s)/G(i) in controlling adenylyl cyclase activity, elevations of intracellular cAMP levels enhance TRPM7 activity and prevent receptor-mediated modulation of TRPM7 activity by muscarinic and adrenergic agonists. This cAMP-dependent effect requires the functional integrity of both protein kinase A (PKA) and the endogenous kinase domain of TRPM7 because cAMP-mediated effects are abolished when treating cells with the PKA inhibitors H89 or KT5720 as well as in cells expressing phosphotransferase-deficient TRPM7 constructs. These mutant channels are also much less susceptible to GTPgammaS-mediated inhibition, suggesting that the main regulatory effect occurs through G(i)- and G(s)-mediated changes in cAMP. Taken together, our results demonstrate that TRPM7 activity is up- and down-regulated through its endogenous kinase in a cAMP- and PKA-dependent manner.
瞬时受体电位阳离子通道亚家族M成员7（TRPM7）是一种广泛表达且组成性激活的二价阳离子选择性离子通道，其基础活性受细胞内镁离子（Mg²⁺）和Mg.ATP水平的调节。我们研究了可能主动调节TRPM7活性的受体介导机制。我们在此报告，细胞内GTPγS可抑制TRPM7电流，提示异源三聚体G蛋白参与其中。刺激内源性毒蕈碱受体也可抑制TRPM7电流，这是由G(i)介导的，因为百日咳毒素可减弱这种抑制作用。相反，刺激内源性G(s)偶联的β-肾上腺素能受体可增强TRPM7电流，而G(q)偶联的凝血酶受体几乎没有影响。与G(s)/G(i)参与控制腺苷酸环化酶活性一致，细胞内cAMP水平升高可增强TRPM7活性，并阻止毒蕈碱和肾上腺素能激动剂对TRPM7活性的受体介导调节。这种cAMP依赖性效应需要蛋白激酶A（PKA）和TRPM7内源性激酶结构域的功能完整性，因为用PKA抑制剂H89或KT5720处理细胞以及在表达磷酸转移酶缺陷型TRPM7构建体的细胞中，cAMP介导的效应会被消除。这些突变通道对GTPγS介导的抑制也更不敏感，表明主要调节作用是通过G(i)和G(s)介导的cAMP变化发生的。综上所述，我们的结果表明，TRPM7活性通过其内源性激酶以cAMP和PKA依赖的方式上调和下调。
