Regulation of the novel Mg2+ transporter transient receptor potential melastatin 7 (TRPM7) cation channel by bradykinin in vascular smooth muscle cells

Background Transient receptor potential melastatin 7 (TRPM7) channels have been identified in the vasculature. However, their regulation and function remain unclear. Methods Here, we tested the hypothesis that bradykinin and its second messenger cAMP upregulate TRPM7, which stimulates activation of annexin-1 (TRPM7 substrate) and increases transmembrane Mg2+ transport and vascular smooth muscle cell (VSMC) migration. Human and rat VSMCs were studied. TRPM7 phosphorylation was assessed by immunoprecipitation: immunoblotting using antiphospho-serine/threonine and anti-TRPM7 antibodies. [Mg2+](i) was measured by mag-fura-2. TRPM7 was downregulated by small interfering RNA and 2-aminoethoxydiphenyl borate. Annexin-1 activity was assessed by cytosol-to-membrane translocation. Cell migration and invasion, functional responses to bradykinin, were assessed in transwell chambers. Results Bradykinin increased expression of TRPM7 and annexin-1. TRPM7 was rapidly (5 min) phosphorylated on serine/threonine but not on tyrosine residues by bradykinin. [Mg2+](i) was increased in bradykinin-stimulated cells (0.53 versus 0.68 mmol/l, basal versus bradykinin, P<0.01). Annexin-1 activation was increased by bradykinin and inhibited by 2-aminoethoxydiphenyl borate. Although Hoe 140 (B-2 receptor antagonist), U-73122 (phospholipase C inhibitor), 4-amino-5-(4-chlorophenyl)-7-(t-butyl) pyrazolo[3,4-d]pyrimidine (c-Src inhibitor) and chelerythrine (protein kinase C inhibitor) blocked bradykinin actions, dibutyryl-c-AMP was without effect. In small interfering RNA-transfected and in 2-aminoethoxydiphenyl borate-treated cells, bradykinin-induced Mg2+ influx and VSMC migration were reduced. Conclusion Our results demonstrate that bradykinin regulates TRPM7 and its downstream target annexin-1 through phospholipase C-dependent, protein kinase C-dependent and c-Src-dependent and cAMP-independent pathways; effects are mediated through bradykinin type 2 receptor; and bradykinin regulates VSMC [Mg2+](i) and migration through TRPM7. These data identify TRPM7/annexin-1/Mg2+ as a novel pathway in bradykinin signaling. J Hypertens 27: 155-166 (C) 2009 Wolters Kluwer Health vertical bar Lippincott Williams & Wilkins.