ATP and PIP2 dependence of the magnesium-inhibited, TRPM7-like cation channel in cardiac myocytes

The Mg²⁺-inhibited cation (MIC) current (I_MIC) in cardiac myocytes biophysically resembles currents of heterologously expressed transient receptor potential (TRP) channels, particularly TRPM6 and TRPM7, known to be important in Mg²⁺ homeostasis. To understand the regulation of MIC channels in cardiac cells, we used the whole cell voltage-clamp technique to investigate the role of intracellular ATP in pig, rat, and guinea pig isolated ventricular myocytes. I_MIC, studied in the presence or absence of extracellular divalent cations, was sustained for 50 min after patch rupture in ATP-dialyzed cells, whereas in ATP-depleted cells I_MIC exhibited complete rundown. Equimolar substitution of internal ATP by its nonhydrolyzable analog adenosine 5'-(,-imido)triphosphate failed to prevent rundown. In ATP-depleted cells, inhibition of lipid phosphatases by fluoride + vanadate + pyrophosphate prevented I_MIC rundown. In contrast, under similar conditions neither the inhibition of protein phosphatases 1, 2A, 2B or of protein tyrosine phosphatase nor the activation of protein kinase A (forskolin, 20 µM) or protein kinase C (phorbol myristate acetate, 100 nM) could prevent rundown. In ATP-loaded cells, depletion of phosphatidylinositol 4,5-bisphosphate (PIP₂) by prevention of its resynthesis (10 µM wortmannin or 15 µM phenylarsine oxide) induced rundown of I_MIC. Finally, loading ATP-depleted cells with exogenous PIP₂ (10 µM) prevented rundown. These results suggest that PIP₂, likely generated by ATP-utilizing lipid kinases, is necessary for maintaining cardiac MIC channel activity. cation channels; hydrolysis; phosphoinositides; rundown