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

Abstract

The Mg 2ϩ -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 Mg 2ϩ 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 Ն50 min after patch rupture in ATP-dialyzed cells, whereas in ATPdepleted cells I MIC exhibited complete rundown. Equimolar substitution of internal ATP by its nonhydrolyzable analog adenosine 5Ј-(␤,␥-imido)triphosphate failed to prevent rundown. In ATP-depleted cells, inhibition of lipid phosphatases by fluoride ϩ vanadate ϩ 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 M) or protein kinase C (phorbol myristate acetate, 100 nM) could prevent rundown. In ATP-loaded cells, depletion of phosphatidylinositol 4,5-bisphosphate (PIP 2) by prevention of its resynthesis (10 M wortmannin or 15 M phenylarsine oxide) induced rundown of I MIC. Finally, loading ATP-depleted cells with exogenous PIP 2 (10 M) prevented rundown. These results suggest that PIP 2, likely generated by ATP-utilizing lipid kinases, is necessary for maintaining cardiac MIC channel activity. cation channels; hydrolysis; phosphoinositides; rundown SEVERAL ION CHANNELS PARTICIPATE in ion fluxes that are critical for cardiac function. Apart from the well-characterized cationselective voltage-gated, ligand-gated, and background ion channels as well as the pacemaker channels, the nature of other cardiac cation-permeable channels remains unclear. In cardiac and other tissues, transient receptor potential (TRP) proteins are molecular candidates for cation-permeable channels (36). The TRP proteins are members of three main (TRPC, TRPV, and TRPM) and some more distantly related subfamilies. They form channels gated by stimuli such as voltage, chemical, or physical factors and are involved in both physiological and pathological functions (36).We previously described (10, 31, 48) a Mg 2ϩ -inhibited cation (MIC) channel in cardiac myocytes that displays pharmacological and pore properties resembling those of TRPM6 and TRPM7 channels. Native TRPM7-like currents such as cardiac MIC are also referred to as magnesium-nucleotideregulated metal ion (MagNuM) currents (32). TRPM6 and TRPM7 are closely related members of the TRPM subfamily, which form channels with very similar properties (29, 45). TRPM6 is mainly expressed in the kidney and small intestines, and its mutation causes hypomagnesemia with secondary hypocalcemia (5, 41, 46). TRPM7 is widely expressed with high levels in the kidney and heart. It is essential for cell viability and prolife...

The Charge Properties of Phospholipid Nanodiscs

“…5). Other studies also have shown that PIP 2 Nanodiscs bind Ca 2þ and Mg 2þ differently (61), but they conflict in that some report that PIP 2 lipids bind Ca 2þ more strongly and others report that PIP 2 lipids bind Mg 2þ more strongly (61,62). For example, Ca 2þ has been observed to induce stronger aggregation behavior than Mg 2þ (57,61); however, we did not observe Ca 2þ -induced aggregation with PIP 2 Nanodiscs.…”

Isoform-specific Inhibition of TRPC4 Channel by Phosphatidylinositol 4,5-Bisphosphate

“…Kir, KCNQ1, and P/Q-type Ca 2ϩ channels) regulated by PIP 2 (41). Although PIP 2 and related phosphoinositides exert positive effects on the activity of many TRP channels, such as TRPV5 (32), TRPM4 (35,44), TRPM5 (45), TRPM7 (46,47), TRPM8 (33,34), and TRPC6 (7,48), it is inhibitory to few others, such as dTRPL (3) and TRPV1 (42, 49). Ironically, the inhibitory effect of PIP 2 on TRPV1 has been challenged by more recent studies showing the activating effect of PIP 2 on TRPV1 in inside-out patches (50,51).…”

C-terminal Di-arginine Motif of Cdc42 Protein Is Essential for Binding to Phosphatidylinositol 4,5-Bisphosphate-containing Membranes and Inducing Cellular Transformation

“…One involved examining the effects of phenylarsine oxide on Cdc42-mediated transformation, given the reports that it inhibits PIP 2 production in cells by blocking the actions of phosphatidylinositol 4-phosphate kinase (27,28). Supplemental Fig.…”