Mechanism of TRPM7 Channel Inhibition by 2-Aminoethyl Diphenyl Borinate (2-APB)

Abstract

2-APB is a widely used chemical compound in ion channel research. It inhibits numerous channels that include inositol 1,4,5-trisphosphate receptors, store-operated calcium channels, connexins and TRP family cation channels TRPC3, TRPC5, TRPC6, TRPM2, TRPM3 and TRPM7. Native and overexpressed TRPM7 channels are inhibited by 2-APB with IC50-s in the 70-170 μM range. A characteristic of TRPM7 channels is their inhibition by intracellular Mg2+ and acidic pH. using patch-clamp electrophysiology, we recorded native TRPM7 channel activity in Jurkat T lymphocytes and tested 2-APB at 10-300 μM for its ability to inhibit TRPM7 currents. When internal HEPES buffer concentration was low (1 mM), 100-300 μM 2-APB inhibited 60-70% of TRPM7 current with a slow time course. This inhibition was voltage-independent and reversible. Increasing the pH buffering capacity of internal solution to 140 mM HEPES abolished the inhibitory 2-APB effect. Simply making the internal recording solution alkaline, greatly diminished 2-APB inhibition. using single-cell pHi imaging with the fluorescent pH indicator BCECF, we found that at concentrations of 100 μM and higher, 2-APB potently acidifies the cytoplasm. A similar 2-APB-induced acidification was also observed in HEK293 cells, often used for TRPM7 channel overexpression. In contrast to 2-APB, its analog 2,2-diphenyltetrahydrofuran (DPTHF) did not produce cytosolic acidification, when applied at concentrations of 150-300 μM. We also found that Kv1.3 channels expressed in Jurkat T cells are modulated by 2-APB: the apparent voltage dependence of these channels was reversibly altered by the compound. Interestingly, this 2-APB effect on Kv1.3 channels did not depend on cytosolic pH. In summary, these experiments suggest that inhibition of TRPM7 channels by 2-APB is not direct but rather, can be explained by cytoplasmic acidification and resulting channel inhibition.