Single-Cell Na+ Flux Assay for Measurement of TRPM7 Channel Activity

Abstract

TRPM7 is a Ca2+/Mg2+ permeable ion channel highly expressed in immune cells. Like many other TRP superfamily members, it conducts physiologically important monovalent cations Na+ and K+. Na+ influx through most TRP channels has not been measured directly, however. We set out to quantify Na+ influx through TRPM7 channels by using fluorescence microscopy and SBFI ratiometric Na+ indicator dye in HEK293 cells overexpressing mTRPM7. Kv1.3 was co-expressed in order to make the membrane more hyperpolarized. In the presence of divalent cations TRPM7 current is strongly outwardly rectifying, and in their absence becomes semi-ohmic with a large inward component. In TRPM7-expressing HEK cells, removal of external divalents resulted in consistently steep increases in intracellular Na+ signal detected by SBFI. Spermine blocks monovalent TRPM7 currents with IC50 of 2 µM. Accordingly, Na+ flux was reduced by ∼50% when 3 µM spermine was included in the divalent-free buffer. Higher spermine concentrations (10 and 30 µM) resulted in progressively stronger inhibition of Na+ flux. Similar results for free Mg2+,which dose-dependently reduced Na+ flux at 31, 62 and 100 µM, suggested that Na+ flux occurs through TRPM7 channels. The phenolic compound carvacrol, previously reported to be a blocker of TRPM7 channels, potently suppressed the Na+ signal at 150 µM but not at 30 µM. In TRPM7-overexpressing cells, switching from no divalents to a 100 mM Ca2+ containing buffer caused Ca2+ elevation, measured by Fura-2, but it was not significantly different from control untransfected HEK cells. Cytoplasmic alkalinization, which activates TRPM7 channels, was effective in increasing the Na+ flux through these channels. Na+ flux could also be measured in Jurkat T cells, which express TRPM7 and Kv1.3 channels endogenously. The presented Na+ flux assay will be useful for measurement of TRPM7 channel activity in intact cells.