Type 2 melastatin-related transient receptor potential channel (TRPM2), a member of the melastatin-related TRP (transient receptor potential) subfamily is a Ca2+-permeable channel activated by hydrogen peroxide (H2O2). We have investigated the role of TRPM2 channels in mediating the H2O2-induced increase in the cytoplasmic free Ca2+ concentration ([Ca2+]i) in insulin-secreting cells. In fura-2 loaded INS-1E cells, a widely used model of β-cells, and in human β-cells, H2O2 increased [Ca2+]i, in the presence of 3 mM glucose, by inducing Ca2+ influx across the plasma membrane. H2O2-induced Ca2+ influx was not blocked by nimodipine, a blocker of the L-type voltage-gated Ca2+ channels nor by 2-aminoethoxydiphenyl borate, a blocker of several TRP channels and store-operated channels, but it was completely blocked by N-(p-amylcinnamoyl)anthranilic acid (ACA), a potent inhibitor of TRPM2. Adenosine diphosphate phosphate ribose, a specific activator of TRPM2 channel and H2O2, induced inward cation currents that were blocked by ACA. Western blot using antibodies directed to the epitopes on the N-terminal and on the C-terminal parts of TRPM2 identified the full length TRPM2 (TRPM2-L), and the C-terminally truncated TRPM2 (TRPM2-S) in human islets. We conclude that functional TRPM2 channels mediate H2O2-induced Ca2+ entry in β-cells, a process potently inhibited by ACA.

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BK channels, which are comprised of pore forming α and accessory β‐subunits, have been shown to eliminate intracellular K in response to hypo‐osmotic swelling. However, the role of the β subunits in this process has not been determined. We used HEK293 cells stably expressing BK‐α and examined a role for transiently expressed BK‐β4 (90% expression rate) to modulate the response of BK to cell swelling. Using patch clamp analysis, we determined NPo (cell attached, ‐Vp = −40 mV) and the % of observed channels per patch. The NPo and % channels per patch of BK‐α expressed alone in cells in isotonic (290 mOsm) solution was 0.71±0.05 (n=3) and 53%, respectively. This value was not significantly different from the NPo and % channels per patch for BK‐α in hypotonic (200 mOsm) solution (0.62±0.17, n=3 and 41%, respectively). The NPo and % channels per patch of BK‐α plus BK–β4 was significantly greater in hypotonic solution (1.32±0.15, n=3 and 76%, respectively) than in isotonic solution (0.70±0.15, n=8 and 41%, respectively). Compared to isotonic solution, when cells expressing BK‐α/β4 were in hypotonic solution, BK‐α exhibited more intense immuno‐staining at the plasma membrane and BK‐β4 was increased at the plasma membrane (western blot; OD = 0.20 in isotonic and 0.49 in hypotonic). These data suggest that the BK‐β4 subunit is necessary for trafficking of BK‐α and BK‐β4 to the plasma membrane in response to hypo‐osmotic stress.

Large conductance Ca2+‐sensitive K+ channels (BK) reside in the distal nephron where they secrete K+ when the rate of distal flow is increased. A recent study showed that aldosterone (aldo) stimulated BK‐mediated K+ secretion in an isolated mouse distal colon preparation. We used HEK293 cells, expressing BK channels, to determine whether aldo increases the number of BK channels in the plasma membrane by a non‐genomic mechanism. Immunocytochemistry, immunoblotting and RT‐PCR confirmed that HEK293 cells contain mineralocorticoid receptors (MR). Using single channel patch clamp analysis, we determined the 30 min. application of aldo (10 nM) on the NPo (total open time of all channels in a patch), and the percent of observed channels per patch. In cell attached patches (−Vp = −40 mV), aldo increased the NPo of BK from 0.71±0.05 to 1.84±0.41 (p=<0.05, n=3) and the percent of observed BK per patch from 58% to 71% (p<0.05, n=4). Treating cells with aldo plus spironolactone, a MR antagonist, reduced the number of detected BK channels to 50% (p<0.05, n=3). The aldo‐treated cells showed an increased quantity of cell surface biotinylated BK (OD, 1.46±0.19 in control and 3.87±0.76 with aldo, p<0.05, n=3) that was inhibited to OD, 1.32±0.35 by 5 μg/ml brefeldin‐A (p<0.05, n=3). These data suggest that aldosterone, via MR, increases the trafficking of expressed BK channels from the ER to the apical membrane of HEK293 cells. Grant support: NIH RO1‐DK49561 and AHA 0610059Z.