Insulin secretion is initiated by activation of voltage-gated Ca2+ channels (VGCC) to trigger Ca2+-mediated insulin vesicle fusion with the β-cell plasma membrane. The firing of VGCC requires β-cell membrane depolarization, which is regulated by a balance of depolarizing and hyperpolarizing ionic currents. Here, we show that SWELL1 mediates a swell-activated, depolarizing chloride current (ICl,SWELL) in both murine and human β-cells. Hypotonic and glucose-stimulated β-cell swelling activates SWELL1-mediated ICl,SWELL and this contributes to membrane depolarization and activation of VGCC-dependent intracellular calcium signaling. SWELL1 depletion in MIN6 cells and islets significantly impairs glucose-stimulated insulin secretion. Tamoxifen-inducible β-cell-targeted Swell1 KO mice have normal fasting serum glucose and insulin levels but impaired glucose-stimulated insulin secretion and glucose tolerance; and this is further exacerbated in mild obesity. Our results reveal that β-cell SWELL1 modulates insulin secretion and systemic glycaemia by linking glucose-mediated β-cell swelling to membrane depolarization and activation of VGCC-triggered calcium signaling.

Transient receptor potential melastatin-7 (TRPM7) channels have been recently reported in human atrial fibroblasts and are believed to mediate fibrogenesis in human atrial fibrillation. The present study investigates whether TRPM7 channels are expressed in human atrial myocytes using whole-cell patch voltage-clamp, RT-PCR and Western blotting analysis. It was found that a gradually activated TRPM7-like current was recorded with a K+- and Mg2+-free pipette solution in human atrial myocytes. The current was enhanced by removing extracellular Ca2+ and Mg2+, and the current increase could be inhibited by Ni2+ or Ba2+. The TRPM7-like current was potentiated by acidic pH and inhibited by La3+ and 2-aminoethoxydiphenyl borate. In addition, Ca2+-activated TRPM4-like current was recorded in human atrial myocytes with the addition of the Ca2+ ionophore A23187 in bath solution. RT-PCR and Western immunoblot analysis revealed that in addition to TRPM4, TRPM7 channel current, mRNA and protein expression were evident in human atrial myocytes. Interestingly, TRPM7 channel protein, but not TRPM4 channel protein, was significantly increased in human atrial specimens from the patients with atrial fibrillation. Our results demonstrate for the first time that functional TRPM7 channels are present in human atrial myocytes, and the channel expression is upregulated in the atria with atrial fibrillation.

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Obesity is associated with a loss of insulin-sensitivity and systemic dysglycemia, resulting in Type 2 diabetes, however the molecular mechanisms underlying this association are unclear. Through adipocyte patch-clamp studies, we recently showed that SWELL1 is required for the Volume-Regulated Anion Current (VRAC) in adipocytes and that SWELL1-mediated VRAC is activated by both mechanical and pathophysiological adipocyte expansion. We also demonstrated that adipocyte SWELL1 is required for maintaining insulin signaling and glucose homeostasis, particularly in the setting of obesity. Here we show that SWELL1 protein expression is induced in subcutaneous fat, visceral fat and liver in the setting of obesity. Long- term AAV/rec2-shRNA mediated SWELL1 knock-down in both fat and liver are associated with increased weight gain, increased adiposity and exacerbated insulin resistance in mice raised on a high-fat diet. These data further support the notion that SWELL1 induction occurs in insulin- sensitive tissues (liver and adipose) in the setting of over-nutrition and contributes to improved systemic glycemia by supporting enhanced insulin-sensitivity.

The natural flavone acacetin has been demonstrated to inhibit transient outward potassium current (Ito) in human atrial myocytes. However, the molecular determinants of acacetin for blocking Ito are unknown. The present study was designed to investigate the properties and molecular determinants of this compound for blocking hKv4.3 channels (coding Ito) stably expressed in HEK 293 cells using the approaches of whole-cell patch voltage-clamp technique and mutagenesis. It was found that acacetin inhibited hKv4.3 current by binding to both the closed and open channels, and decreased the recovery from inactivation. The blockade of hKv4.3 channels by acacetin was use- and frequency-dependent, and IC50s of acacetin for inhibiting hKv4.3 were 7.9, 6.1, 3.9, and 3.2 µM, respectively, at 0.2, 0.5, 1, and 3.3 Hz. The mutagenesis study revealed that the hKv4.3 mutants T366A and T367A in the P-loop helix, and V392A, I395A and V399A in the S6-segment had a reduced channel blocking efficacy of acacetin (IC50, 44.5 µM for T366A, 25.8 µM for T367A, 17.6 µM for V392A, 16.2 µM for I395A, and 19.1 µM for V399A). These results demonstrate the novel information that acacetin may inhibit the closed channels and block the open state of the channels by binding to their P-loop filter helix and S6 domain. The use- and rate-dependent blocking of hKv4.3 by acacetin is likely beneficial for managing atrial fibrillation.

Transient receptor potential (TRP) channels are not well understood in human atrium, and the present study was therefore designed to investigate whether TRPC channels would mediate the nonselective cation current reported previously and are involved in the formation of store-operated Ca(2+) entry (SOCE) channels in human atrial myocytes using approaches of whole-cell patch voltage-clamp, RT-PCR, Western blotting, co-immunoprecipitation, and confocal scanning approaches, etc. We found that a nonselective cation current was recorded under K(+)-free conditions in human atrial myocytes, and the current was inhibited by the TRP channel blocker La(3+). Thapsigargin enhanced the current, and its effect was suppressed by La(3+) and prevented by pipette inclusion of anti-TRPC1 antibody. Endothlin-1 and angiotensin II enhanced the current that could be inhibited by La(3+). Gene and protein expression of TRPC1 channels were abundant in human atria. In addition, mRNA and protein of STIM1 and Orai1, components of SOCE channels, were abundantly expressed in human atria. Co-immunoprecipitation analysis demonstrated an interaction of TRPC1 with STIM1 and/or Orai1. Ca(2+) signaling mediated by SOCE channels was detected by a confocal microscopy technique. These results demonstrate the novel evidence that TRPC1 channels not only mediate the nonselective cation current, but also form SOCE channels in human atria as a component. TRPC1 channels can be activated by endothelin-1 or angiotensin II, which may be involved in the atrial electrical remodeling in patients with atrial fibrillation.

Our results demonstrate that human atrial I(to) and cloned hKv4.3 channels are modulated by EGFR kinase via phosphorylation of the Y136 residue and by Src-family kinases via phosphorylation of the Y108 residue; tyrosine phosphorylation of the channel may be involved in regulating cardiac electrophysiology.

BACKGROUND AND PURPOSEThe ultra-rapidly activating delayed rectifier K + current I Kur (encoded by K v 1.5 or KCNA5) plays an important role in human atrial repolarization. The present study investigates the regulation of this current by protein tyrosine kinases (PTKs). EXPERIMENTAL APPROACHWhole-cell patch voltage clamp technique and immunoprecipitation and Western blotting analysis were used to investigate whether the PTK inhibitors genistein, tyrphostin AG556 (AG556) and PP2 regulate human atrial I Kur and hKv1.5 channels stably expressed in HEK 293 cells. KEY RESULTSHuman atrial I Kur was decreased by genistein (a broad-spectrum PTK inhibitor) and AG556 (a highly selective EGFR TK inhibitor) in a concentration-dependent manner. Inhibition of I Kur induced by 30 μM genistein or 10 μM AG556 was significantly reversed by 1 mM orthovanadate (a protein tyrosine phosphatase inhibitor). Similar results were observed in HEK 293 cells stably expressing hK v 1.5 channels. On the other hand, the Src family kinase inhibitor PP2 (1 μM) slightly enhanced I Kur and hK v 1.5 current, and the current increase was also reversed by orthovanadate. Immunoprecipitation and Western blotting analysis showed that genistein, AG556, and PP2 decreased tyrosine phosphorylation of hK v 1.5 channels and that the decrease was countered by orthovanadate. CONCLUSION AND IMPLICATIONSThe PTK inhibitors genistein and AG556 decrease human atrial I Kur and cloned hK v 1.5 channels by inhibiting EGFR TK, whereas the Src kinase inhibitor PP2 increases I Kur and hK v 1.5 current. These results imply that EGFR TK and the soluble Src kinases may have opposite effects on human atrial I Kur .Abbreviations EGFR, EGF receptor; I Kur , ultra-rapidly activating delayed rectifier potassium current; PP2, 3-(4-chlorophenyl) 1-(1,1-dimethylethyl)-1H-pyrazolo [3,4-d] pyrimidin-4-amine; PTK, protein tyrosine kinase; TK, protein kinase; tyrphostin AG556, AG556