Calmodulin Inhibitors Directly Block Potassium Channels & Ion Channels And Their Role In Cell Proliferation Of Preadipocytes

Part I The Calmodulin inhibitors directly blocks potassium channels(hERG, hKvl.5 and Kir 2.1) stably expressed in HEK 293 cellsBackground and purpose:N-(6-Aminohexyl)-5-chloro-l-naphthalenesulfonamide (W-7) is a well-known calmodulin inhibitor used as a tool to study calmodulin regulation of intracellular Ca2+ signaling-related process. The present study was to determine whether W-7 would inhibit human ether a-go-go-related gene (hERG or Kv11.1) potassium channel, hKv1.5 channel or hKir2.1 channel expressed in HEK 293 cells. The effect of W-7 on WT hERG channel and three hERG mutants were proformed to identify the molecular determinants of W-7 block of hERG channels.We also investigate the effect of W-13, another camlodulin inhibitor on hERG, hKv1.5 or hKir2.1 potassium channels. The hERG channel current, hKvl.5 channel current or hKir2.1 channel current was recorded with a whole-cell patch-clamp technique.Results:It was found that the calmodulin inhibitor W-7 blocked hERG, hKv1.5, and hKir2.1 channels. W-7 decreased the hERG current (IhERG) in a concentration-dependent manner (IC50:3.5μM) and voltage-dependent manner, and the inhibition was more significant at depolarization potentials between+10 and+60 mV. The stedy-state activation and inactivation curve of hERG current were negatively shifted and the inactivation of hERG channel was accerlated by W-7. Pipette inclusion of W-7 induced a slow reduction of IhERG.tail, and the inhibitory effect was weaker than bath application of W-7. We included 500 nM calmodulin in an EGTA-free pipette solution (to ensure intracellular free Ca2+ at physiological level) and no significant change in the current was observed in dialysized cells with calmodulin. The hERG mutations in the S6 region Y652A and F656V, and in the pore helix S631A, had the IC50S of 5.5μM,9.8μM, and 25.4μM, respectively. In addition, the compound inhibited hKvl.5 and hKir2.1 channels with IC50S of 6.5 and 13.4μM respectively. W-13 also suppressed these K+ currents; however, the inhibitory effect was remarkably reduced, compared with W-7 with IC50s of 19.8,75.54 and 43.75μM.Conclusion and implication:These results indicate that the calmodulin inhibitor W-7 exerts a direct channel blocking effect on hERG, hKv1.5 and hKir2.1 channels stably expressed in HEK 293 cells. The caution should be taken in the interpretation of calmodulin regulation of ion channels with W-7. Part II Ion channels and their role in cell proliferation of 3T3-L1 preadipocytesBackground and Purpose:Mouse 3T3-L1 peradipocytes are widely used for metabolic study; however, cellular physiology (e.g. functional ion channel expression) is not fully understood. The present study was to investigate ion channel expression and functional role of them in regulating cell proliferation using whole cell patch voltage clamp technique, RT-PCR, Western blot, and cell proliferation assay in undifferentiated 3T3-L1 preadipocytes.Results:We found that three types of ionic currents were present in 3T3-L1 preadi pocytes, including a Ca2+-activated K+ current (IKCa) in 39% cells, an inwardly-rectifying K+ current (IKir) in 15% cells, and a chloride current (ICl) only in 8% cells under isotonic conditions. Interestingly, ICl was observed in all cells with hypotonic (0.8T) insult, suggesting that it is a volume-sensitive ICl (ICl.vol).IKir was inhibited by Ba2+, and IKCa was inhibited by the intermediate conductance IKCa channel blocker clotrimazole. ICl was reduced by the chloride channel blockers DIDS. RT-PCR revealed significant expression of mRNAs:KCa3.1 for IKCa, Kir2.1 for IKir, and Clcn3 for ICl.vol.Proteins of these channels were detected using western blot analysis. Proliferation assay demonstrated that blockade of IKCa with clotrimazole or ICl.vol with DIDS inhibited cell proliferation in a concentration-dependent manner. Knockdown of KCa3.1 or Clcn3 with specific short interference (si)RNAs significantly reduced IKCa or ICl.vol mRNA and channel protein and produced a remarkable suppression of cell proliferation (by 12.76.29±2.7% and 11.29±2.5%, respectively, P<0.05 vs control). Flowcytometry analysis showed that clotrimazole (3μM) and DIDS (200μM) accumulated the cells at G0/G1 phase (from control 50.93±1.64% to 55.29±3.13% for clotrimazole, P<0.01; to 70.21±4.09% for DIDS, P< 0.01). Similar results were obtained with specific siRNAs targeting to KCa3.1 and Clcn3. Specific siRNA increased the cell number of G0/G1 phase(from control siRNA 44.38±4.5% to 48.56±4.89% for KCa3.1 siRNA, P<0.05; from control siRNA 53.64±0.68% to 60.54±0.82% for Clcn3 siRNA, P<0.01).Conclusions:These results demonstrate the first information that three types of functional ion channel currents, including intermediate-conductance IKCa, ICl.vol, and IKir, are heterogeneously present in 3T3-L1 preadipocytes. IKCa and ICl.vol participate in the regulation of cell proliferation.