Inhibitory Effects Of A TRPM4 Inhibitor 9-phenanthrol On Electrical Activity Of Pancreatic β-cells

Objectives:The goal of this study was to validate TRPM4 channel as a potential target in pancreaticβ-cells for regulation of insulin secretion.To carry out this study,we aimed to establish a method for evaluating the effect of small molecule compounds on pancreaticβ-cells,and also to examine the effect of TRPM4 channel inhibition on electrical activities of pancreaticβ-cells.Methods:Western blot method was used to examine TRPM4 expression in the pancreaticβ-cells;Knockdown efficiency of TRPM4 m RNA by q RT-PCR analysis;Whole-cell patch clamp was used to record the TRPM4 current of pancreaticβ-cells and the inhibitory effect of the TRPM4 inhibitor 9-phenanthrol;Whole-cell current clamp was used to record action potentials of pancreaticβ-cells.Results:TRPM4 channel protein is expressed in the pancreaticβ-cells line INS-1.TRPM4 sh RNA knockdown efficiency in pancreaticβ-cells is 71.0±11.0%;Whole-cell patch clamp recordings confirmed that 9-phenanthrol inhibited TRPM4 channels expressed in HEK-293 cells,and it can also inhibit endogenous TRPM4 current in pancreaticβ-cells.Whole-cell current clamp recordings further confirmed that9-phenanthrol inhibited action potentials in dose-dependent manner with an IC₅₀ value of15.0±7.9μM,the inhibition rates of 9-phenanthrol to tolubutamide-stimulated pancreaticβ-cells in different concentrations of 10 n M,100 n M,1μM,10μM,30μM,50μM,100μM and 1 m M are16.4±6.2%,31.6±3.8%,25.9±9.7%,57.0±15.2%,69.9±19.8%,80.0±8.0%,89.6±13.4%and 84.8±12.0%,respectively.The inhibition rates of 1μM,10μM and 50μM 9-phenanthrol on action potentials induced by electrical-stimulation of pancreaticβ-cells are 16.6±6.4%,65.1±7.2%and 92.0±5.3%,respectively.To further validate the inhibition effect of 9-phenanthrol on electrical activities of pancreaticβ-cells,we analyzed the effect of 9-phenanthrol on the action potential peak and action potential amplitude of pancreaticβ-cells.The results showed that 10μM 9-phenanthrol reduced the action potential peak from 17.5±5.6 m V to 8.5±2.9 m V,a decrease of 8.5±3.1 m V,thus by 50.1±7.6%;The action potential amplitude from 58.7±8.1 m V to 43.7±11.0m V,a decrease of 15.4±5.0 m V,thus by 26.2±9.7%.Conclusions:1.TRPM4 channel protein is functionally expressed in pancreaticβ-cells line INS-1.2.TRPM4 channel inhibitor 9-phenanthrol can inhibit the endogenous TRPM4 current in pancreaticβ-cells,indicating that 9-phenanthrol provides essential tools for further understanding of TRPM4 channel physiology and pharmacology in pancreaticβ-cells.3.The action potentials induced by tolbutamide-and electrical-stimulation of pancreatic β-cells are dose-dependently inhibited by TRPM4 channel inhibitor 9-phenanthrol,indicating that 9-phenanthrol can inhibit electrical activity of pancreaticβ-cells.4.Inhibition of TRPM4 by 9-phenanthrol can reduce the action potential peak and shorten the action potential amplitude of pancreaticβ-cells.