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1 Department of Pharmacology, New York Medical College, Valhalla, NY, USA
* To whom correspondence should be addressed. E-mail: wenhui_wang{at}nymc.edu.
We used confocal microscopy, patch clamp and biotin labeling techniques to examine the role of soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) proteins in mediating the effect of inhibition of protein tyrosine kinase (PTK) on the ROMK1 trafficking in HEK293 cells transfected with c-Src and GFP-ROMK1. Inhibition of c-Src with herbimycin A significantly decreased the tyrosine phosphorylation level of ROMK1. Patch clamp studies demonstrated that addition of herbimycin A increased the activity of ROMK1 in cell-attached patches. Confocal microscope imaging showed that herbimycin A increased intracellular intensity of GFP-ROMK1. The biotin labeling technique demonstrated that the inhibition of c-Src increased the surface ROMK1 by 110%. In contrast, inhibition of c-Src did not increase the K channel number in HEK cells transfected with R1Y337A, a ROMK1 mutant in which tyrosine residue 337 was mutated to alanine. This suggests that tyrosine residue 337 is essential for the herbimycin A-induced increase in the surface ROMK1 channels. To determine whether SNARE proteins are involved in mediating exocytosis of ROMK1 induced by the inhibition of c-Src, we examined the effect of herbimycin A on ROMK1 trafficking in the cells treated with tetanus toxin. The incubation of cells in a media containing tetanus toxin abolished the herbimycin A-induced increase in surface numbers of ROMK1. In contrast, inhibition of c-Src still increased the surface numbers of ROMK1 in cells treated with the boiled tetanus toxin. We conclude that tyrosine dephosphorylation enhances the exocytosis of ROMK1 and that SNARE proteins are required for exocytosis induced by inhibition of PTK.
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