Tetanus toxin abolishes exocytosis of ROMK1 induced by inhibition of protein tyrosine kinase

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 PTK on ROMK1 trafficking in HEK-293 cells transfected with c-Src and green fluorescent protein (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 microscopic imaging showed that herbimycin A decreased the intracellular intensity of GFP-ROMK1. The biotin-labeling technique demonstrated that the inhibition of c-Src increased 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 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 cells treated with tetanus toxin. The incubation of cells in a medium containing tetanus toxin abolished the herbimycin A-induced increase in the number of surface ROMK1. In contrast, inhibition of c-Src still increased the numbers of surface ROMK1 in cells treated with 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.