The affinity of the organic cation transporter rOCT1 is increased by protein kinase C-dependent phosphorylation

Members of the organic cation transporter (OCT) family are mainly expressed in kidney, liver, intestine, and brain. The regulation of the OCT type 1 f rom rat (rOCT1) stably transfected in HEK293 cells was examined using a flu orimetric technique, 1-[H-3]methyl-4-phenylpyridinium uptake studies, and f ast-whole-cell patch-clamp recordings. For the fluorescence measurements, t he cation 4-(4-(dimethylamino)styryl)-N-methylpyridinium iodide (ASP(+)) wa s used as substrate. Uptake of ASP(+) via rOCT1 was electrogenic, and its i nhibition by other organic cations was consistent with previously reported radioactive tracer flux measurements. The inhibitor quinine was not translo cated by the organic cation transporter in contrast to tetraethylammonium. Stimulation of diacyl glycerol-dependent protein kinase C (PKC) by sn-1,2-d ioctanoyl glycerol (1 mu M) resulted in an increase in initial ASP(+) uptak e rate by 216 +/- 28% (n = 29). The effect was completely antagonized by th e PKC inhibitor tamoxifen (20 mu M, n = 22). Forskolin (1 mu M), which acti vates adenylate cyclase and thereby protein kinase A (PKA), stimulated the initial rate of ASP(+) accumulation by 51 +/- 6% (n = 19), This effect was inhibited by the specific PKA inhibitor KT5720 (1 mu M, n = 12). Inhibition of tyrosine kinases by aminogenestein (10 mu M) reduced ASP(+) uptake by 6 3 +/- 7% (n = 7), while genestein or tyrphostin AG1295 (each 10 mu M) were without significant effects. incubation of the cells with sn-1,2-dioctanoyl glycerol (1 mu M) increased the affinities of the transporter to tetraethy lammonium, tetrapenthylammonium, and quinine by a factor of 58, 14.5, and 2 .4, respectively. Western blot analysis revealed that rOCT1 protein was pho sphorylated at a serine residue upon stimulation of PKC. In conclusion, it has been demonstrated that the organic cation transport by rOCT1 is stimula ted by PKC, PKA, and endogenous tyrosine kinase activation. The PKC phospho rylates rOCT1 and leads to a conformational change at the substrate binding site.