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The affinity of the organic cation transporter rOCT1 is increased by protein kinase C-dependent phosphorylation
Thomas Mehrens1, Silke Lelleck1, Ibrahim Çetinkaya1
1Medizinische Poliklinik, Experimentelle Nephrologie, Westfälische Wilhelms-Universität Münster, Germany.
Abstract:
Members of the organic cation transporter (OCT) family are mainly expressed in kidney, liver, intestine, and brain. The regulation of the OCT type 1 from rat (rOCT1) stably transfected in HEK293 cells was examined using a fluorimetric technique, 1-[(3)H]methyl-4-phenylpyridinium uptake studies, and fast-whole-cell patch-clamp recordings. For the fluorescence measurements, the cation 4-(4-(dimethylamino)styryl)-N-methylpyridinium iodide (ASP(+)) was used as substrate. Uptake of ASP(+) via rOCT1 was electrogenic, and its inhibition by other organic cations was consistent with previously reported radioactive tracer flux measurements. The inhibitor quinine was not translocated by the organic cation transporter in contrast to tetraethylammonium. Stimulation of diacyl glycerol-dependent protein kinase C (PKC) by sn-1,2-dioctanoyl glycerol (1 microM) resulted in an increase in initial ASP(+) uptake rate by 216 +/- 28% (n = 29). The effect was completely antagonized by the PKC inhibitor tamoxifen (20 microM, n = 22). Forskolin (1 microM), which activates 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 microM, n = 12). Inhibition of tyrosine kinases by aminogenestein (10 microM) reduced ASP(+) uptake by 63 +/- 7% (n = 7), while genestein or tyrphostin AG1295 (each 10 microM) were without significant effects. Incubation of the cells with sn-1, 2-dioctanoyl glycerol (1 microM) increased the affinities of the transporter to tetraethylammonium, tetrapenthylammonium, and quinine by a factor of 58, 14.5, and 2.4, respectively. Western blot analysis revealed that rOCT1 protein was phosphorylated at a serine residue upon stimulation of PKC. In conclusion, it has been demonstrated that the organic cation transport by rOCT1 is stimulated by PKC, PKA, and endogenous tyrosine kinase activation. The PKC phosphorylates rOCT1 and leads to a conformational change at the substrate binding site.
Insights
Regulation of organic cation transporter 1 (OCT1) is stimulated by protein kinase C (PKC), protein kinase A (PKA), and tyrosine kinases. PKC-mediated phosphorylation of OCT1 alters its substrate binding site, enhancing organic cation transport.
Area of Science:
- Pharmacology
- Cell Biology
- Biochemistry
Background:
- Organic cation transporters (OCTs) are crucial for drug and metabolite transport, primarily in the kidney, liver, and brain.
- Understanding OCT regulation is vital for optimizing drug delivery and efficacy.
- Rat OCT type 1 (rOCT1) is a key transporter involved in these physiological processes.
Purpose of the Study:
- To investigate the regulatory mechanisms of rat OCT type 1 (rOCT1) activity.
- To determine the roles of protein kinase C (PKC), protein kinase A (PKA), and tyrosine kinases in rOCT1 regulation.
- To elucidate the molecular changes in rOCT1 upon stimulation by these kinases.
Main Methods:
- Utilized HEK293 cells stably expressing rOCT1.
- Employed fluorimetric assays with 4-(4-(dimethylamino)styryl)-N-methylpyridinium iodide (ASP(+)) as a substrate.
- Conducted 1-[(3)H]methyl-4-phenylpyridinium uptake studies and fast-whole-cell patch-clamp recordings.
- Applied specific kinase inhibitors (tamoxifen, KT5720) and activators (sn-1,2-dioctanoyl glycerol, forskolin).
- Performed Western blot analysis to detect protein phosphorylation.
Main Results:
- PKC stimulation increased ASP(+) uptake by 216%, an effect reversed by tamoxifen.
- PKA activation by forskolin enhanced ASP(+) uptake by 51%, inhibited by KT5720.
- Tyrosine kinase inhibition by aminogenestein reduced uptake by 63%.
- PKC activation increased rOCT1 affinity for several organic cations.
- Western blot confirmed rOCT1 phosphorylation at a serine residue upon PKC stimulation.
Conclusions:
- Organic cation transport via rOCT1 is significantly stimulated by PKC, PKA, and endogenous tyrosine kinases.
- PKC-mediated phosphorylation of rOCT1 induces a conformational change in the substrate binding site.
- These findings highlight key signaling pathways that modulate OCT1 function, impacting its physiological and pharmacological roles.