Regulation of human organic cation transporter hOCT2 by PKA, PI3K, and calmodulin-dependent kinases.
Properties and regulation of the human organic cation (OC) transporter type 2 (hOCT2) expressed in HEK-293 cells were extensively characterized using the fluorescent OC 4-[4-(dimethylamino)styryl]-N-methylpyridinium (ASP(+)). ASP(+) uptake was electrogenic and inhibited by TPA(+) (EC(50) = 2.7 microM), tetraethylammonium (EC(50) = 35 microM), cimetidine (EC(50) = 36 microM), or quinine (EC(50) = 6.7 microM). Stimulation with carbachol or ATP decreased initial uptake by 44 +/- 3 (n = 14) and 34 +/- 4% (n = 21), respectively, independently of PKC but dependent on phosphatidylinositol 3-kinase (PI3K). PKA stimulation decreased uptake by 18 +/- 4% (n = 40). Inhibition of calmodulin (CaM), Ca(2+)/CaM-dependent kinase II, or myosin light chain kinase decreased uptake by 63 +/- 2 (n = 15), 40 +/- 4 (n = 30), and 31 +/- 4% (n = 16), respectively. Inhibition of CaM resulted in a significant change in the EC(50) value for the inhibition of ASP(+) uptake by tetraethylammonium. In conclusion, we demonstrate that the hOCT2 is inhibited by PI3K and PKA and activated by a CaM-dependent signaling pathway, probably via a change in substrate affinity.
Cetinkaya
Ibrahim
I
Medizinische Klinik und Poliklinik D, Experimentelle Nephrologie, and Klinik und Poliklinik für Kinderheilkunde, Universitätsklinikum Münster, D-48149 Münster, Germany.
Ciarimboli
Giuliano
G
Yalçinkaya
Gülay
G
Mehrens
Thomas
T
Velic
Ana
A
Hirsch
Jochen R
JR
Gorboulev
Valentin
V
Koepsell
Hermann
H
Schlatter
Eberhard
E
D001667:Binding, Competitive; D002118:Calcium / Q000502:physiology; D017871:Calcium-Calmodulin-Dependent Protein Kinases / Q000502:physiology*; D002460:Cell Line; D017868:Cyclic AMP-Dependent Protein Kinases / Q000502:physiology*; D004563:Electrochemistry; D004789:Enzyme Activation / Q000502:physiology; D006801:Humans; D027701:Organic Cation Transport Proteins / Q000037:antagonists & inhibitors / Q000378:metabolism*; D000075102:Organic Cation Transporter 2; D019869:Phosphatidylinositol 3-Kinases / Q000502:physiology*; D011493:Protein Kinase C / Q000378:metabolism; D011726:Pyridinium Compounds / Q000493:pharmacokinetics; D013379:Substrate Specificity
C032315:4-(4-dimethylaminostyryl)-1-methylpyridinium; D027701:Organic Cation Transport Proteins; D000075102:Organic Cation Transporter 2; D011726:Pyridinium Compounds; C491013:SLC22A2 protein, human; D019869:Phosphatidylinositol 3-Kinases; D017868:Cyclic AMP-Dependent Protein Kinases; D011493:Protein Kinase C; D017871:Calcium-Calmodulin-Dependent Protein Kinases; D002118:Calcium