Regulation of human organic cation transporter hOCT2 by PKA, PI3K, and calmodulin-dependent kinases

Abstract

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+ (EC50 = 2.7 μM), tetraethylammonium (EC50 = 35 μM), cimetidine (EC50 = 36 μM), or quinine (EC50 = 6.7 μM). 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), Ca2+/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 EC50 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.

Contributions of phosphorylation to regulation of OCTN2 uptake of carnitine are minimal in BeWo cells

“…Regulation of OCTN2 by phosphorylation/dephosphorylation mechanisms was expected due to previous reports regarding such effects on OCT1, OCT2, and OCT3 [5][6][7] and the presence of putative phosphorylation sites on the OCTN2 protein (see Figure 1). Nevertheless, the results gathered while testing this hypothesis suggest that the contributions of phosphorylation in the regulation of OCTN2 are minimal in this model of the placental barrier.…”

“…Ciarimboli et al report that OCT1 activity decreased upon protein kinase A (PKA) activation and upon inhibition of calmodulin, calmodulin-dependent kinase II, and p56 lck tyrosine kinase [5]. This same group also determined that OCT2 uptake is inhibited by phosphatidylinositol 3-kinase (PI3K) and PKA and activated by calmodulin-dependent signaling [6]. OCT3 regulation by phosphorylation/dephosphorylation mechanisms was investigated by Martel et al, who observed reduced MPP + uptake in the presence of Ca 2+ /calmodulin pathway inhibitors, phosphodiesterase inhibitors, and an alkaline phosphatase (ALP) inhibitor [7].…”

Individual PKC-Phosphorylation Sites in Organic Cation Transporter 1 Determine Substrate Selectivity and Transport Regulation

“…This can explain why different isoforms are differently regulated. hOCT1 (11), hOCT2 (10), and hOCT3 (12), for example, are not activated by PKC stimulation, but by various other regulatory pathways such as PKA or Ca 2ϩ -calmodulin (10,12). In this study, we attempted to determine which of the described five putative PKC sites (23) is responsible for PKC-mediated regulation of rOCT1 and whether transporter phosphorylation changes substrate affinities.…”

“…PKC has been shown to directly phosphorylate rOCT1, leading to a conformational change at the substrate binding site (8): PKCmediated phosphorylation increased the apparent affinities of rOCT1 for various substrates. However, in HEK293 cells stably expressing the hOCT2, PKC seemed not to stimulate the transporter, whereas its activity was reduced by PI3K and PKA activation and was stimulated by an endogenously active calmodulin-dependent signaling pathway (10). Similar results have been obtained with HEK293 cells stably expressing hOCT1 (11) or the human extraneuronal monoamine transporter (or hOCT3) (12), which were not affected by PKC activation but again were inhibited by PKA and stimulated by an endogenously active calmodulin-dependent signaling path-way.…”

Most CML patients who have a suboptimal response to imatinib have low OCT-1 activity: higher doses of imatinib may overcome the negative impact of low OCT-1 activity

“…13,14 Post-transcriptional regulation of OCT-1 by phosphorylation status 15 and such compounds as protein kinase A (PKA), Src-like p56, and CaM also have been demonstrated. [16][17][18] Both quantitative and qualitative changes in OCT-1 may have an impact on imatinib uptake. We hypothesized that a functional assay of imatinib uptake into patient cells may provide the most clinically predictive assay of OCT-1 function.…”