Calmodulin-associated post-translational regulation of rat organic cation transporter 2 in the kidney is gender dependent

Abstract

In this work, regulation of organic cation transporter type 2 from rat (rOCT2) stably transfected in HEK293 cells was investigated by microfluorimetry with 4-(4-(dimethylamino)styryl)-N-methylpyridinium as substrate. The transport mediated by rOCT2 was specifically stimulated by PKA, phosphatidylinositol-3-kinase, p56(lck) tyrosine kinase, mitogen-extracellular-signal-regulated-kinase-1/2, calmodulin (CaM), and CaM-kinase-II. The regulatory pattern of rOCT2 differs markedly quantitatively and qualitatively from that of other OCT isoforms. Only CaM-dependent upregulation is conserved throughout the OCT family. For this reason, CaM regulation of rOCT2 was also investigated in isolated S3-segments (known to express only rOCT2) of male and female rat proximal tubules. Inhibition of CaM by calmidazolium significantly decreased rOCT2 activity (-49.0 +/- 13.6%, n = 4) in male but not female (9.0 +/- 13.0%, n = 4) rats. Real-time PCR and Western blot investigations of CaM expression in rat kidneys showed that male animals have significantly higher CaM expression. This is the first study describing post-translational gender-dependent rOCT2 regulation.

Xenobiotic, Bile Acid, and Cholesterol Transporters: Function and Regulation

“…In contrast to what has been reported for rat Oct1, protein kinase A activation reduces transport mediated by human OCT1 and OCT2 in HEK293 cells (Cetinkaya et al, 2003;Ciarimboli et al, 2004). Although the exact mechanism underlying this species difference is not entirely clear, it has been proposed that protein kinase A activation differentially affects the association of rat and human Oct2/OCT2 with additional proteins (such as calmodulin) that can influence substrate affinity (Wilde et al, 2009).…”

“…Transport in rat Oct1-and Oct2-HEK293-overexpressing cells is stimulated by pharmacological activation of protein kinases A TRANSPORTER FUNCTION AND REGULATION and C (Mehrens et al, 2000;Wilde et al, 2009). Specifically, protein kinase C phosphorylates rat Oct1 protein at certain serine residues (Mehrens et al, 2000;Ciarimboli et al, 2005a).…”

LAPTM4A interacts with hOCT2 and regulates its endocytotic recruitment

“…Organic cation uptake by hOCT2-expressing cells was measured using a fluorescent plate reader (Infinity M200; Tecan, Crailsheim, Germany), according to the method described by us previously [22]. As substrate for OCTs, the fluorescent organic cation 4-[4-(dimethylamino)styryl]-Nmethylpyridinium (ASP ? )…”

“…This effect seems to be mediated by stimulation of androgen responsive stimulated promoter activity [16]. Rapid regulation of OCTs can be mediated by several kinases in an isoform-specific fashion [17][18][19][20][21], whereas the regulation via calmodulin (CaM) is conserved through the OCT family but gender dependent [22].…”

Properties and regulation of organic cation transport in freshly isolated mouse proximal tubules analyzed with a fluorescence reader-based method

“…The aim of this study cannot be to obtain information on specific OCT isoforms, which can only be obtained in cell systems overexpressing single OCT isoforms, or to see if specific inhibitors of individual OCT isoforms are available. To obtain such functional data from the ex vivo situation of isolated mouse proximal tubules, the microtiter plate fluorescence reader-based analysis method, which we developed for the analysis of OCT transport in cell cultures [43], was adapted to analyze OC transport properties of proximal tubules isolated from mouse kidneys. This method allows increasing the number of data which can be obtained from one animal as compared to the microfluorimetrical analysis method applied to individual tubules that we had used before [33,43].…”