The organic cation transporter 3 (OCT3) as molecular target of psychotropic drugs: transport characteristics and acute regulation of cloned murine OCT3

Abstract

The organic cation transporter 3 (OCT3) is a widely expressed transporter for endogenous and exogenous organic cations. Of particular interest is OCT3 expression and function in the brain, where it plays a role in serotonin clearance and influences mood and behavior. Protein kinase signaling mediates rapid modulation of cerebral processes, but little is known about acute regulation of OCT3 by protein kinases. Therefore, we cloned mouse OCT3 (mOCT3) and generated a human embryonic kidney cell line stably expressing the transporter to study transport characteristics, acute regulation by protein kinases, and interaction with psychotropic drugs. Uptake measurement was performed using the fluorescent cation 4-(4-(dimethylamino)styryl)-N-methylpyridinium iodide (ASP(+), 1 μM) as a substrate. The translational value of these findings was determined by comparing results obtained with cloned mouse and human OCT3. mOCT3-mediated transport is membrane potential dependent and pH independent. ASP(+) uptake by mOCT3 and human OCT3 (hOCT3) was efficiently inhibited by 1-methyl-4-phenylpyridinium, tetrapentylammonium (TPA(+)), corticosterone, serotonin, and histamine and by the drugs ketamine, fluoxetine, and diazepam. The half maximal inhibitory concentrations of mOCT3 and hOCT3 for TPA(+), serotonin, diazepam, and ketamine are significantly different. Diazepam is a non-transported inhibitor. Furthermore, the activities of mOCT3 and hOCT3 are acutely regulated by the p56 (lck) tyrosine kinase by decreasing their V max. Studies with freshly isolated renal proximal tubules from mOCT1/2(-/-) mice, in which mOCT3 is the only OCT present, confirmed this regulation pathway. Only the activity of hOCT3 is regulated by calmodulin. These findings suggest that even though many transport properties of mOCT3 and hOCT3 are similar, there are also species-specific aspects of OCT3 function.

Expression of xenobiotic transporters in the human renal proximal tubule cell line RPTEC/TERT1

“…The functionality and directionality of organic cation transport was proven by using the fluorescent cation 4-Di-1-ASP. 4-Di-1-ASP was previously shown to be a substrate of OCT2 and OCT3 using HEK293 cells transfected with the respective transporter (Biermann et al, 2006;Cetinkaya et al, 2003;Ciarimboli et al, 2004;Massmann et al, 2014). RPTEC/TERT1 cells took up the dye preferentially from the basolateral side and we could also demonstrate a net transport across the cells to the luminal compartment.…”

“…Several studies have shown ASP to be a substrate for different OCTs and MATE isoforms (Biermann et al, 2006;Cetinkaya et al, 2003;Ciarimboli et al, 2005Ciarimboli et al, , 2004Maekawa et al, 2007;Massmann et al, 2014). RPTEC/TERT1 cells exhibited significantly higher uptake of 4-Di-1-ASP if applied from the basolateral than apical side (p = 0.008, Fig.…”

What do drug transporters really do?

“…For example, mice lacking both SLC22A1 and SLC22A2 exhibit defective secretion of organic cations by the kidney 109 . SLC22A3 (OCT3) seems to have a key role in neurotransmitter uptake in the nervous system 10,110,111 . OCTNs, which include SLC22A4 and SLC22A5 (also known as OCTN1 and OCTN2, respectively), are highly expressed in muscle and in many other tissues, playing a crucial part in metabolism involving carnitine 10 (TABLE 3).…”

“…Within various cells and structures of the CNS, there are also many SLC and ABC transporters, including those related to the drug transporters discussed here. SLC22A3 (OCT3), for instance, is expressed in the brain, where it is involved in neurotransmitter uptake, and it may be a therapeutic target for certain neuro-psychiatric disorders 111 . Thus, it will be very interesting to trace, in detail, the movement of substrates through the blood–brain barrier–CNS–choroid plexus axis.…”

The pharmacokinetics of ketamine following intramuscular injection to F344 rats

“…Ketamine and its active metabolite norketamine undergo metabolism via hepatic Cytochrome P450s primarily via CYP2B6 and CYP3A4, which may be impacted with the co‐administration of other pharmaceuticals, such as rifampicin, itraconazole, and ticolpidine, though none of the SOC agents are predicted to have effects on ketamine metabolism . In addition to metabolic clearance, ketamine is a substrate of the rat and human uptake organic cation ion transporters (OCT1, OCT2, OCT3) that mediate biliary and renal elimination of organic bases . It is known that 2‐PAM is a substrate in rats and mice of OCT1 and OCT2, atropine is a potent inhibitor of OCT1 and OCT3, while midazolam is an OCT1 inhibitor .…”