OCT3 in the rat sympathetic nervous system

The advent of molecular biology has led to the cloning of the three transporter proteins that sequester the neurotransmitter noradrenaline (NA) in the sympathetic nervous system: the transmembrane NA transporter (NAT), the vesicular monoamine transporter (VMAT), and most recently the organic cation transporter 3 (OCT3), better known in the classical literature as uptake2. Uptake2 has always been considered a non-neuronal clearance mechanism for NA, but recent evidence based on in situ hybridization suggests a neuronal localization for OCT3 in the retina and as well as in distinct nerve cells in the central nervous system. To date, however, the gene product has not been identified in neurons, neither has it been attempted to attribute a function to the OCT3 in nerve cells. In a preparatory series of experiments with RT-PCR we were able to show mRNA of OCT3 (but not OCT1 and OCT2) in intact ganglia, conventional cell cultures, and neuron-enriched cell cultures of the rat superior cervical ganglion. Furthermore, single-cell RT-PCR of neurons identified by phase contrast microscopy suggests a neuronal localization of the transporter. Guanidine, a substrate of OCT3, induced inwardly-directed currents when applied to individual neurons, and in superfusion experiments the release of [3H] from cultures preloaded with [3H]MPP+. These observations are sufficiently encouraging to check on the gene product and its potential function in nerve cells. We will first verify our initial results and extend it by Northern blotting. We will then generate OCT3- specific antibodies in order to identify the protein by means of Western blots and learn about its tissue and subcellular distribution with immunohistochemistry. Potential functions will be studies by uptake experiments with established substrates of OCT3/uptake2 such as isoprenaline and MPP+ in the presence and absence of inhibitors; by release experiments of preloaded substrate on grounds of trans-stimulation; and by patch clamp recordings that indicate net charge transfer across the membrane and permit insight into the voltage and ion dependence of the transport. Upon the successful completion of this project we expect not only solid confirmation for the presence of OCT3, but also an indication for a potential role of the transporter in nerve cells.