Expression of a potassium channel -reporter gene fusion protein in cultured hippocampal neurons

Voltage-gated potassium channels are an important detenrtinan: of the maximum firing rate of neurons. Overexpression of potassium channels could provide an important therapy for neurological disorders characterized by increased neuronal excitability such as focal epilepsies. The shaker-type potassium channels such as Kv1.4 are thought to be important in determining the maximum firing rate of neurons. We have produced a viral vector capable of expressing a functonal potassium channel-fusion protein in cultured hippocampal neurons. The small protein, green fluorescent protein is used as a fluorescent reporter to verify expression of the gene using fluorescent microscopy. The HSV-Amplicon system was used to produce a mammalian expression vector package within a herpes virus capsid. The gene for Kv1.4-EGFP fusion protein was a gift of J. Levitan at the University of Pittsburgh and has been previously shown to produce a functional iron channel in non-neuronal cultured cell lines. We used standard techniques of molecular biology to insert this gene into a plasmid containing sequences enabling replication in a specially engineered cell line (E-5) and subsequent packaging as a viral particle. Fluorescent microscopy of the infected E-5 cell line suggests that the gene product is being packaged in the Golgi apparatus and transported to the cell membrane. The packaged viral vector has been successful in transfecting cultured hippocampal neurons as determined by fluorescence microscopy. Ongoing studies of these transfected neurons are being analyzed using single cell patch clamp electiophysiology. It is our hypothesis that tnmsfected neurons will have an altered electrophy siological property, particularly a reduced maximum firing rate. If this is the case, this viral vector may prove successful in treatment of focal epilepsies. Further studies will involve in vivo injection of this viral vector in a rodent model of temporal lobe epilepsy.