Biophysical And Pharmacological Profiling Of Multiple NaV Subtypes On QPatch HT

Abstract

The voltage dependent sodium channel is responsible for the upstroke and directed propagation of action potentials in nerve and muscle cells, and is therefore a central ion channel in excitable tissues. The implication of voltage gated sodium channels in pain mediation, and diseases such as epilepsy and cardiac arrythmia has made them very important targets for drug discovery. Nine functional mammalian isoforms have been discovered so far with different functional and pharmacological properties. In our study, eight subtypes of the voltage gated sodium channel were tested in parallel on the automated patch clamp system QPatch HT. The new clone screening feature developed for QPatch 16 and QPatch HT allows running up to eight different cell lines (clones or subtypes) at the same time, thus ensuring that the exact same conditions (temperature, Ringer's, pH etc.) are applied for each of the cell lines tested. Nav1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7 and 1.8 were tested together in a range of experiments on QPatch HT. All but Nav1.2 and Nav1.8 were co-expressed with the β1 subunit. Three types of experiments were designed to explore 1) TTX sensitivity, 2) IV-relationship for activation and inactivation, and 3) recovery from inactivation, for the entire panel of Nav channel subtypes in a single experiment. It was shown that QPatch experiments using the cell clone screening feature together with the QPatch Assay Software data analysis package, enables the experimenter to obtain IC50 values for TTX, IV-relationships and time constants for recovery from inactivation which are very similar to manual patch clamp data, for all Nav subtypes, thus successfully distinguishing one subtype from another.