Electrophysiological characterisation of the human N-type Ca2+ channel III: pH-dependent inhibition by a synthetic macrocyclic polyamine.

ABSTRACT

The effects of a novel synthetic macrocyclic polyamine (LY310315) were investigated on recombinant human N-type Ca2+ channels stabley expressed in HEK293 cells. LY310315 proved to be a potent and reversible N-type Ca2+ channel antagonist. Inhibition by this compound was dose-dependent with an IC50 of approximately 0.4 microM at pH 7.35. LY310315 blocked very rapidly at all concentrations tested. Upon washout, recovery of the Ca2+ current developed with a time constant of approximately 30 s. Use-dependence in the development of block indicated that voltage-dependent transitions in the channel protein were required to permit significant inhibition. Application of > 100 times the IC50 dose of LY310315 to the interior of the cell produced no detectable Ca2+ current inhibition. LY310315 had no effects on the kinetics of channel activation or deactivation but did slightly slow the rate of macroscopic inactivation observed during a 300 ms test depolarisation. In the presence of LY310315 the activation curve was significantly shallower. This resulted in a shift in the activation midpoint voltage to a more depolarised levels. LY310315-induced inhibition of human N-type channels was strongly dependent on the extracellular pH, with increased potency seen upon extracellular acidification. Although most effective against N-type Ca2+ channels, LY310315 was also found to inhibit both P-type and L-type Ca2+ channels. LY310315 proved to be a weak blocker of Na+ currents, but produced approximately 50% of the K+ currents of AtT20 cells at a concentration of 0.5 microM.