Divalent ion dependent inactivation of L-type calcium channel when barium(2+) is the charge carrier and asymmetric contribution of the pore glutamates to charged DHP interaction

It is widely accepted that inactivation of L-type calcium channel is exclusively dependent on membrane potential when Ba2+ is the charge carrier. Evidence supporting the presence of Ba2+ dependent inactivation in L-type calcium channel has been subject to many criticisms because of endogenous Ca2+ and technical difficulties in measuring monovalent ion conduction through calcium channels. In this study, we used an alternative approach to solve this controversy. We altered Ba2+ conductivity of L-type calcium channel by neutralizing the glutamate residues in the pore region. We observed that the reduction of Ba2+ permeability correlated with the decrease in channel inactivation suggesting that inactivation is associated with Ba2+ entry. This hypothesis was further confirmed by the removal of inactivation upon Na+ substitution in the bath solution, as well as the parallel increase of inactivation with current amplitude. Internal perfusion of BAPTA also produced significant reduction in Ba2+ inactivation suggesting the presence of a divalent ion dependent inactivation process. We further demonstrated that divalent ion dependent inactivation was also present when Ca2+ or Sr2 is the charge carrier. The molecular determinants of divalent ion dependent inactivation are yet to be identified. Our results show that it is not mediated by calmodulin-IQ interaction or E462 in the I-II linker of L-type calcium channel.; We also investigated the effect of the glutamate mutations on the interaction between DHPs and L-type calcium channel. Mutations in the glutamate residues reduced the potency of neutral DHP while exerted location specific effect on charged DHP. In particular, the glutamate residue in domain IV stood out to be important in determining the difference in the potencies of charged and neural DHPs, confirming an asymmetric contribution of the pore glutamate residues.