Molecular mechanism of use-dependent calcium channel block by phenylalkylamines: Role of inactivation

Abstract

The role of channel inactivation in the molecular mechanism of calcium (Ca channel block by PAA is, however, still not understood. High affinity determinants of the PAA-receptor site of L-type Ca 2ϩ channels have recently been identified on transmembrane segment IVS6 by mutating putative pore-orientated amino acids and subsequent screening of the resulting mutants for PAA-sensitivity (8) or by transferring the responsible L-type amino acids into the ␣ 1A subunit of P͞Q-type channels thereby introducing PAA sensitivity (10, 11). The resulting highly PAA sensitive triple ␣ 1A mutant AL25 (11) displayed significantly faster inactivation kinetics than the wild-type channel. Furthermore, an even more rapid inactivation and higher apparent PAA sensitivity was induced into ␣ 1A if an additional phenylalanine in position 1,805 of AL25 was mutated to the corresponding L-type methionine in IVS6 suggesting a close link between inactivation properties of the channel and use-dependent block by PAA.However, because mutations of the PAA-receptor site itself or of closely located amino acids may not only affect channel inactivation but also the interaction of PAAs with their binding site on transmembrane segment IVS6, no unequivocal conclusions about the role of inactivation in channel block could be drawn (11). To answer this question we designed Ca 2ϩ channels that inactivate at different rates by site directed mutagenesis of segment IIIS6.We report here that substitutions of putative pore-lining amino acids that are highly conserved between ␣ 1A , ␣ 1S , and ␣ 1C gradually reduce the rate of channel inactivation while also gradually reducing use-dependent block by (Ϫ)D600. Our data indicate that changes in Ca 2ϩ channel inactivation induced by site directed mutagenesis modulate use-dependent Ca 2ϩ channel block by PAA. This suggests that channel inactivation is an important determinant of use-dependent Ca 2ϩ channel block by these drugs.

Block of the lymphocyte K+ channel mKv1.3 by the phenylalkylamine verapamil: Kinetic aspects of block and disruption of accumulation of block by a single point mutation

“…Several possibilities could account for this faster inactivation rate with verapamil. (i) In analogy to D600 action on L-type Ca 2+ channels Hering et al (1997) proposed that for block of the L-type Ca 2+ channel by the PAA D600 the faster inactivation observed with D600, in combination with the fact that deactivation in the presence of D600 was impossible, was due to the drug keeping the channel in an open-blocked conformation (see Figure 1, scheme I). Therefore, in the case for D600 action on L-type Ca 2+ channels, inactivation rate did increase with an unchanged inactivation-rate constant because the probability increased to be in a state, from which inactivation can occur.…”

“…Therefore the time course for recovery from block even after short depolarizations is similar to the time course for recovery from inactivation observed only after long depolarizations. In addition, in Ltype Ca 2+ channels the interaction of the PAA with the inactivated channel is discussed controversely because anities for the states were found to be dependent on the PAA species (Johnson et al, 1996) and even on the permeating ions (Nawrath & Wegener, 1997;Hering et al, 1997). However, a trapping of the PAA in the inactivated mKv1.3 channel, as reported for L-type Ca 2+ channels (Hering et al, 1997), might be ruled out by our experiments, because in Figure 4 the I-state was thought to provide access to the verapamil.…”

“…In addition, in Ltype Ca 2+ channels the interaction of the PAA with the inactivated channel is discussed controversely because anities for the states were found to be dependent on the PAA species (Johnson et al, 1996) and even on the permeating ions (Nawrath & Wegener, 1997;Hering et al, 1997). However, a trapping of the PAA in the inactivated mKv1.3 channel, as reported for L-type Ca 2+ channels (Hering et al, 1997), might be ruled out by our experiments, because in Figure 4 the I-state was thought to provide access to the verapamil. Similarly, simulations of recovery from verapamil block resulted in a slower time course compared to recovery from inactivation, even if anities of verapamil to states O and I were identical, as was expected for the verapamil block of mKv1.3 according to scheme I (Figure 1).…”

“…Hering et al (1997) presented a model for PAA interaction that did not allow access to the binding site as long as the activation gate was closed. In addition, Johnson et al (1996) simulated the open block by PAA assuming a K d *20 ± 30 times higher for the closed state than for open and inactivated states.…”

“…Johnson et al (1996) proposed a model with weak block of closed channels and similar anities for activated and inactivated states. Also Hering et al (1997) pointed out the importance of the inactivation for PAA-block of L-type channels reaching a`deep' inactivated channel conformation responsible for accumulation of block.…”