Molecular mechanism of use-dependent calcium channel block by phenylalkylamines: Role of inactivation (Ca21 channelyphenylalkylaminesystate-dependent blockyantiarrhythmic action)

The role of channel inactivation in the mo- lecular mechanism of calcium (Ca 21 ) channel block by phe- nylalkylamines (PAA) was analyzed by designing mutant Ca 21 channels that carry the high affinity determinants of the PAA receptor site (Hockerman, G. H., Johnson, B. D., Scheuer, T., and Catterall, W. A. (1995) J. Biol. Chem. 270, 22119-22122) but inactivate at different rates. Use-dependent block by PAAs was studied after expressing the mutant Ca 21 channels in Xenopus oocytes. Substitution of single putative pore- orientated amino acids in segment IIIS6 by alanine (F-1499-A, F-1500-A, F-1510-A, I-1514-A, and F-1515-A) gradually slowed channel inactivation and simultaneously reduced inhibition of barium currents (IBa )b y (2)D600 upon depolarization by 100 ms steps at 0.1 Hz. This apparent reduction in drug sensitivity was only evident if test pulses were applied at a low frequency of 0.1 Hz and almost disappeared at the frequency of 1 Hz. (2)D600 slowed IBa recovery after maintained membrane depolarization (1-3 sec) to a comparable extent in all channel constructs. A drug-induced delay in the onset of IBa recovery from inactivation suggests that PAAs promote the transition to a deep inactivated channel conformation. These findings indicate that apparent PAA sensitivity of Ca 21 channels is not