title: 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 (Ca2+) channel block by phenylalkylamines (PAA) was analyzed by designing mutant Ca2+ 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 Ca2+ 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 (I(Ba)) by (-)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. (-)D600 slowed I(Ba) recovery after maintained membrane depolarization (1-3 sec) to a comparable extent in all channel constructs. A drug-induced delay in the onset of I(Ba) recovery from inactivation suggests that PAAs promote the transition to a deep inactivated channel conformation. These findings indicate that apparent PAA sensitivity of Ca2+ channels is not only defined by drug interaction with its receptor site but also crucially dependent on intrinsic gating properties of the channel molecule. A molecular model for PAA-Ca2+ channel interaction that accounts for the relationship between drug induced inactivation and channel block by PAA is proposed.
authors.affiliation: Institut für Biochemische Pharmakologie, Innsbruck, Austria. Steffen.Hering@uibk.ac.at
authors.forename: S
authors.initials: S
authors.lastname: Hering
authors.forename: S
authors.initials: S
authors.lastname: Aczél
authors.forename: R L
authors.initials: RL
authors.lastname: Kraus
authors.forename: S
authors.initials: S
authors.lastname: Berjukow
authors.forename: J
authors.initials: J
authors.lastname: Striessnig
authors.forename: E N
authors.initials: EN
authors.lastname: Timin
mesh_terms: D000588:Amines / Q000494:pharmacology*; D000595:Amino Acid Sequence; D000818:Animals; D002121:Calcium Channel Blockers / Q000494:pharmacology*; D015220:Calcium Channels / Q000187:drug effects / Q000235:genetics / Q000502:physiology; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D017386:Sequence Homology, Amino Acid; D014981:Xenopus
chemical_list: D000588:Amines; D002121:Calcium Channel Blockers; D015220:Calcium Channels