title: Speeding the recovery from ultraslow inactivation of voltage-gated Na+ channels by metal ion binding to the selectivity filter: a foot-on-the-door?
abstract: Slow inactivated states in voltage-gated ion channels can be modulated by binding molecules both to the outside and to the inside of the pore. Thus, external K(+) inhibits C-type inactivation in Shaker K(+) channels by a "foot-in-the-door" mechanism. Here, we explore the modulation of a very long-lived inactivated state, ultraslow inactivation (I(US)), by ligand binding to the outer vestibule in voltage-gated Na(+) channels. Blocking the outer vestibule by a mutant mu-conotoxin GIIIA substantially accelerated recovery from I(US). A similar effect was observed if Cd(2+) was bound to a cysteine engineered to the selectivity filter (K1237C). In K1237C channels, exposed to 30 microM Cd(2+), the time constant of recovery from I(US) was decreased from 145.0 +/- 10.2 s to 32.5 +/- 3.3 s (P < 0.001). Recovery from I(US) was only accelerated if Cd(2+) was added to the bath solution during recovery (V = -120 mV) from I(US), but not when the channels were selectively exposed to Cd(2+) during the development of I(US) (-20 mV). These data could be explained by a kinetic model in which Cd(2+) binds with high affinity to a slow inactivated state (I(S)), which is transiently occupied during recovery from I(US). A total of 50 microM Cd(2+) produced an approximately 8 mV hyperpolarizing shift of the steady-state inactivation curve of I(S), supporting this kinetic model. Binding of lidocaine to the internal vestibule significantly reduced the number of channels entering I(US), suggesting that I(US) is associated with a conformational change of the internal vestibule of the channel. We propose a molecular model in which slow inactivation (I(S)) occurs by a closure of the outer vestibule, whereas I(US) arises from a constriction of the internal vestibule produced by a widening of the selectivity filter region. Binding of Cd(2+) to C1237 promotes the closure of the selectivity filter region, thereby hastening recovery from I(US). Thus, Cd(2+) ions may act like a foot-on-the-door, kicking the I(S) gate to close.
authors.affiliation: Center for Biomolecular Medicine and Pharmacology, Institute of Pharmacology, Medical University of Vienna, Vienna, Austria.
authors.forename: Julia
authors.initials: J
authors.lastname: Szendroedi
authors.forename: Walter
authors.initials: W
authors.lastname: Sandtner
authors.forename: Touran
authors.initials: T
authors.lastname: Zarrabi
authors.forename: Eva
authors.initials: E
authors.lastname: Zebedin
authors.forename: Karlheinz
authors.initials: K
authors.lastname: Hilber
authors.forename: Samuel C
authors.initials: SC
authors.lastname: Dudley
authors.forename: Harry A
authors.initials: HA
authors.lastname: Fozzard
authors.forename: Hannes
authors.initials: H
authors.lastname: Todt
mesh_terms: D000818:Animals; D001665:Binding Sites; D002104:Cadmium / Q000378:metabolism*; D002478:Cells, Cultured; D015640:Ion Channel Gating / Q000502:physiology*; D007700:Kinetics; D008564:Membrane Potentials / Q000502:physiology*; D009865:Oocytes / Q000502:physiology*; D011485:Protein Binding; D012964:Sodium / Q000378:metabolism*; D015222:Sodium Channels / Q000502:physiology*; D014982:Xenopus laevis
chemical_list: D015222:Sodium Channels; D002104:Cadmium; D012964:Sodium