Speeding the Recovery from Ultra-Slow Inactivation of Voltage-Gated Na⁺ Channels by Metal Ion Binding to the Selectivity Filter: A Foot-on-the-Door?

¹ Center for Biomolecular Medicine and Pharmacology, Medical University of
² Center for Biomolecular Medicine and Pharmacology, Institute of Pharmacology, Medical University of
³ Division of Cardiology, Emory University, Atlanta, GA
⁴ University of Chicago, Chicago, IL, USA

^* To whom correspondence should be addressed. E-mail: hannes.todt{at}meduniwien.ac.at.

Submitted on January 23, 2007
Revised on March 5, 2007
Accepted on 10 August 2007

	Abstract

Slow inactivated states in voltage-gated ion channels can be modulated by binding of 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, ultra-slow inactivation (I_US) by ligand-binding to the outer vestibule in voltage-gated Na⁺ channels. Blocking of the outer vestibule by a mutant µ-conotoxin GIIIA substantially accelerated recovery from I_US. A similar effect was observed if Cd²⁺ was bound to a cysteine engineered to the selectivity filter (K1237C). In K1237C channels, exposed to 30 µM Cd²⁺, 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 IUS was only accelerated if Cd²⁺ was added to the bath solution during recovery (V = -120 mV) from IUS, but not when the channels were selectively exposed to Cd²⁺ during development of I_US (-20 mV). These data could be explained by a kinetic model in which Cd²⁺ binds with high affinity to a slow inactivated state (I_S) which is transiently occupied during recovery from I_US. 50 µM Cd²⁺ produced a ~8 mV hyperpolarizing shift of the steady-state inactivation curve of IS, 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 Cd²⁺ to the C1237 promotes the closure of the selectivity filter region, thereby hastening recovery from I_US. Thus, Cd²⁺ ions may act like a foot-on-the-door, kicking the I_S gate to close.

Key Words: Cadmium, Na channel, recovery from inactivation, selectivity filter, slow inactivation, µ