TY - JOUR
T1 - Speeding the recovery from ultraslow inactivation of voltage-gated Na + channels by metal ion binding to the selectivity filter
T2 - A foot-on-the-door?
AU - Szendroedi, Julia
AU - Sandtner, Walter
AU - Zarrabi, Touran
AU - Zebedin, Eva
AU - Hilber, Karlheinz
AU - Dudley, Samuel C.
AU - Fozzard, Harry A.
AU - Todt, Hannes
N1 - Funding Information:
This work was supported by grants P13961-B05 and P17509-B11 provided by the Austrian Science Fund.
PY - 2007/12/15
Y1 - 2007/12/15
N2 - 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 (IUS), by ligand binding to the outer vestibule in voltage-gated Na+ channels. Blocking the outer vestibule by a mutant μ-conotoxin GIIIA substantially accelerated recovery from IUS. 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 Cd 2+, the time constant of recovery from IUS 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 the development of IUS (-20 mV). These data could be explained by a kinetic model in which Cd2+ binds with high affinity to a slow inactivated state (IS), which is transiently occupied during recovery from I US. A total of 50 μM Cd2+ produced an ∼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 IUS, suggesting that IUS is associated with a conformational change of the internal vestibule of the channel. We propose a molecular model in which slow inactivation (IS) occurs by a closure of the outer vestibule, whereas IUS arises from a constriction of the internal vestibule produced by a widening of the selectivity filter region. Binding of Cd2+ to C1237 promotes the closure of the selectivity filter region, thereby hastening recovery from IUS. Thus, Cd2+ ions may act like a foot-on-the-door, kicking the IS gate to close.
AB - 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 (IUS), by ligand binding to the outer vestibule in voltage-gated Na+ channels. Blocking the outer vestibule by a mutant μ-conotoxin GIIIA substantially accelerated recovery from IUS. 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 Cd 2+, the time constant of recovery from IUS 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 the development of IUS (-20 mV). These data could be explained by a kinetic model in which Cd2+ binds with high affinity to a slow inactivated state (IS), which is transiently occupied during recovery from I US. A total of 50 μM Cd2+ produced an ∼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 IUS, suggesting that IUS is associated with a conformational change of the internal vestibule of the channel. We propose a molecular model in which slow inactivation (IS) occurs by a closure of the outer vestibule, whereas IUS arises from a constriction of the internal vestibule produced by a widening of the selectivity filter region. Binding of Cd2+ to C1237 promotes the closure of the selectivity filter region, thereby hastening recovery from IUS. Thus, Cd2+ ions may act like a foot-on-the-door, kicking the IS gate to close.
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U2 - 10.1529/biophysj.107.104794
DO - 10.1529/biophysj.107.104794
M3 - Article
C2 - 17720727
AN - SCOPUS:37349113858
SN - 0006-3495
VL - 93
SP - 4209
EP - 4224
JO - Biophysical journal
JF - Biophysical journal
IS - 12
ER -