Aims: 

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. 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.

Methods: 

rNav1.4 channels were heterologously expressed in Xenopus laevis oocytes and examined by means of the two electrode voltage clamp technique.

Results: 

Binding of Cd²⁺(30 mM) to a cysteine engineered to the selectivity filter (K1237C) substantially accelerated recovery from I_US. (time constant of recovery = 145 ± 10 s at control; 2.5 ± 3 s during superfusion with Cd²⁺; P < 0.001). Cd²⁺only accelerated recovery from I_US at -120 mV but did not affect development of I_US at -20 mV. On the other hand, I_US was also modified by binding of the local anaesthetic lidocaine to the internal vestibule. These effects could be simulated 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. In support of this model, 50 mM Cd²⁺produced a ~8 mV hyperpolarizing shift of the steady-state inactivation curve of I_S.

Conclusion: 

We propose a molecular model in which binding of Cd²⁺to C1237 promotes the closure of the selectivity filter region (=I_S gate), thereby hastening recovery from I_US. Thus, Cd²⁺ ions may act like a foot-on-the-door, kicking the I_S gate to close.

Support: Austrian Science Fund P17509-B11.