Voltage-Sensing Mechanism of Neuronal KCNQ Channels

Abstract

The voltage-gated potassium channel KCNQ2/KCNQ3 (KCNQ2/3) mediates the M-current (IKM) in neurons. The IKM channel contributes to maintaining the resting membrane potential and impedes repetitive neuronal firing, therefore playing a crucial role in controlling neuronal excitability. Mutations within the voltage-sensor (S4) of the IKM channel have been linked to Benign Familial Neonatal Seizures (BFNS) and epileptic encephalopathies, conditions that currently affect 50 million people worldwide. However, how KCNQ2/3 channels function remains largely unknown. Here, we use two-electrode voltage clamp and inside-out patch clamp in a Xenopus oocyte expression system to assess external and internal accessibility of S4 cysteine-substituted residues to the membrane-impermeable thiol reagent (MTSET) in homomeric and heteromeric KCNQ channels. We find that in homomeric KCNQ2 (and in KCNQ3) channels, extracellular MTSET labels residues N190C (Q218C) with a similar time course in both closed and open states. By contrast, extracellular MTSET modifies residues A193 to R201 in KCNQ2 (G219-S228 in KCNQ3) at depolarized potentials, but not at hyperpolarized potentials, suggesting that these residues likely lie buried in the membrane in the closed state. We also use voltage clamp fluorometry (VCF) to determine S4 movement in KCNQ2/3 channels. In homomeric KCNQ channels, the time course and voltage dependence of fluorescence F(V) and ionic current G(V) correlate, as if the S4 and the gate motion were directly coupled. Moreover, in KCNQ2, the F(V) follows the voltage dependence of the modification rate of residue A193C. Our data also indicate that the presence of KCNQ3 subunits affects the S4 and gate of KCNQ2 in heteromeric KCNQ2/3 channels.