The voltage-gated potassium channel Kv7.2/7.3 mediates the M-current (I KM ) in neurons. The I KM 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) and/or the gating regions of the I KM channel have been linked to Benign Familial Neonatal Seizures (BFNS) and neonatal epileptic encephalopathies, conditions that currently affect 3.5 million people in the United States. A complete molecular understanding of how the Kv7.2/7.3 channel works has not been established. Here, we combine cysteine accessibility (patch and voltage clamp) with voltage clamp fluorometry to understand the molecular mechanisms of Kv7.2/7.3 channel gating. We substitute different S4 residues for a cysteine in Kv7.2 or Kv7.3 channels and assess the state-dependent intracellular and extracellular accessibility to the membrane-impermeable thiol reagent MTSET. We find that in homomeric Kv7.2 channels, membrane depolarization increases accessibility to the extracellular solution of amino acids located at the N-terminal portion of the S4 region. Interestingly, coexpression of Kv7.3 significantly increases the rate of extracellular cysteine modification of these S4 amino acids in Kv7.2, as if Kv7.3 speeds up the kinetics of S4 movement of heterotetrameric Kv7.2/7.3 channels. We also use voltage clamp fluorometry (VCF) to further understand how Kv7.3 affects the voltage sensor and the gate of Kv7.2 channels. VCF shows that in homomeric Kv7.2 channels the time courses and voltage dependency of fluorescence and ionic current correlate, as if the S4 and the gate motion were directly coupled. We also find that the voltage dependencies of the G(V) and F(V) correlate with that of the cysteine modification rates of membrane-buried S4 residues in Kv7.2 and these correlations change upon co-expression with Kv7.3 channel.
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