Using Voltage Clamp Fluorometry to Track Voltage Sensor Movement in a Mammalian Kv1.2 Channel in the Presence of the Kvbeta1.2 Subunit

Abstract

The N-termini of Kv1 α-subunits bind co-translationally with cytosolic Kvβ-subunits, with 1:1 symmetry. Kvβ-subunits of three distinct families have been found in neural and cardiac tissue, and members of the Kvβ1 family confer fast inactivation and slowed deactivation when co-assembled with Kv1 α-subunits. These effects may be due to a blocking action by the Kvβ1 N-terminus. Kvβ1 subunits also cause an apparent hyperpolarizing shift in the activation curve of Kv1 channels, which may be a consequence of block by the Kvβ1 N-terminus, due to premature saturation of deactivating tail currents, or alternately may be due to an allosteric interaction between Kvβ1 and Kv1 α-subunits, modifying voltage sensor movement. Here, we use voltage clamp fluorometry to directly track the movement of the Kv1.2 voltage sensor in the absence or presence of the Kvβ1.2 subunit, or an N-terminally-truncated Kvβ1.2 subunit which does not produce fast inactivation. While Kvβ1.2 led to a spike-and-decay current waveform and a hyperpolarized shift in ionic current activation, the voltage dependence of ON gating charge movements were unaffected. Kvβ1.2 also slowed Kv1.2 fluorescence and current deactivation, implying that the return of the voltage sensor to its pre-activation position followed the closing of the activation gate. These findings suggest that the hyperpolarizing shift in channel activation is a consequence of pore block by the Kvβ1.2 N-terminus, and not an allosteric effect on the Kv1.2 voltage sensor, and that block prevents both closure of the activation gate and the return of the S4 helix upon repolarization.