An optical study of potassium ion channel inactivation: Modulation by balls and beta subunits

K+ channels undergo a series of steps in the process of inactivation. The process involves fast inactivation due to block by an internal “ball” of either the alpha or beta subunit, followed by one or more slow inactivation components involving rearrangements of the selectivity filter and outer mouth of the pore. Block by the ball accelerates slow inactivation, an effect that has been attributed to an indirect effect of K+ occupancy, where inactivation is faster when binding sites in the selectivity filter are vacant. Co-assembly with the beta subunit also accelerates slow inactivation by a separate mechanism, even in the absence of the beta ball. Such modulation is likely due to either an allosteric effect of beta subunit binding, or possible beta enzymatic related actions. We have used voltage clamp fluorometry (VCF) to examine the effect of K+ ions, alpha and beta balls, and the ball-deleted beta subunit on both inactivation of the ionic current, and on a structural rearrangement which is tightly associated with slow inactivation. Using varying external K+ concentrations and domain-specific cysteine mutations we address whether K+ occupancy is sufficient to account for the coupling between ball and slow inactivation, or if an allosteric mechanism is additionally involved. In addition, we describe the steps of the inactivation process that are modulated by the alpha ball, the beta ball and by the beta subunit without the ball, and explore the possible enzymatic function of the beta subunit using mutants in its cofactor and substrate binding sites and catalytic sites.