UBC Theses and Dissertations
Regulation of slow inactivation in Kv1.5 by the turret Eduljee, Cyrus
In Kv channels, slow, P/C-type, inactivation is thought to eliminate ionic current via a concerted structural rearrangement of the outer pore mouth. However, little is known regarding the contributions of other pore regions to the regulation of slow inactivation. Our experiments now indicate that the turret region of Kv1.5 can be an important determinant of slow inactivation. Application of extracellular protons, or divalent cations such as Zn²⁺ or Ni²⁺, was found to reduce the peak current amplitude, and with protons, also increase the rate of depolarization-induced inactivation. These effects were alleviated by the substitution of residue H463 in the turret with a glutamine, indicating that the interaction of this turret residue with extracellular ligands facilitated the inhibition of current. Based on macroscopic, unitary and gating current analyses, we have attributed the effects of protonation and divalent cations to an enhancement of depolarization-induced and closed-state inactivation. These findings provide strong evidence that the turret region contributes to the regulation of slow inactivation in Kv1.5. To determine which properties of position 463 were important in modulating inactivation, various side chain substitutions of this position were examined and several were found to mimic the effects of proton or divalent cation binding. However, the properties of the substituted side chains were not well correlated with the effects on channel function, and it appeared that the turret and inactivation gate did not interact strictly via an electrostatic interaction, as suggested by others. A SCAM analysis was subsequently conducted by substituting cysteine residues into each turret position to determine which regions of the turret were important for regulating slow inactivation. Modification of substituted cysteine residues in the distal turret by MTS reagents produced an inhibition that could also be related to an enhancement of slow inactivation and demonstrated that position 463 was contained within a distinct locus of turret residues that could influence slow inactivation. These results indicate that in Kv1.5 a specific region of the turret modulates the state of the inactivation gate and provide constraints on the mechanisms of interaction between the turret and inactivation gate.