Awakening Loss-of-Function KATP Channel Mutants with an Engineered ‘Forced Gating’ Approach

Abstract

Regulation of inwardly-rectifying potassium channels by intracellular ligands couples membrane excitability to important signaling cascades and metabolic pathways. A significant barrier to understand coupling mechanisms between ligand binding and gating is that many functionally important channel motifs are highly sensitive to mutagenesis, resulting in a loss of function phenotype. We have developed a ‘forced gating’ approach that rescues function in electrically silent channel mutants, enabling characterization of channel motifs that are otherwise intractable to electrophysiological recording. This approach involves substitution of a glutamate in the hydrophobic Kir channel bundle crossing (F168E mutation in Kir6.2), generating channels that are pH sensitive and open upon alkalization, due to mutual repulsion of introduced negatively charged side chains in the channel gate. We have implemented this ‘forced gating’ approach in mutagenic scans of the Kir channel slide helix and G-loop, two motifs proposed to play a role in ligand dependent gating of Kir channels. Both motifs are also highly sensitive to mutagenesis, with alanine mutations causing nearly complete loss-of-function at 7/20 slide helix positions, and 8/13 G-loop positions. Without exception, expression of silent mutants on the Kir6.2[F168E] background permitted activation of functional channels in alkaline pH, and measurement of kinetics and potency of ATP inhibition. Our results highlight an essential ‘aspartate anchor’ (Kir6.2 residue D58) that bridges the slide helix and multiple interacting residues in the cytoplasmic domain. Disruption of the highly conserved ‘aspartate anchor’ uncouples the transmembrane and cytoplasmic domains, reducing ATP sensitivity of Kir6.2 far more than any other G-loop or slide helix mutants. These findings indicate a central role for the ‘aspartate anchor’ in coupling ligand binding to Kir gating, and also emphasize the potential general utility of this ‘forced gating’ method to study loss-of-function channel mutants.