Linkage analysis reveals allosteric coupling in Kir2.1 channels

Potassium-selective inward rectifier (Kir) channels are a class of membrane proteins necessary for maintaining stable resting membrane potentials, controlling excitability, and shaping the final repolarization of action potentials in excitable cells. In addition to the strong inward rectification of the ionic current caused by intracellular blockers, Kir2.1 channels possess "weak" inward rectification observed in inside-out patches after prolonged washout of intracellular blockers. The mechanisms underlying strong inward rectification have been attributed to voltage-dependent block by intracellular Mg²⁺ and polyamines; however, the mechanism responsible for weak rectification remains elusive. Hypotheses include weak voltage-dependent block and intrinsic voltage-dependent gating. Here, we performed a conductance Hill analysis of currents recorded with a double-ramp protocol to evaluate different mechanisms proposed for weak inward rectification of Kir2.1 channels. Linkage analysis in the form of a Hill plot revealed that the ramp currents could be best explained by allosteric coupling between a mildly voltage-dependent pore gate (gating charge ∼0.18 e_o) and a voltage sensor (gating charge ∼1.7 e_o). The proposed voltage sensor stabilized the closing of the pore gate (coupling factor ∼31). We anticipate that the use of linkage analysis will broaden understanding of functional coupling in ion channels and proteins in general.