KirBac1.1: It's An Inward Rectifying Potassium Channel

Abstract

The structures of KirBac1.1 and KirBac3.1 have been used extensively to generate in silico homology models of eukaryotic Kir channels and to explore ion permeation, gating and drug-channel interactions by computational approaches. Functional studies of KirBac1.1 have been limited to 86Rb+ flux assays, but we have now succeeded in measuring voltage-clamp currents of KirBac1.1 reconstituted in giant liposomes. Using the patch-clamp technique, we recapitulate the results of 86Rb+ flux experiments, showing that KirBac1.1 currents are potassium-selective, blocked by barium, and inhibited by PIP2. These findings suggest that KirBac1.1 channels are functionally similar to eukaryotic Kir channels. Like the weak inward rectifiers Kir1.1 and Kir6.2, introduction of a negative charge at the “rectification controller” residue in the inner cavity of KirBac1.1 (I138D) confers strong inward rectification (i.e. steep voltage-dependent block by spermine). Steady state single channel currents of KirBac1.1 show multiple subconductance levels and gating modes in 150 mM symmetrical K+. However, similar to eukaryotic Kir channels, single channel amplitudes exhibit mild intrinsic inward rectification, with a maximum conductance at ∼56 pS (-100 mV), and open probability is higher at positive potentials. Multiple conductance states are still present in single channel currents of other permeant ions such as Rb+ and Tl+. However, similar to many K+ channels, including KcsA, Rb+ and Tl+ single channel currents show increased mean open time and decreased conductance. We find that KirBac1.1(T142C), equivalent to the Kir6.2 high P(o) mutant L164C, also has a high open probability and is effectively blocked by Cd+. These electrophysiological results confirm that KirBac1.1 is a bona fide inward rectifying K+ channel and a tractable model for study of the molecular basis of inward rectification, permeation and gating in eukaryotic Kir channels.