A single residue contributes to the difference between Kir4.1 and Kir1.1 channels in pH sensitivity, rectification and single channel conductance.

Kir1.1 and Kir4.1 channels may be involved in the maintenance of pH and K+ homeostasis in renal epithelial cells and CO2 chemoreception in brainstem neurons. To understand the molecular determinants for their characteristic differences, the structure-function relationship was studied using site-directed mutagenesis. According to previous studies, Glu158 in Kir4.1 is likely to be the major rectification controller. This was confirmed in both Kir1.1 and Kir4.1. Mutation of Gly210, the second potential rectification controller, to glutamate did not show any additional effect on the inward rectification. More interestingly, we found that Glu158 in Kir4.1 was also an important residue contributing to single channel conductance and pH sensitivity. The E158N Kir4.1 mutant had a unitary conductance of 35 pS and a midpoint pH for channel inhibition (pKa) value of 6.72, both of which were almost identical to those of the wild-type (WT) Kir1.1. Flickering channel activity was clearly seen in the E158N mutant at positive membrane potentials, which is typical in the WT Kir1.1 but absent in the WT Kir4.1. Reverse mutation in Kir1.1 (N171E) reduced the unitary conductance to 27 pS (23 pS in WT Kir4.1). However, the pH sensitivity of this mutant did not show a marked difference from the WT Kir1.1. Therefore, it is possible that a residue(s) in addition to Asn171 is also involved. Thus we studied several other residues in both M2 and H5 regions. We found that joint mutations of Val140 and Asn171 to residues seen in Kir4.1 greatly reduced the pH sensitivity (pKa 6. 08). The V140T mutation in Kir1.1 led to a unitary conductance of approximately 70 pS, and the G210E mutation in Kir4.1 caused a decrease in pH sensitivity of 0.4 pH units. These results indicate that the pore-forming sequences are targets for modulations of multiple channel-biophysical properties and demonstrate a site contributing to rectification, unitary conductance and proton sensitivity in these Kir channels.