Gating of inwardly rectifying potassium (Kir) channels by intracellular pH is important for many aspects of channel physiology and pathophysiology. In mammalian Kir1.1 (ROMK) an intra-subunit H-bond between a lysine in TM1 (K80) and a backbone carbonyl group in TM2 (A177) at the helix bundle crossing is thought to control the kinetics of the gating motion of the TM helices in response to pH and PIP2. However, the exact pH gating mechanism is not fully understood and further studies are needed to determine which other residues are important for Kir1.1 pH sensitivity. In this study, we exploited a yeast genetic complementation assay to screen for gain of function (GoF) mutations in Kir1.1 which might alter pH gating, given that the intracellular pH in yeast is highly acidic and predicted to inhibit Kir1.1 function. However, the screening of a random-mutated Kir1.1 library in SGY1528 K+ uptake deficient yeast yielded only two mutants (K80M and K80I) which were already known to decrease pH sensitivity. We therefore used a chimeric channel (30C) which contained the transmembrane domain of Kir1.1 and the cytoplasmic domain of Kir4.1. This chimera showed no complementation in yeast even with the mutation K80M. However, upon screening of a randomly mutated library a large number of novel gain of function (GoF) mutations were found. Positive GoF mutant clones were verified and sequenced. These mutations were found to occur in many regions of the Kir1.1 transmembrane domain, including known gating sensitive regions and those that may interact with PIP2, with only one mutation identified in the Kir4.1 section of the 30C chimera. Because these mutations might permit channel function in yeast by reducing pH sensitivity these GoF mutations were made individually in the Kir1.1 channel and their pH-sensitivity measured electrophysiologically. Many mutants had only a modest effect on Kir1.1 pH-sensitivity. However, one novel mutant in TM2 had a dramatic effect reducing pH sensitivity to a similar extent to the K80M mutation. In addition, the whole cell currents of each GoF mutation were recorded in neutral conditions and correlated with the screening results. These results showed the validity of this yeast genetic complementation assay to screen for pH-gating mutations in K+ channels. Further electrophysiological studies on these mutations would help to understand the mechanism by which these mutations affect pH-gating.