Kir7.1 is a potassium channel expressed in a variety of epithelial tissues prominent among which is the retinal pigment epithelium of the eye. Mutations in KCNJ13, the gene coding Kir7.1, cause eye diseases in humans. An example is snowflake vitreoretinal degeneration (SVD), an autosomal dominant inherited pathology characterized by retinal and vitreous deterioration associated to an R162 to W mutation in Kir7.1 (Hejtmancik et al., 2008). The mechanism by which this mutation produces its dominant negative effect is not clear. This C-terminus located arginine is speculated to be part of a ligand site for PIP2 which would be necessary for stabilizing the open state of the channel and its neutralization in the R162W mutant would lead to channel silencing (Pattnaik et al., 2013; Zhang et al., 2013). We are now using site directed mutagenesis and functional analysis after transfection into HEK-293 cells to test this hypothesis. Cells transfected with the R162W mutant of Kir7.1 do not express any specific current as expected for an inactivating mutation and consistent with the loss of charge and PIP2 interaction hypothesis. We then assayed an R162C mutant of Kir7.1, that given the neutral nature of the cysteine side-chain was expected to behave as Kir7.1-R162W. Kir7.1-R162C, however, showed robust activity measured as inward Rb+ whole cell current at -160 mV (-8.8 ± 1.5 nA, compared to -3.6 ± 0.7 nA in WT Kir7.1). Experiments using Kir7.1-R162Q gave a current of -2.0 ± 0.6 nA whilst the R162F mutant of Kir7.1 completely lacked channel activity. These data suggest a relation between the volume of the amino acid side chain (Cys 39.7, Gln 85.6, Phe 129.7, Trp 167.9 Å3) and the current through the channel. To emulate a large neutral amino acid side chain in the background of the Kir7.1-R162C mutant we used BMTS benzylmethanethiosulfonate (10 µM) to attempt to modify C162. These experiments were conducted in excised inside-ourt patches. BMTS had no effect on WT Kir7.1 activity but elicited a rapid and complete suppression of current in the Kir7.1-R162C mutant. The smaller MMTS methylmethanethiosulfonate also inhibited the current through Kir7.1-R162C but to a lesser extent. These results suggest that the volume of the side chain at position 162 of Kir7.1 is an important determinant of activity. The increase in side chain volume that occurs when replacing arginine by a tryptophan residue in SVD might be at the root of the dominant-negative effect behind the pathology.