Inwardly rectifying potassium channel Kir4.1 is critical for glial function, control of neuronal excitability, and systemic K(+) homeostasis. Novel mutations in Kir4.1 have been associated with EAST/SeSAME syndrome, characterized by mental retardation, ataxia, seizures, hearing loss, and renal salt waste. Patients are homozygous for R65P, G77R, C140R or T164I; or compound heterozygous for A167V/R297C or R65P/R199Stop, a deletion of the C-terminal half of the protein. We investigated the functional significance of these mutations by radiotracer efflux and inside-out membrane patch clamping in COSm6 cells expressing homomeric Kir4.1 or heteromeric Kir4.1/Kir5.1 channels. All of the mutations compromised channel function, but the underlying mechanisms were different. R65P, T164I, and R297C caused an alkaline shift in pH sensitivity, indicating that these positions are crucial for pH sensing and pore gating. In R297C, this was due to disruption of intersubunit salt bridge Glu(288)-Arg(297). C140R breaks the Cys(108)-Cys(140) disulfide bond essential for protein folding and function. A167V did not affect channel properties but may contribute to decreased surface expression in A167V/R297C. In G77R, introduction of a positive charge within the bilayer may affect channel structure or gating. R199Stop led to a dramatic decrease in surface expression, but channel activity was restored by co-expression with intact subunits, suggesting remarkable tolerance for truncation of the cytoplasmic domain. These results provide an explanation for the molecular defects that underlie the EAST/SeSAME syndrome.