*,p< 0.05 as compared with the WT (one-way analysis of variance and Kruskal-Wallis test). We then determined the effect of EAST/SeSAME Maxacalcitol mutations in heteromeric Kir4.1/Kir5.1 channels (Fig. for pH sensing and pore gating. In R297C, this was due to disruption of intersubunit salt bridge Glu288Arg297. C140R breaks the Cys108Cys140disulfide 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. Keywords:Brain, Genetic Diseases, Ion Channels, Kidney, Potassium Channels, EAST/SeSAME Syndrome, Inward Rectifying Potassium Channels, KCNJ10, Kir4.1, Kir4.1/Kir5.1 == Introduction == Kir4.1 (encoded by geneKCNJ10), a member of the inwardly rectifying potassium (Kir) channel family, is essential for the control of glial function and neuronal excitability, systemic K+homeostasis, and renal salt exchange (1,2). Kir4.1 subunits form homotetrameric channels (seeFig. 1A) or co-assemble with Kir5.1 (KCNJ16) in heterotetramers with distinct physiological properties (36). In renal tubular epithelia, these channels are responsible for maintenance of a negative membrane potential that drives ion pumps and exchangers (7,8). In astrocytic glia, Kir4.1-containing channels account for the spatial buffering of K+released by neurons during action potential propagation (1,912). Kir4.1 is predominant in Mller glial cells, where it contributes to normal retinal physiology (1318), and in satellite glial cells of sensory ganglia (19). Kir4.1 channels in oligodendrocytes are critical for myelination Maxacalcitol (20,21), and in various cell types of the Rabbit polyclonal to JAK1.Janus kinase 1 (JAK1), is a member of a new class of protein-tyrosine kinases (PTK) characterized by the presence of a second phosphotransferase-related domain immediately N-terminal to the PTK domain.The second phosphotransferase domain bears all the hallmarks of a protein kinase, although its structure differs significantly from that of the PTK and threonine/serine kinase family members. inner ear they play a role in K+regulation and generation of the endocochlear potential, essential for normal development of the cochlea and audition (2225). == FIGURE 1. == Molecular basis of the EAST/SeSAME syndrome.Patients are homozygous for Kir4.1 mutation R65P, G77R, C140R or T164I; or compound heterozygous for R65P and R199Stop (a deletion of the C-terminal half of the protein) or A167V and R297C.A, predicted location of modified residues in the Kir4.1 channel, modeled on the crystal structure of chicken Kir2.2 (42) (Swiss-PdbViewer 4.0.1). Kir4.1 subunits consist of two membrane-spanning domains (TM1andTM2) that flank a signature K+-selective pore (F), and functional channels are formed by the assembly of four subunits. Viewed in the plane of the membrane, only alternate transmembrane or cytoplasmic regions (uniquely colored) are displayed for clarity.Cyan spheresrepresent K+ions. The area missing in R199Stop is shown ingray. B, close-up views showing predicted intrasubunit disulfide bond between Cys108and Cys140(top) and predicted salt bridge between Glu288and Arg297from adjacent subunits (bottom). The side chains are shown assticksand colored according to atom type: oxygen,red; carbon,yellow; nitrogen,blue; sulfur,green. C, sequence alignment of human Kir4.1 (SwissProt accession numberP78508.1) with hKir1.1 (ROMK,P48048.1), hKir2.1 (P63252.1), hKir3.1 (P48549.1), hKir5.1 (Q9NPI9.1), hKir6.2 (Q14654.2), and hKir7.1 (O60928.1) reveals different degrees of conservation of the positions mutated in EAST/SeSAME syndrome throughout the Kir channel family. Polymorphisms in theKCNJ10gene have been associated with decreased Maxacalcitol astrocytic Kir channel currents (26), seizure susceptibility, and epilepsy (2730), but biophysical characterization of these variants failed to show effects on Kir4.1 channel properties (31). More recently, two independent studies described a novel syndrome, termed EAST or SeSAME, that presents with a unique set of symptoms including epilepsy, ataxia, mental retardation, hearing loss, and electrolyte imbalance related to renal salt loss (32,33). Genetic screening revealed that EAST/SeSAME patients are homozygous or compound heterozygous for novel mutations inKCNJ10that result in amino acid substitutions in Kir4.1 (R65P, G77R, C140R, T164I, A167V, or R297C) or a deletion of the C-terminal half of the protein (R199Stop; seeFig. 1,AandC). Loss of Kir4.1 function associated with these mutations has been reported (34,35). Here, we used radiotracer efflux and electrophysiological measurements to investigate the functional significance of EAST/SeSAME mutations expressed heterologously in COSm6 cells. Our data show that each of these mutations compromises the function Maxacalcitol of both homomeric Kir4.1 and heteromeric Kir4.1/Kir5.1 channels and can account for the clinical phenotype. Kir4.1 function is reduced by distinct mechanisms, and collectively our findings provide novel insights into the critical role played by these residues in Kir4.1 channel function. == EXPERIMENTAL PROCEDURES == == == == == == Expression of Wild-type and Mutant Kir4.1 Channels in COSm6 Cells == pEGFP-rKir4.1, in which rat Kir4.1 cDNA is fused to a N-terminal EGFP2tag (36), was used as template into which EAST/SeSAME mutations were introduced, by means of the QuikChange II site-directed mutagenesis kit (Stratagene). The integrity of all of the constructs was verified by sequencing. COSm6 cells were.