Channelopathies have provided an unique window on the fundamental biology and functions of various channels in diverse cell types [26
]. Kir-associated channelopathies have been linked to diseases affecting primarily renal, pancreatic or cardiac function [26
]. The SeSAME syndrome is characterized by a constellation of seemingly unrelated multi-organ dysfunctions [17
]. Patients with sensorineural deafness, epilepsy, ataxia and a renal salt-losing symptoms possess homozygous mutations in KCNJ10
. The wide range of tissues affected in SeSAME syndrome reflects the extensive pattern of Kir4.1 channel tissue expression [27
Using an amenable heterologous expression system we found that various SeSAME mutations affect Kir4.1 channel function dissimilarly. Two mutations (R65P and A167V) yielded productive Kir4.1 channel currents with overall properties similar to wild-type. Thus these mutant channels were sensitive to extracellular blockade by barium ions and were selective for K+
. These currents were, however, considerably smaller than those measured in the wild-type channel. The remaining SeSAME mutations, R297C, C120R, R199X and T164I yielded currents too small to resolve in our electrophysiological assays. Even by raising extracellular K+
to 50 mM to maximize the magnitude of inward currents we were not able to detect the expression of these mutant channels (data not shown). Previous studies performed on Kir4.1 channels or other members of Kir channel family provide some insights into the functional consequences of these mutations. Neutralization of positively charged residues at R65 and R297 is likely to affect the PIP2
binding to the channel, an essential cofactor for gating in the Kir channel family[28
]. The more mild effect of R65 neutralization may indicate that residual PIP2
binding persists in these mutant channels. A step subsequent to PIP2
binding may be affected in the T164I mutant as this particular mutation prevents the formation of H-bonds between the channel two transmembrane helices (TM)[29
] and affect channel gating [29
]. The cysteine residue at position 120 in Kir4.1 is conserved across other Kir channel family members and mutation of this residue in the homologous Kir2.3 channel induced a loss of channel function [30
In SeSAME syndrome, the wide array of tissue dysfunctions is only manifested in homozygous patients and such lack of dominant negative effect of the disease-causing mutations was recapitulated in our study where co-expression of mutant and wild-type channels resulted in wild-type like currents. The apparent haplosufficiency of KCNJ10 is also seen in Kir4.1 knockout mouse models in which the heterozygous animals do not bear gross behavioral or reproductive abnormalities [15
]. It is somewhat surprising that we did not observe a dominant-negative effect for the C120R mutation in our experiments as expression of the analogous mutation in Kir2.3 channels was reported to show a dominant negative inhibition of wild-type currents in Xenopus
]. We speculate that differences in post-translational processing of proteins in these two expression systems might account for these discrepancies.
Our assessment of cell surface expression of the Kir4.1 channel mutants also reveal that missense mutations (R65P, A167V, R297C, C140R and T164I) did not drastically impair the ability of these channels to be expressed at the plasma membrane. Agents that induce or maintain the opening of Kir4.1 channels may therefore offer a potential avenue of therapy for patients bearing such mutations. However the diminished or lack of plasma membrane expression for the nonsense mutation R199X makes clear that development of novel therapies should be tailored to individual patients.
It is intriguing that SeSAME mutations which failed to yield functional expression in our studies do not cause early postnatal lethality in patients [17
] while mutant mice with genetic inactivation of Kir4.1 do not survive beyond two or three weeks of age [15
]. Species differences might account for such drastic differences in mortality rates or perhaps the inactive channels in many SeSAME syndrome patients may still have a functional role. Conceivably Kir4.1 mutants in the native tissue may be rescued partially by another accessory protein or even another Kir channel subunit. Further investigations using animal models of SeSAME syndrome might provide insights to these questions.