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Nephron Physiol
2013 Jan 01;1233-4:7-14. doi: 10.1159/000356353.
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KCNJ10 mutations display differential sensitivity to heteromerisation with KCNJ16.
Parrock S
,
Hussain S
,
Issler N
,
Differ AM
,
Lench N
,
Guarino S
,
Oosterveld MJ
,
Keijzer-Veen M
,
Brilstra E
,
van Wieringen H
,
Konijnenberg AY
,
Amin-Rasip S
,
Dumitriu S
,
Klootwijk E
,
Knoers N
,
Bockenhauer D
,
Kleta R
,
Zdebik AA
.
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BACKGROUND/AIMS: Mutations in the inwardly-rectifying K(+)-channel KCNJ10/Kir4.1 cause autosomal recessive EAST syndrome (epilepsy, ataxia, sensorineural deafness and tubulopathy). KCNJ10 is expressed in the distal convoluted tubule of the kidney, stria vascularis of the inner ear and brain glial cells. Patients diagnosed clinically with EAST syndrome were genotyped and mutations in KCNJ10 were studied functionally.
METHODS: Patient DNA was amplified and sequenced, and new mutations were identified. Mutant and wild-type KCNJ10 constructs were cloned and heterologously expressed in Xenopus oocytes. Whole-cell K(+) currents were measured by 2-electrode voltage clamping and channel expression was analysed by Western blotting.
RESULTS: We identified 3 homozygous mutations in KCNJ10 (p.F75C, p.A167V and p.V91fs197X), with mutation p.A167V previously reported in a compound heterozygous state. Oocytes expressing wild-type human KCNJ10 showed inwardly rectified currents, which were significantly reduced in all of the mutants (p < 0.001). Specific inhibition of KCNJ10 currents by Ba(2+) demonstrated a large residual function in p.A167V only, which was not compatible with causing disease. However, co-expression with KCNJ16 abolished function in these heteromeric channels almost completely.
CONCLUSION: This study provides an explanation for the pathophysiology of the p.A167V KCNJ10 mutation, which had previously not been considered pathogenic on its own. These findings provide evidence for the functional cooperation of KCNJ10 and KCNJ16. Thus, in vitro ascertainment of KCNJ10 function may necessitate co-expression with KCNJ16.
Fig. 1. a Averaged current/voltage curves obtained in Xenopus oocytes injected with 2 ng WT or mutant KCNJ10 cRNA. Oocytes expressing KCNJ10 F75C and V91fs197X are indistinguishable from water-injected oocytes. Oocytes were held in 20 mM K+ at -40 mV and clamped from +100 to -100 mV in 20 mV steps. Note that currents reversed around -40 mV due to the 20-mM K+ solution used. Three batches of oocytes were analysed.
Fig. 1. b. Analysis of the chord conductance between -100 and 0 mV
Fig. 1. c. Barium (100 μM) inhibited more strongly the inward component of WT and A167V mutant currents.
Fig. 3. a Averaged current/voltage curves obtained in Xenopus oocytes expressing WT and KCNJ10 A167V (1 ng) with and without KCNJ16 (3 ng) in the same batch of oocytes. Again, altered rectification for WT and almost complete loss of current for KCNJ10 A167V upon co-expression with KCNJ16 was observed. b Analysis of the chord conductance between -100 and 0 mV. Conductances as a percentage of WT KCNJ10 alone: KCNJ10/KCNJ16, 64%; KCNJ10 A167V alone, 58%; KCNJ10 A167V/KCNJ16, 0.8%.
Fig. 4. a KCNJ10 WT, A167V, F75C and V91fs197X expressed in oocytes, separated by SDS-PAGE and probed for KCNJ10. Note that V91fs197X cannot be detected with our antibody as it recognizes an epitope lost through frameshift in this mutation, suggesting the antibody is specific. b, c Time-course of expression of 1 ng KCNJ10 and 3 ng KCNJ16 cRNA co-expressed in oocytes over 48 h. Detection in membrane preparations with a monoclonal antibody against KCNJ10 (b), and with a polyclonal antibody against KCNJ16 (c). d Western blot probed for KCNJ10 prepared from oocytes expressing KCNJ10 A167V with and without KCNJ16. e Same membrane as in d, probed for KCNJ16. Expression levels appear somewhat lower for KCNJ10 A167V, but the co-expression with KCNJ16 does not alter expression significantly, suggesting that overall stability of KCNJ10 A167V is unaltered by heteromerisation with KCNJ16. KCNJ10 is known to produce multiple bands on Western blotting, depending on the preparation [34]. Note that the lane description applies for d and e. Arrows indicate specific bands throughout the figure, and M indicates molecular weight marker lanes with the approximate molecular weight given in kDa.
