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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 Ba2+ 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. © 2013 S. Karger AG, Basel [ABSTRACT FROM AUTHOR]

Background/Aims: Mutations in the inwardly-rectifying K+ channel KCNJ10/Kir4.1 cause an autosomal recessive disorder characterized by epilepsy, ataxia, sensorineural deafness and tubulopathy (EAST syndrome). KCNJ10 is expressed in the kidney distal convoluted tubule, cochlear stria vascularis and brain glial cells. Patients clinically diagnosed with EAST syndrome were genotyped to identify and study mutations in KCNJ10. Methods: Patient DNA was sequenced and new mutations identified. Mutant and wild-type KCNJ10 constructs were cloned and heterologously expressed in Xenopus oocytes. Whole-cell K+ currents were measured by two-electrode voltage clamping. Results: Three new mutations in KCNJ10 (p.R65C, p.F75L and p.V259fs259X) were identified, and mutation p.R297C, previously only seen in a compound heterozygous patient, was found in a homozygous state. Wild-type human KCNJ10-expressing oocytes showed strongly inwardly-rectified currents, which by comparison were significantly reduced in all the mutants (p < 0.001). Specific inhibition of KCNJ10 currents by Ba2+ demonstrated residual function in all mutant channels (p < 0.05) but V259X. Conclusion: This study confirms that EAST syndrome can be caused by many different mutations in KCNJ10 that significantly reduce K+ conductance. EAST syndrome should be considered in any patient with a renal Gitelman-like phenotype with additional neurological signs and symptoms like ataxia, epilepsy or sensorineural deafness. Copyright © 2011 S. Karger AG, Basel [ABSTRACT FROM AUTHOR]

Short QT Syndrome (SQTS) is a novel clinical entity characterized by markedly rapid cardiac repolarization and lethal arrhythmias. A mutation in the Kir2.1 inward rectifier K+ channel (D172N) causes one form of SQTS (SQT3). Pharmacologic block of Kir2.1 channels may hold promise as potential therapy for SQT3. We recently reported that the anti-malarial drug chloroquine blocks Kir2.1 channels by plugging the cytoplasmic pore domain. In this study, we tested whether chloroquine blocks D172N Kir2.1 channels in a heterologous expression system and if chloroquine normalizes repolarization properties using a mathematical model of a human ventricular myocyte. Chloroquine caused a dose- and voltage-dependent reduction in wild-type (WT), D172N and WT-D172N heteromeric Kir2.1 current. The potency and kinetics of chloroquine block of D172N and WT-D172N Kir2.1 current were similar to WT. In silico modeling of the heterozygous WT-D172N Kir2.1 condition predicted that 3 μM chloroquine normalized inward rectifier K+ current magnitude, action potential duration and effective refractory period. Our results suggest that therapeutic concentrations of chloroquine might lengthen cardiac repolarization in SQT3. Copyright © 2009 S. Karger AG, Basel [ABSTRACT FROM AUTHOR]

Inwardly-rectifying K+ channel subunits are not homogenously expressed in different cardiac tissues. In ventricular myocytes (VM) the background current-voltage relation is dominated by IK1, carried by channels composed of Kir2.x subunits, which is less important in atrial myocytes (AM). On the other hand in AM a large G protein gated current carried by Kir3.1/3.4 complexes can be activated by stimulation of muscarinic M2 receptors (IK(ACh)), which is assumed to be marginal in VM. Recent evidence suggests that total current carried by cardiac inward-rectifiers (IK(ATP), IK(ACh), IK1) in both, AM and VM is limited, due to K+ accumulation/depletion. This lead us to hypothesize that in conventional whole celI recordings IK(ACh) in VM is underestimated as a consequence of constitutive IK1. In that case a reduction in density of IK1 should be paralleled by an increase in density of IK(ACh). Three different experimental strategies have been used to test for this hypothesis: (i) Adenovirus-driven expression of a dominant-negative mutant of Kir2.1, one of the subunits supposed to form IK1 channels, in VM caused a reduction in IK1-density by about 80 %. In those cells IK(ACh) was increased about 4 fold. (ii) A comparable increase in IK(ACh) was observed upon reduction of IK1 caused by adenovirus-mediated RNA interference.(iii) Ba2+ in a concentration of 2 μM blocks IK1 in VM by about 60 % without affecting atrial IK(ACh). The reduction in IK1 by 2 μM Ba2+ is paralleled by a reversible increase in IK(ACh) by about 100%. These data demonstrate that the increase in K+ conductance underlying ventricular IK(ACh) is largely underestimated, suggesting that it might be of greater physiological relevance than previously thought. Copyright © 2008 S. Karger AG, Basel [ABSTRACT FROM AUTHOR]

Inwardly-rectifying K+ channel subunits are not homogenously expressed in different cardiac tissues. In ventricular myocytes (VM) the background current-voltage relation is dominated by IK1, carried by channels composed of Kir2.x subunits, which is less important in atrial myocytes (AM). On the other hand in AM a large G protein gated current carried by Kir3.1/3.4 complexes can be activated by stimulation of muscarinic M2 receptors (IK(ACh)), which is assumed to be marginal in VM. Recent evidence suggests that total current carried by cardiac inward-rectifiers (IK(ATP), IK(ACh), IK1) in both, AM and VM is limited, due to K+ accumulation/depletion. This lead us to hypothesize that in conventional whole celI recordings IK(ACh) in VM is underestimated as a consequence of constitutive IK1. In that case a reduction in density of IK1 should be paralleled by an increase in density of IK(ACh). Three different experimental strategies have been used to test for this hypothesis: (i) Adenovirus-driven expression of a dominant-negative mutant of Kir2.1, one of the subunits supposed to form IK1 channels, in VM caused a reduction in IK1-density by about 80 %. In those cells IK(ACh) was increased about 4 fold. (ii) A comparable increase in IK(ACh) was observed upon reduction of IK1 caused by adenovirus-mediated RNA interference.(iii) Ba2+ in a concentration of 2 μM blocks IK1 in VM by about 60 % without affecting atrial IK(ACh). The reduction in IK1 by 2 μM Ba2+ is paralleled by a reversible increase in IK(ACh) by about 100%. These data demonstrate that the increase in K+ conductance underlying ventricular IK(ACh) is largely underestimated, suggesting that it might be of greater physiological relevance than previously thought. Copyright © 2008 S. Karger AG, Basel [ABSTRACT FROM AUTHOR]

The weak inward rectifier potassium channel ROMK is important for water and salt reabsorption in the kidney. Here we identified Golgin-160 as a novel interacting partner of the ROMK channel. By using yeast two-hybrid assays and co-immunoprecipitations from transfected cells, we demonstrate that Golgin-160 associates with the ROMK C-terminus. Immunofluorescence microscopy confirmed that both proteins are co-localized in the Golgi region. The interaction was further confirmed by the enhancement of ROMK currents by the co-expressed Golgin-160 in Xenopus oocytes. The increase in ROMK current amplitude was due to an increase in cell surface density of ROMK protein. Golgin-160 also stimulated current amplitudes of the related Kir2.1, and of voltage-gated Kv1.5 and Kv4.3 channels, but not the current amplitude of co-expressed HERG channel. These results demonstrate that the Golgi-associated Golgin-160 recognizes the cytoplasmic C-terminus of ROMK, thereby facilitating the transport of ROMK to the cell surface. However, the stimulatory effect on the activity of more distantly-related potassium channels suggests a more general role of Golgin-160 in the trafficking of plasma membrane proteins. [ABSTRACT FROM AUTHOR]