SeSAME/EAST syndrome is a channelopathy consisting of a hypokalemic, hypomagnesemic, metabolic alkalosis associated with seizures, sensorineural deafness, ataxia, and developmental abnormalities. This disease links to autosomal recessive mutations in KCNJ10, which encodes the Kir4.1 potassium channel, but the functional consequences of these mutations are not well understood. In Xenopus oocytes, all of the disease-associated mutant channels (R65P, R65P/R199X, G77R, C140R, T164I, and A167V/R297C) had decreased K ϩ current (0 to 23% of wild-type levels). Immunofluorescence demonstrated decreased surface expression of G77R, C140R, and A167V expressed in HEK293 cells. When we coexpressed mutant and wild-type subunits to mimic the heterozygous state, R199X, C140R, and G77R currents decreased to 55, 40, and 20% of wild-type levels, respectively, suggesting that carriers of these mutations may present with an abnormal phenotype. Because Kir4.1 subunits can form heteromeric channels with Kir5.1, we coexpressed the aforementioned mutants with Kir5.1 and found that currents were reduced at least as much as observed when we expressed mutants alone. Reduction of pH i from approximately 7.4 to 6.8 significantly decreased currents of all mutants except R199X but did not affect wild-type channels. In conclusion, perturbed pH gating may underlie the loss of channel function for the disease-associated mutant Kir4.1 channels and may have important physiologic consequences.

Background β-adrenergic stimulation is the main trigger for cardiac events in type-1 long QT syndrome (LQT1). We evaluated a possible association between ion channel response to β-adrenergic stimulation and clinical response to β-blocker therapy according to mutation location. Methods and Results The study sample comprised 860 patients with genetically-confirmed mutations in the KCNQ1 channel. Patients were categorized into carriers of missense mutations located in the cytoplasmic loops (C-loops), membrane spanning domain, C/N-terminus, and non-missense mutations. There were 27 aborted cardiac arrest [ACA] and 78 sudden cardiac death [SCD] events from birth through age 40 years. After multivariable adjustment for clinical factors, the presence of C-loop mutations was associated with the highest risk for ACA or SCD (hazard ratio [95% confidence interval] vs. non-missense mutations = 2.75 [1.29-5.86, P=0.009]). β-blocker therapy was associated with a significantly greater reduction in the risk of ACA or SCD among patients with C-loop mutations than among all other patients (hazard ratios = 0.12 [0.02-0.73, P=0.02] and 0.82 [0.31-2.13, P=0.68], respectively; P-for interaction = 0.04). Cellular expression studies showed that membrane spanning and C-loop mutations produced a similar decrease in current, but only C-loop mutations showed a pronounced reduction in channel activation in response to β-adrenergic stimulation. Conclusions Patients with C-loop missense mutations in the KCNQ1 channel exhibit a high-risk for life-threatening events and derive a pronounced benefit from treatment with β-blockers. Reduced channel activation following sympathetic activation can explain the increased clinical risk and response to therapy in patients with C-loop mutations.

Gray, Daniel A., Gustavo Frindt, and Lawrence G. Palmer. Quantification of K ϩ secretion through apical low-conductance K channels in the CCD. Am J Physiol Renal Physiol 289: F117-F126, 2005. First published February 22, 2005 doi:10.1152/ajprenal.00471.2004.-Outward and inward currents through single small-conductance K ϩ (SK) channels were measured in cell-attached patches of the apical membrane of principal cells of the rat cortical collecting duct (CCD). Currents showed mild inward rectification with high [K ϩ ] in the pipette (K p ϩ ), which decreased as K p ϩ was lowered. Inward conductances had a hyperbolic dependence on K p ϩ with half-maximal conductance at ϳ20 mM. Outward conductances, measured near the reversal potential, also increased with K p ϩ from 15 pS (K p ϩ ϭ 0) to 50 pS (K p ϩ ϭ 134 mM). SK channel density was measured as the number of conducting channels per patch in cell-attached patches. As reported previously, channel density increased when animals were on a high-K diet for 7 days. Addition of 8-cpt-cAMP to the bath at least 5 min before making a seal increased SK channel density to an even greater extent, although this increase was not additive with the effect of a high-K diet. In contrast, increases in Na channel activity, assessed as the whole cell amiloride-sensitive current, due to K loading and 8-cpt-cAMP treatment were additive. Singlechannel conductances and channel densities were used as inputs to a simple mathematical model of the CCD to predict rates of transepithelial Na ϩ and K ϩ transport as a function of apical Na ϩ permeability and K ϩ conductance, basolateral pump rates and K ϩ conductance, and the paracellular conductance. With measured values for these parameters, the model predicted transport rates that were in good agreement with values measured in isolated, perfused tubules. The number and properties of SK channels account for K ϩ transport by the CCD under all physiological conditions tested.ROMK; epithelial sodium channels; high-potassium diet; aldosterone; cAMP; epithelial transport model SMALL CONDUCTANCE (SK) channels are the most abundant K channel type observed in patch-clamp studies of the apical membrane of the mammalian cortical collecting duct (CCD) and connecting tubule (CNT) (6,7,34). These channels are believed to be encoded by the ROMK gene (16, 36), a conclusion that was recently directly confirmed by the absence of the channels in a ROMK knockout mouse strain (13). They are thought to be the predominant route for K ϩ secretion by the distal nephron (10). However, the ROMK knockout animals showed net secretion of K ϩ into the urine. They had high rates of K ϩ excretion without the hyperkalemia that would be expected from elimination of the major secretory system, implying that other pathways exist (12,13). A quantitative assessment of the role of the SK channels in secretion is therefore useful. The properties of the SK/ROMK channels have been studied extensively both in the CCD and in heterologous expression systems but typically these studies h...

Key Words: mitochondrial ATP-sensitive potassium channel Ⅲ ischemia/reperfusion Ⅲ ischemic preconditioning Ⅲ fluoxetine T he mitochondrial ATP-sensitive potassium channel (mK ATP ) is thought to be essential for cardioprotection recruited by ischemic preconditioning (IPC), 1,2 but despite intense research the molecular identity of this channel remains unclear. The simplest thesis is that mK ATP channels are derivative of surface K ATP channels, and thus composed of inward rectifying K ϩ channels (K IR ) and sulfonylurea receptors (SURs). The cardiomyocyte surface K ATP channel is comprised of K IR 6.2 and SUR2A isoforms, 3 but efforts to conclusively assign these proteins to the cardiac mK ATP have been unsuccessful to date.Neither K IR 6 nor SUR genes contain mitochondrial target sequences, and K IR 6/SUR proteins are not found in mitochondrial proteome databases or prediction engines. 4,5 Furthermore, immune-based methods to detect K IR /SUR subunits in mitochondria are plagued by issues of antibody specificity 6 and mitochondrial purity/contamination. Several of the key pharmacological reagents used to study mK ATP channels (eg, the agonist diazoxide and antagonist 5-hydroxydecanoate) are also known to exhibit off-target effects. 7,8 Targeted gene deletion in mice to identify the mK ATP channel involved in IPC has proven futile, because of the confounding cardiovascular effects of knocking out K IR 6 and SUR genes (Kcnj8, Kcnj11, Abcc8, and Abcc9) on surface K ATP channel function. In general, K IR and SUR knockouts exhibit profound defects in glucose/insulin handling, 9 -12 which impacts the response to IPC. 13 A recent study using custom-made antibodies and SUR knockout mice identified short-form splice variants of SUR2 in mitochondria. 14 Furthermore, recent pharmacological evidence suggests that complex II of the respiratory chain (succinate dehydrogenase) may be a regulatory component of the mK ATP channel. 15,16 However, both these findings leave the identity of the K ϩ channel-forming subunit of mK ATP unknown. In this regard, mK ATP is similar to other mitochondrial ion channels which exist at a phenomenological level but have not been molecularly identified (eg, the mitochondrial Ca 2ϩ uniporter). A major obstacle in studying the mK ATP channel has been the availability of a reliable assay. Most studies to date have used an isolated mitochondrial rapid swelling assay, in which Original received October 20, 2009; resubmission received December 18, 2009; revised resubmission received January 28, 2010; accepted February 12, 2010. From K ϩ uptake into mitochondria is followed by osmotically-obliged water, leading to mild swelling that is assayed as light scattering in a spectrophotometer. 17,18 This assay has been criticized as irreproducible by some laboratories, 19 with the precise timing of mitochondrial isolation appearing to be a critical factor. 20 Studying the literature on surface K ATP channels, two key biochemical properties that appeared to have been overlooked in the mK ATP channel field...