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Dendrites are injured in a variety of clinical conditions such as traumatic brain and spinal cord injuries and stroke. How neurons detect injury directly to their dendrites to initiate a pro-regenerative response has not yet been thoroughly investigated. Calcium plays a critical role in the early stages of axonal injury detection and is also indispensable for regeneration of the severed axon. Here, we report cell and neurite type-specific differences in laser injury-induced elevations of intracellular calcium levels. Using a human KCNJ2 transgene, we demonstrate that hyperpolarizing neurons only at the time of injury dampens dendrite regeneration, suggesting that inhibition of injury-induced membrane depolarization (and thus early calcium influx) plays a role in detecting and responding to dendrite injury. In exploring potential downstream calcium-regulated effectors, we identify L-type voltage-gated calcium channels, inositol triphosphate signaling, and protein kinase D activity as drivers of dendrite regeneration. In conclusion, we demonstrate that dendrite injury-induced calcium elevations play a key role in the regenerative response of dendrites and begin to delineate the molecular mechanisms governing dendrite repair.

The Kir4.1 channel, an inwardly rectifying potassium ion (K+) channel, is located in the hair cells of the organ of Corti as well as the intermediate cells of the stria vascularis. The Kir4.1 channel has a crucial role in the generation of endolymphatic potential and maintenance of the resting membrane potential. However, the role and functions of the Kir4.1 channel in the progenitor remain undescribed. To observe the role of Kir4.1 in the progenitor treated with the one-shot ototoxic drugs (kanamycin and furosemide), we set the proper condition in culturing Immortomouse-derived HEI-OC1 cells to express the potassium-related channels well. And also, that was reproduced in mice experiments to show the important role of Kir4.1 in the survival of hair cells after treating the ototoxicity drugs. In our results, when kanamycin and furosemide drugs were cotreated with HEI-OC1 cells, the Kir4.1 channel did not change, but the expression levels of the NKCC1 cotransporter and KCNQ4 channel are decreased. This shows that inward and outward channels were blocked by the two drugs (kanamycin and furosemide). However, noteworthy here is that the expression level of Kir4.1 channel increased when kanamycin was treated alone. This shows that Kir4.1, an inwardly rectifying potassium channel, acts as an outward channel in place of the corresponding channel when the KCNQ4 channel, an outward channel, is blocked. These results suggest that the Kir4.1 channel has a role in maintaining K+ homeostasis in supporting cells, with K+ concentration compensator when the NKCC1 cotransporter and Kv7.4 (KCNQ4) channels are deficient.

Oxytocin (OXT) and OXT receptor (OXTR)-mediated signaling control excitability, firing patterns, and plasticity of hippocampal CA2 pyramidal neurons, which are pivotal in generation of brain oscillations and social memory. Nonetheless, the ionic mechanisms underlying OXTR-induced effects in CA2 neurons are not fully understood. Using slice physiology in a reporter mouse line and interleaved current-clamp and voltage-clamp experiments, we systematically identified the ion channels modulated by OXT signaling in CA2 pyramidal cells (PYRs) in mice of both sexes and explored how changes in channel conductance support altered electrical activity. Activation of OXTRs inhibits an outward potassium current mediated by inward rectifier potassium channels (I Kir) and thus favoring membrane depolarization. Concomitantly, OXT signaling also diminishes inward current mediated by hyperpolarization-activated cyclic-nucleotide-gated (HCN) channels (I h), providing a hyperpolarizing drive. The combined reduction in both I Kir and I h synergistically elevate the membrane resistance and favor dendritic integration while the membrane potential is restrained from quickly depolarizing from rest. As a result, the responsiveness of CA2 PYRs to synaptic inputs is highly sharpened during OXTR activation. Unexpectedly, OXTR signaling also strongly enhances a tetrodotoxin-resistant (TTX-R), voltage-gated sodium current that helps drive the membrane potential to spike threshold and thus promote rhythmic firing. This novel array of OXTR-stimulated ionic mechanisms operates in close coordination and underpins OXT-induced burst firing, a key step in CA2 PYRs' contribution to hippocampal information processing and broader influence on brain circuitry. Our study deepens our understanding of underpinnings of OXT-promoted social memory and general neuropeptidergic control of cognitive states.SIGNIFICANCE STATEMENT Oxytocin (OXT) plays key roles in reproduction, parenting and social and emotional behavior, and deficiency in OXT receptor (OXTR) signaling may contribute to neuropsychiatric disorders. We identified a novel array of OXTR-modulated ion channels that operate in close coordination to retune hippocampal CA2 pyramidal neurons, enhancing responsiveness to synaptic inputs and sculpting output. OXTR signaling inhibits both potassium conductance (I Kir) and mixed cation conductance (I h), engaging opposing influences on membrane potential, stabilizing it while synergistically elevating membrane resistance and electrotonic spread. OXT signaling also facilitates a tetrodotoxin-resistant (TTX-R) Na+ current, not previously described in hippocampus (HP), engaged on further depolarization. This TTX-R current lowers the spike threshold and supports rhythmic depolarization and burst firing, a potent driver of downstream circuitry.

Weak electromagnetic fields (WEF) alter Ca2+ handling in skeletal muscle myotubes. Owing to the involvement of Ca2+ in muscle development, we investigated whether WEF affects fusion of myoblasts in culture. Rat primary myoblast cultures were exposed to WEF (1.75 µT, 16 Hz) for up to six days. Under control conditions, cell fusion and creatine kinase (CK) activity increased in parallel and peaked at 4-6 days. WEF enhanced the extent of fusion after one and two days (by ~40%) vs. control, but not thereafter. Exposure to WEF also enhanced CK activity after two days (almost four-fold), but not afterwards. Incorporation of 3H-thymidine into DNA was enhanced by one-day exposure to WEF (~40%), indicating increased cell replication. Using the potentiometric fluorescent dye di-8-ANEPPS, we found that exposure of cells to 150 mM KCl resulted in depolarization of the cell membrane. However, prior exposure of cells to WEF for one day followed by addition of KCl resulted in hyperpolarization of the cell membrane. Acute exposure of cells to WEF also resulted in hyperpolarization of the cell membrane. Twenty-four hour incubation of myoblasts with gambogic acid, an inhibitor of the inward rectifying K+ channel 2.1 (Kir2.1), did not affect cell fusion, WEF-mediated acceleration of fusion or hyperpolarization. These data demonstrate that WEF accelerates fusion of myoblasts, resulting in myotube formation. The WEF effect is associated with hyperpolarization but WEF does not appear to mediate its effects on fusion by activating Kir2.1 channels.

Mutations in KCNJ13 are associated with two retinal disorders; Leber congenital amaurosis (LCA) and snowflake vitreoretinal degeneration (SVD). We describe a novel fibrovascular proliferation in the retina of two affected members of a KCNJ13-related LCA family with a homozygous c.458C > T, p.(Thr153Ile) missense mutation. Optical coherence tomography retinal imaging of the kcnj13 mutant zebrafish (obelixtd15 c.502T > C, p.[Phe168Leu]) revealed a late onset retinal degeneration at 12 months, with retinal thinning and associated retinovascular changes, including increased vessel calibre and vitreous deposits. Both human and zebrafish variants are missense and located within the conserved transmembrane M2 protein domain, suggesting that disruption of this region may contribute to retinovascular changes as an additional feature to the previously described LCA phenotype. Close monitoring of other patients with similar mutations may be required to minimise the ensuing retinal damage.

A key assumption of optogenetics is that light only affects opsin-expressing neurons. However, illumination invariably heats tissue, and many physiological processes are temperature-sensitive. Commonly used illumination protocols increased the temperature by 0.2-2 °C and suppressed spiking in multiple brain regions. In the striatum, light delivery activated an inwardly rectifying potassium conductance and biased rotational behavior. Thus, careful consideration of light-delivery parameters is required, as even modest intracranial heating can confound interpretation of optogenetic experiments.

Glial support is critical for normal axon function and can become dysregulated in white matter (WM) disease. In humans, loss-of-function mutations of KCNJ10, which encodes the inward-rectifying potassium channel KIR4.1, causes seizures and progressive neurological decline. We investigated Kir4.1 functions in oligodendrocytes (OLs) during development, adulthood and after WM injury. We observed that Kir4.1 channels localized to perinodal areas and the inner myelin tongue, suggesting roles in juxta-axonal K+ removal. Conditional knockout (cKO) of OL-Kcnj10 resulted in late onset mitochondrial damage and axonal degeneration. This was accompanied by neuronal loss and neuro-axonal dysfunction in adult OL-Kcnj10 cKO mice as shown by delayed visual evoked potentials, inner retinal thinning and progressive motor deficits. Axon pathologies in OL-Kcnj10 cKO were exacerbated after WM injury in the spinal cord. Our findings point towards a critical role of OL-Kir4.1 for long-term maintenance of axonal function and integrity during adulthood and after WM injury.

Microglia show a rich repertoire of activation patterns regulated by a complex ensemble of surface ion channels, receptors, and transporters. We and others have investigated whether microglia vary their K+ channel expression as a means to achieve functional diversity. However, most of the prior studies were conducted using in vitro models such as BV2 cells, primary microglia, or brain slices in culture, which may not accurately reflect microglia physiology in adult individuals. Here we employed an in vivo mouse model of selective innate immune activation by intracerebroventricular injection of lipopolysaccharides (ICV-LPS) to determine the role of the voltage-gated Kv1.3 channel in LPS-induced M1-like microglial activation. Using microglia acutely isolated from adult brains, we detected Kv1.3 and Kir2.1 currents, and found that ICV-LPS increased the current density and RNA expression of Kv1.3 but did not affect those of Kir2.1. Genetic knockout of Kv1.3 abolished LPS-induced microglial activation exemplified by Iba-1 immunoreactivity and expression of pro-inflammatory mediators such as IL-1β, TNF-α, IL-6, and iNOS. Moreover, Kv1.3 knockout mitigated the LPS-induced impairment of hippocampal long-term potentiation (hLTP), suggesting that Kv1.3 activity regulates pro-inflammatory microglial neurotoxicity. Pharmacological intervention using PAP-1, a small molecule that selectively blocks homotetrameric Kv1.3 channels, achieved anti-inflammatory and hLTP-recovery effects similar to Kv1.3 knockout. We conclude that Kv1.3 is required for microglial M1-like pro-inflammatory activation in vivo. A significant implication of our in vivo data is that Kv1.3 blockers could be therapeutic candidates for neurological diseases where microglia-mediated neurotoxicity is implicated in the pathogenesis.

There have been no published reports of central respiratory control abnormalities in pediatric patients with UNC80 or KCNJ11 mutations which cause neurologic channelopathies. We describe an 8-year-old male with a pathogenic UNC80 mutation, intellectual disability, hypotonia and epilepsy with severe central sleep apnea (213.5 events/h) on polysomnography (PSG). We also describe a 20-month-old female with a KCNJ11 mutation, neonatal diabetes and developmental delay who had severe central sleep apnea (131.1 events/h). Both patients had irregular respiratory patterns during sleep and wakefulness and were placed on empiric bilevel positive airway pressure therapy, which was well tolerated with resolution of abnormal respiratory control and hypercapnia. Patients with UNC80 and KCNJ11 gene mutations may have abnormal respiratory rhythm during sleep and wakefulness, mirroring animal models. We recommend routine PSG tests and further investigation into the respiratory control of patients with pediatric channelopathies involved in chemoreceptor function or central integration of respiratory control.

Diversified neurons are essential for sensorimotor function, but whether astrocytes become specialized to optimize circuit performance remains unclear. Large fast α-motor neurons (FαMNs) of spinal cord innervate fast-twitch muscles that generate peak strength. We report that ventral horn astrocytes express the inward-rectifying K+ channel Kir4.1 (a.k.a. Kcnj10) around MNs in a VGLUT1-dependent manner. Loss of astrocyte-encoded Kir4.1 selectively altered FαMN size and function and led to reduced peak strength. Overexpression of Kir4.1 in astrocytes was sufficient to increase MN size through activation of the PI3K/mTOR/pS6 pathway. Kir4.1 was downregulated cell autonomously in astrocytes derived from amyotrophic lateral sclerosis (ALS) patients with SOD1 mutation. However, astrocyte Kir4.1 was dispensable for FαMN survival even in the mutant SOD1 background. These findings show that astrocyte Kir4.1 is essential for maintenance of peak strength and suggest that Kir4.1 downregulation might uncouple symptoms of muscle weakness from MN cell death in diseases like ALS.

Alzheimer's disease (AD) is a leading cause of mortality among the elderly. We performed a whole-genome sequencing study of AD in the Chinese population. In addition to the variants identified in or around the APOE locus (sentinel variant rs73052335, P = 1.44 × 10-14), two common variants, GCH1 (rs72713460, P = 4.36 × 10-5) and KCNJ15 (rs928771, P = 3.60 × 10-6), were identified and further verified for their possible risk effects for AD in three small non-Asian AD cohorts. Genotype-phenotype analysis showed that KCNJ15 variant rs928771 affects the onset age of AD, with earlier disease onset in minor allele carriers. In addition, altered expression level of the KCNJ15 transcript can be observed in the blood of AD subjects. Moreover, the risk variants of GCH1 and KCNJ15 are associated with changes in their transcript levels in specific tissues, as well as changes of plasma biomarkers levels in AD subjects. Importantly, network analysis of hippocampus and blood transcriptome datasets suggests that the risk variants in the APOE, GCH1, and KCNJ15 loci might exert their functions through their regulatory effects on immune-related pathways. Taking these data together, we identified common variants of GCH1 and KCNJ15 in the Chinese population that contribute to AD risk. These variants may exert their functional effects through the immune system.

BackgroundIn human cardiac ventricle, IK1 is mainly comprised Kir2.1, but Kir2.2 and Kir2.3 heterotetramers occur and modulate IK1. Long-QT syndrome-9-associated CAV3 mutations cause decreased Kir2.1 current density, but Kir2.x heterotetramers have not been studied. Here, we determine the effect of long-QT syndrome-9-CAV3 mutation F97C on Kir2.x homo- and heterotetramers and model-associated arrhythmia mechanisms.Methods and resultsSuper-resolution microscopy, co-immunoprecipitation, cellular electrophysiology, on-cell Western blotting, and simulation of Purkinje and ventricular myocyte mathematical models were used. Kir2.x isoforms have unique subcellular colocalization in human cardiomyocytes and coimmunoprecipitate with Cav3. F97C-Cav3 decreased peak inward Kir2.2 current density by 50% (-120 mV; P=0.019) and peak outward by 75% (-40 mV; P

The ATP-sensitive potassium channel (KATP) functions as a metabo-electric transducer in regulating insulin secretion from pancreatic β-cells. The pancreatic KATP channel is composed of a pore-forming inwardly-rectifying potassium channel, Kir6.2, and a regulatory subunit, sulphonylurea receptor 1 (SUR1). Loss-of-function mutations in either subunit often lead to the development of persistent hyperinsulinemic hypoglycemia of infancy (PHHI). PHHI is a rare genetic disease and most patients present with immediate onset within the first few days after birth. In this study, we report an unusual form of PHHI, in which the index patient developed hyperinsulinemic hypoglycemia after 1 year of age. The patient failed to respond to routine medication for PHHI and underwent a complete pancreatectomy. Genotyping of the index patient and his immediate family members showed that the patient and other family members with hypoglycemic episodes carried a heterozygous novel mutation in KCNJ11 (C83T), which encodes Kir6.2 (A28V). Electrophysiological and cell biological experiments revealed that A28V hKir6.2 is a dominant-negative, loss-of-function mutation and that KATP channels carrying this mutation failed to reach the cell surface. De novo protein structure prediction indicated that this A28V mutation reoriented the ER retention motif located at the C-terminal of the hKir6.2, and this result may explain the trafficking defect caused by this point mutation. Our study is the first report of a novel form of late-onset PHHI that is caused by a dominant mutation in KCNJ11 and exhibits a defect in proper surface expression of Kir6.2.

Gain-of-function mutations in KCNJ2-encoded Kir2.1 channels underlie variant 3 (SQT3) of the short QT syndrome, which is associated with atrial fibrillation (AF). Using biophysically-detailed human atria computer models, this study investigated the mechanistic link between SQT3 mutations and atrial arrhythmogenesis, and potential ion channel targets for treatment of SQT3. A contemporary model of the human atrial action potential (AP) was modified to recapitulate functional changes in IK1 due to heterozygous and homozygous forms of the D172N and E299V Kir2.1 mutations. Wild-type (WT) and mutant formulations were incorporated into multi-scale homogeneous and heterogeneous tissue models. Effects of mutations on AP duration (APD), conduction velocity (CV), effective refractory period (ERP), tissue excitation threshold and their rate-dependence, as well as the wavelength of re-entry (WL) were quantified. The D172N and E299V Kir2.1 mutations produced distinct effects on IK1 and APD shortening. Both mutations decreased WL for re-entry through a reduction in ERP and CV. Stability of re-entrant excitation waves in 2D and 3D tissue models was mediated by changes to tissue excitability and dispersion of APD in mutation conditions. Combined block of IK1 and IKr was effective in terminating re-entry associated with heterozygous D172N conditions, whereas IKr block alone may be a safer alternative for the E299V mutation. Combined inhibition of IKr and IKur produced a synergistic anti-arrhythmic effect in both forms of SQT3. In conclusion, this study provides mechanistic insights into atrial proarrhythmia with SQT3 Kir2.1 mutations and highlights possible pharmacological strategies for management of SQT3-linked AF.

PurposeTo test the effects of rearing light intensity on retinal function and morphology in the retinoschisis knockout (Rs1-KO) mouse model of X-linked retinoschisis, and whether it affects functional outcome of RS1 gene replacement.MethodsSeventy-six Rs1-KO mice were reared in either cyclic low light (LL, 20 lux) or moderate light (ML, 300 lux) and analyzed at 1 and 4 months. Retinal function was assessed by electroretinogram and cavity size by optical coherence tomography. Expression of inward-rectifier K+ channel (Kir4.1), water channel aquaporin-4 (AQP4), and glial fibrillary acidic protein (GFAP) were analyzed by Western blotting. In a separate study, Rs1-KO mice reared in LL (n = 29) or ML (n = 27) received a unilateral intravitreal injection of scAAV8-hRs-IRBP at 21 days, and functional outcome was evaluated at 4 months by electroretinogram.ResultsAt 1 month, no functional or structural differences were found between LL- or ML-reared Rs1-KO mice. At 4 months, ML-reared Rs1-KO mice showed significant reduction of b-wave amplitude and b-/a-wave ratio with no changes in a-wave, and a significant increase in cavity size, compared to LL-reared animals. Moderate light rearing increased Kir4.1 expression in Rs1-KO mice by 4 months, but not AQP4 and GFAP levels. Administration of scAAV8-hRS1-IRBP to Rs1-KO mice showed similar improvement of inner retinal ERG function independent of LL or ML rearing.ConclusionsRearing light conditions affect the development of retinal cavities and post-photoreceptor function in Rs1-KO mice. However, the effect of rearing light intensity does not interact with the efficacy of RS1 gene replacement in Rs1-KO mice.

Microglia are highly plastic cells that can assume different phenotypes in response to microenvironmental signals. Lipopolysaccharide (LPS) and interferon-γ (IFN-γ) promote differentiation into classically activated M1-like microglia, which produce high levels of pro-inflammatory cytokines and nitric oxide and are thought to contribute to neurological damage in ischemic stroke and Alzheimer's disease. IL-4 in contrast induces a phenotype associated with anti-inflammatory effects and tissue repair. We here investigated whether these microglia subsets vary in their K+ channel expression by differentiating neonatal mouse microglia into M(LPS) and M(IL-4) microglia and studying their K+ channel expression by whole-cell patch-clamp, quantitative PCR and immunohistochemistry. We identified three major types of K+ channels based on their biophysical and pharmacological fingerprints: a use-dependent, outwardly rectifying current sensitive to the KV 1.3 blockers PAP-1 and ShK-186, an inwardly rectifying Ba2+ -sensitive Kir 2.1 current, and a Ca2+ -activated, TRAM-34-sensitive KCa 3.1 current. Both KV 1.3 and KCa 3.1 blockers inhibited pro-inflammatory cytokine production and iNOS and COX2 expression demonstrating that KV 1.3 and KCa 3.1 play important roles in microglia activation. Following differentiation with LPS or a combination of LPS and IFN-γ microglia exhibited high KV 1.3 current densities (∼50 pA/pF at 40 mV) and virtually no KCa 3.1 and Kir currents, while microglia differentiated with IL-4 exhibited large Kir 2.1 currents (∼ 10 pA/pF at -120 mV). KCa 3.1 currents were generally low but moderately increased following stimulation with IFN-γ or ATP (∼10 pS/pF). This differential K+ channel expression pattern suggests that KV 1.3 and KCa 3.1 inhibitors could be used to inhibit detrimental neuroinflammatory microglia functions. GLIA 2016;65:106-121.

Short-acting β2-agonist bronchodilators are the most common medications used in treating chronic obstructive pulmonary disease (COPD). Genetic variants determining bronchodilator responsiveness (BDR) in COPD have not been identified. We performed a genome-wide association study (GWAS) of BDR in 5789 current or former smokers with COPD in one African-American and four white populations. BDR was defined as the quantitative spirometric response to inhaled β2-agonists. We combined results in a meta-analysis. In the meta-analysis, single-nucleotide polymorphisms (SNPs) in the genes KCNK1 (P=2.02 × 10(-7)) and KCNJ2 (P=1.79 × 10(-7)) were the top associations with BDR. Among African Americans, SNPs in CDH13 were significantly associated with BDR (P=5.1 × 10(-9)). A nominal association with CDH13 was identified in a gene-based analysis in all subjects. We identified suggestive association with BDR among COPD subjects for variants near two potassium channel genes (KCNK1 and KCNJ2). SNPs in CDH13 were significantly associated with BDR in African Americans.The Pharmacogenomics Journal advance online publication, 27 October 2015; doi:10.1038/tpj.2015.65.

Photostimulation is a noninvasive way to control biological events with excellent spatial and temporal resolution. New methods are desired to photo-regulate endogenous proteins expressed in their native environment. Here, we present an approach to optically control the function of a neuronal protein directly in neurons using a genetically encoded unnatural amino acid (Uaa). By using an orthogonal tRNA/aminoacyl-tRNA synthetase pair to suppress the amber codon, a photo-reactive Uaa 4,5-dimethoxy-2-nitrobenzyl-cysteine (Cmn) is site-specifically incorporated in the pore of a neuronal protein Kir2.1, an inwardly rectifying potassium channel. The bulky Cmn physically blocks the channel pore, rendering Kir2.1 non-conducting. Light illumination instantaneously converts Cmn into a smaller natural amino acid Cys, activating Kir2.1 channel function. We express these photo-inducible inwardly rectifying potassium (PIRK) channels in rat hippocampal primary neurons, and demonstrate that light-activation of PIRK ceases the neuronal firing due to the outflux of K(+) current through the activated Kir2.1 channels. Using in utero electroporation, we also express PIRK in the embryonic mouse neocortex in vivo, showing the light-activation of PIRK in neocortical neurons. Genetically encoding Uaa imposes no restrictions on target protein type or cellular location, and a family of photoreactive Uaas is available for modulating different natural amino acid residues. This technique thus has the potential to be generally applied to many neuronal proteins to achieve optical regulation of different processes in brains. The current protocol presents an accessible procedure for intricate Uaa incorporation in neurons in vitro and in vivo to achieve photo control of neuronal protein activity on the molecular level.

The development of next-generation sequencing (NGS) technologies for HLA and KIR genotyping is rapidly advancing knowledge of genetic variation of these highly polymorphic loci. NGS genotyping is poised to replace older methods for clinical use, but standard methods for reporting and exchanging these new, high quality genotype data are needed. The Immunogenomic NGS Consortium, a broad collaboration of histocompatibility and immunogenetics clinicians, researchers, instrument manufacturers and software developers, has developed the Minimum Information for Reporting Immunogenomic NGS Genotyping (MIRING) reporting guidelines. MIRING is a checklist that specifies the content of NGS genotyping results as well as a set of messaging guidelines for reporting the results. A MIRING message includes five categories of structured information - message annotation, reference context, full genotype, consensus sequence and novel polymorphism - and references to three categories of accessory information - NGS platform documentation, read processing documentation and primary data. These eight categories of information ensure the long-term portability and broad application of this NGS data for all current histocompatibility and immunogenetics use cases. In addition, MIRING can be extended to allow the reporting of genotype data generated using pre-NGS technologies. Because genotyping results reported using MIRING are easily updated in accordance with reference and nomenclature databases, MIRING represents a bold departure from previous methods of reporting HLA and KIR genotyping results, which have provided static and less-portable data. More information about MIRING can be found online at miring.immunogenomics.org.

We present an electronic format for exchanging data for HLA and KIR genotyping with extensions for next-generation sequencing (NGS). This format addresses NGS data exchange by refining the Histoimmunogenetics Markup Language (HML) to conform to the proposed Minimum Information for Reporting Immunogenomic NGS Genotyping (MIRING) reporting guidelines (miring.immunogenomics.org). Our refinements of HML include two major additions. First, NGS is supported by new XML structures to capture additional NGS data and metadata required to produce a genotyping result, including analysis-dependent (dynamic) and method-dependent (static) components. A full genotype, consensus sequence, and the surrounding metadata are included directly, while the raw sequence reads and platform documentation are externally referenced. Second, genotype ambiguity is fully represented by integrating Genotype List Strings, which use a hierarchical set of delimiters to represent allele and genotype ambiguity in a complete and accurate fashion. HML also continues to enable the transmission of legacy methods (e.g. site-specific oligonucleotide, sequence-specific priming, and Sequence Based Typing (SBT)), adding features such as allowing multiple group-specific sequencing primers, and fully leveraging techniques that combine multiple methods to obtain a single result, such as SBT integrated with NGS.

The water channel aquaporin-4 (AQP4) forms supramolecular clusters whose size is determined by the ratio of M1- and M23-AQP4 isoforms. In cultured astrocytes, differences in the subcellular localization and macromolecular interactions of small and large AQP4 clusters results in distinct physiological roles for M1- and M23-AQP4. Here, we developed quantitative superresolution optical imaging methodology to measure AQP4 cluster size in antibody-stained paraffin sections of mouse cerebral cortex and spinal cord, human postmortem brain, and glioma biopsy specimens. This methodology was used to demonstrate that large AQP4 clusters are formed in AQP4(-/-) astrocytes transfected with only M23-AQP4, but not in those expressing only M1-AQP4, both in vitro and in vivo. Native AQP4 in mouse cortex, where both isoforms are expressed, was enriched in astrocyte foot-processes adjacent to microcapillaries; clusters in perivascular regions of the cortex were larger than in parenchymal regions, demonstrating size-dependent subcellular segregation of AQP4 clusters. Two-color superresolution imaging demonstrated colocalization of Kir4.1 with AQP4 clusters in perivascular areas but not in parenchyma. Surprisingly, the subcellular distribution of AQP4 clusters was different between gray and white matter astrocytes in spinal cord, demonstrating regional specificity in cluster polarization. Changes in AQP4 subcellular distribution are associated with several neurological diseases and we demonstrate that AQP4 clustering was preserved in a postmortem human cortical brain tissue specimen, but that AQP4 was not substantially clustered in a human glioblastoma specimen despite high-level expression. Our results demonstrate the utility of superresolution optical imaging for measuring the size of AQP4 supramolecular clusters in paraffin sections of brain tissue and support AQP4 cluster size as a primary determinant of its subcellular distribution.

Weak electric fields guide cell migration, known as galvanotaxis/electrotaxis. The sensor(s) cells use to detect the fields remain elusive. Here we perform a large-scale screen using an RNAi library targeting ion transporters in human cells. We identify 18 genes that show either defective or increased galvanotaxis after knockdown. Knockdown of the KCNJ15 gene (encoding inwardly rectifying K(+) channel Kir4.2) specifically abolishes galvanotaxis, without affecting basal motility and directional migration in a monolayer scratch assay. Depletion of cytoplasmic polyamines, highly positively charged small molecules that regulate Kir4.2 function, completely inhibits galvanotaxis, whereas increase of intracellular polyamines enhances galvanotaxis in a Kir4.2-dependent manner. Expression of a polyamine-binding defective mutant of KCNJ15 significantly decreases galvanotaxis. Knockdown or inhibition of KCNJ15 prevents phosphatidylinositol 3,4,5-triphosphate (PIP3) from distributing to the leading edge. Taken together these data suggest a previously unknown two-molecule sensing mechanism in which KCNJ15/Kir4.2 couples with polyamines in sensing weak electric fields.

Brain edema formation occurs after dysfunctional control of extracellular volume partly through impaired astrocytic ion and water transport. Here, we show that such processes might involve synergistic cooperation between the glial water channel aquaporin 4 (AQP4) and the transient receptor potential isoform 4 (TRPV4), a polymodal swelling-sensitive cation channel. In mouse retinas, TRPV4 colocalized with AQP4 in the end feet and radial processes of Müller astroglia. Genetic ablation of TRPV4 did not affect the distribution of AQP4 and vice versa. However, retinas from Trpv4(-/-) and Aqp4(-/-) mice exhibited suppressed transcription of genes encoding Trpv4, Aqp4, and the Kir4.1 subunit of inwardly rectifying potassium channels. Swelling and [Ca(2+)]i elevations evoked in Müller cells by hypotonic stimulation were antagonized by the selective TRPV4 antagonist HC-067047 (2-methyl-1-[3-(4-morpholinyl)propyl]-5-phenyl-N-[3-(trifluoromethyl)phenyl]-1H-pyrrole-3-carboxamide) or Trpv4 ablation. Elimination of Aqp4 suppressed swelling-induced [Ca(2+)]i elevations but only modestly attenuated the amplitude of Ca(2+) signals evoked by the TRPV4 agonist GSK1016790A [(N-((1S)-1-{[4-((2S)-2-{[(2,4-dichlorophenyl)sulfonyl]amino}-3-hydroxypropanoyl)-1-piperazinyl]carbonyl}-3-methylbutyl)-1-benzothiophene-2-carboxamide]. Glial cells lacking TRPV4 but not AQP4 showed deficits in hypotonic swelling and regulatory volume decrease. Functional synergy between TRPV4 and AQP4 during cell swelling was confirmed in the heterologously expressing Xenopus oocyte model. Importantly, when the swelling rate was osmotically matched for AQP4-positive and AQP4-negative oocytes, TRPV4 activation became independent of AQP4. We conclude that AQP4-mediated water fluxes promote the activation of the swelling sensor, whereas Ca(2+) entry through TRPV4 channels reciprocally modulates volume regulation, swelling, and Aqp4 gene expression. Therefore, TRPV4-AQP4 interactions constitute a molecular system that fine-tunes astroglial volume regulation by integrating osmosensing, calcium signaling, and water transport and, when overactivated, triggers pathological swelling. Significance statement: We characterize the physiological features of interactions between the astroglial swelling sensor transient receptor potential isoform 4 (TRPV4) and the aquaporin 4 (AQP4) water channel in retinal Müller cells. Our data reveal an elegant and complex set of mechanisms involving reciprocal interactions at the level of glial gene expression, calcium homeostasis, swelling, and volume regulation. Specifically, water influx through AQP4 drives calcium influx via TRPV4 in the glial end foot, which regulates expression of Aqp4 and Kir4.1 genes and facilitates the time course and amplitude of hypotonicity-induced swelling and regulatory volume decrease. We confirm the crucial facets of the signaling mechanism in heterologously expressing oocytes. These results identify the molecular mechanism that contributes to dynamic regulation of glial volume but also provide new insights into the pathophysiology of glial reactivity and edema formation.

Key pointsHeart failure leads to dramatic electrophysiological remodelling as a result of numerous cellular and tissue-level changes. Important cellular changes include increased sensitivity of ryanodine receptors (RyRs) to Ca(2+) release and down-regulation of the inward rectifying K(+) current (IK1), both of which contribute to triggered action potentials in isolated cells. We studied the role of increased RyR sensitivity and decreased IK1 in contributing to focal arrhythmia in the intact non-failing rabbit heart using optical mapping and pharmacological manipulation of RyRs and IK1. Neither increased RyR sensitivity or decreased IK1 alone led to significant increases in arrhythmia following local sympathetic stimulation; however, in combination, these two factors led to a significant increase in premature ventricular complexes and focal ventricular tachycardia. These results suggest synergism between increased RyR sensitivity and decreased IK1 in contributing to focal arrhythmia in the intact heart and may provide important insights into novel anti-arrhythmic treatments in heart failure.AbstractHeart failure (HF) results in dramatic electrophysiological remodelling, including increased sensitivity of ryanodine receptors (RyRs) and decreased inward rectifying K(+) current (IK1), which predisposes HF myocytes to delayed afterdepolarizations and triggered activity. Therefore, we sought to determine the role of increased RyR sensitivity and decreased IK1 in contributing to focal arrhythmia in the intact non-failing heart. Optical mapping of transmembrane potential and intracellular Ca(2+) was performed in Langendorff-perfused rabbit hearts (n = 15). Local β-adrenergic receptor stimulation with noradrenaline (norepinephrine; NA, 50 μl, 250 μM) was applied to elicit focal activity (premature ventricular complexes (PVCs) or ventricular tachycardia (VT ≥ 3 beats)). NA was administered under control conditions (CTL) and following pretreatment with 50 μM BaCl2 to reduce IK1, or 200 μM caffeine (Caff) to sensitize RyRs, both alone and in combination. Local NA injection resulted in Ca(2+)-driven PVCs arising from the injection site in all hearts studied. No increase in NA-mediated PVCs was observed following pretreatment with either BaCl2 or Caff alone (CTL: 1.1 ± 0.7, BaCl2: 1.0 ± 0.7, Caff: 1.3 ± 0.8 PVCs/injection, P not significant). However, pretreatment with the combination of BaCl2 + Caff resulted in a significant increase in PVCs (2.3 ± 2.8 PVCs/injection, P < 0.05 vs. CTL, BaCl2, Caff). Additionally, pretreatment with BaCl2 + Caff led to sustained monomorphic VT arising from the NA application site in all hearts studied, which lasted up to 6 min following a single NA injection. VT was never observed under any other condition suggesting synergism between increased RyR sensitivity and decreased IK1 in contributing to focal activity. These findings may have important implications for the understanding and prevention of focal arrhythmia in HF.

IntroductionAndersen-Tawil syndrome (ATS) is a potassium channelopathy affecting cardiac and skeletal muscle. Periodic paralysis is a presenting symptom in some patients, whereas, in others, symptomatic arrhythmias or prolongation of QT in echocardiographic recordings will lead to diagnosis of ATS. Striking intrafamilial variability of expression of KCNJ2 mutations and rarity of the syndrome may lead to misdiagnosis.MethodsWe report 15 patients from 8 Polish families with ATS, including 3 with novel KCNJ2 mutations.ResultsAll patients had dysmorphic features; periodic paralysis affected males more frequently than females (80% vs. 20%), and most attacks were normokalemic. Two patients (with T75M and T309I mutations) had aborted sudden cardiac death. An implantable cardioverter-defibrillator was utilized in 40% of cases.ConclusionsKCNJ2 mutations cause a variable phenotype, with dysmorphic features seen in all patients studied, a high penetrance of periodic paralysis in males and ventricular arrhythmia with a risk of sudden cardiac death.

The principal cell of the kidney collecting duct is one of the most highly regulated epithelial cell types in vertebrates. The effects of hormonal, autocrine, and paracrine factors to regulate principal cell transport processes are central to the maintenance of fluid and electrolyte balance in the face of wide variations in food and water intake. In marked contrast with the epithelial cells lining the proximal tubule, the collecting duct is electrically tight, and ion and osmotic gradients can be very high. The central role of principal cells in salt and water transport is reflected by their defining transporters-the epithelial Na(+) channel (ENaC), the renal outer medullary K(+) channel, and the aquaporin 2 (AQP2) water channel. The coordinated regulation of ENaC by aldosterone, and AQP2 by arginine vasopressin (AVP) in principal cells is essential for the control of plasma Na(+) and K(+) concentrations, extracellular fluid volume, and BP. In addition to these essential hormones, additional neuronal, physical, and chemical factors influence Na(+), K(+), and water homeostasis. Notably, a variety of secreted paracrine and autocrine agents such as bradykinin, ATP, endothelin, nitric oxide, and prostaglandin E2 counterbalance and limit the natriferic effects of aldosterone and the water-retaining effects of AVP. Considerable recent progress has improved our understanding of the transporters, receptors, second messengers, and signaling events that mediate principal cell responses to changing environments in health and disease. This review primarily addresses the structure and function of the key transporters and the complex interplay of regulatory factors that modulate principal cell ion and water transport.

Dietary and metabolic therapies are increasingly being considered for a variety of neurological disorders, based in part on growing evidence for the neuroprotective properties of the ketogenic diet (KD) and ketones. Earlier, we demonstrated that ketones afford hippocampal synaptic protection against exogenous oxidative stress, but the mechanisms underlying these actions remain unclear. Recent studies have shown that ketones may modulate neuronal firing through interactions with ATP-sensitive potassium (KATP) channels. Here, we used a combination of electrophysiological, pharmacological, and biochemical assays to determine whether hippocampal synaptic protection by ketones is a consequence of KATP channel activation. Ketones dose-dependently reversed oxidative impairment of hippocampal synaptic integrity, neuronal viability, and bioenergetic capacity, and this action was mirrored by the KATP channel activator diazoxide. Inhibition of KATP channels reversed ketone-evoked hippocampal protection, and genetic ablation of the inwardly rectifying K+ channel subunit Kir6.2, a critical component of KATP channels, partially negated the synaptic protection afforded by ketones. This partial protection was completely reversed by co-application of the KATP blocker, 5-hydoxydecanoate (5HD). We conclude that, under conditions of oxidative injury, ketones induce synaptic protection in part through activation of KATP channels.

In this Viewpoint, we summarize and discuss the recent serendipitous discovery of an astrocyte Kir4.1 potassium channel dysfunction in two mouse models of Huntington's disease (HD). Restoration of Kir4.1 channels within astrocytes in vivo attenuated neuronal dysfunction, some aspects of motor dysfunction and increased survival time in a HD mouse model. Overall, the data show that aspects of altered neuronal excitability associated with HD may be secondary to changes in astrocyte-mediated K(+) homeostasis, thereby revealing a new striatal neural microcircuit mechanism in HD, and Kir4.1 channels and astrocytes as potential therapeutic targets for drug development.

Huntington's disease (HD) is characterized by striatal medium spiny neuron (MSN) dysfunction, but the underlying mechanisms remain unclear. We explored roles for astrocytes, in which mutant huntingtin is expressed in HD patients and mouse models. We found that symptom onset in R6/2 and Q175 HD mouse models was not associated with classical astrogliosis, but was associated with decreased Kir4.1 K(+) channel functional expression, leading to elevated in vivo striatal extracellular K(+), which increased MSN excitability in vitro. Viral delivery of Kir4.1 channels to striatal astrocytes restored Kir4.1 function, normalized extracellular K(+), ameliorated aspects of MSN dysfunction, prolonged survival and attenuated some motor phenotypes in R6/2 mice. These findings indicate that components of altered MSN excitability in HD may be caused by heretofore unknown disturbances of astrocyte-mediated K(+) homeostasis, revealing astrocytes and Kir4.1 channels as therapeutic targets.

Aims/hypothesisMore than 90% of Chinese familial early-onset type 2 diabetes mellitus is genetically unexplained. To investigate the molecular aetiology, we identified and characterised whether mutations in the KCNJ11 gene are responsible for these families.MethodsKCNJ11 mutations were screened for 96 familial early-onset type 2 diabetic probands and their families. Functional significance of the identified mutations was confirmed by physiological analysis, molecular modelling and population survey.ResultsThree novel KCNJ11 mutations, R27H, R192H and S116F117del, were identified in three families with early-onset type 2 diabetes mellitus. Mutated KCNJ11 with R27H or R192H markedly reduced ATP sensitivity (E23K>R27H>C42R>R192H>R201H), but no ATP-sensitive potassium channel currents were detected in the loss-of-function S116F117del channel in vitro. Molecular modelling indicated that R192H had a larger effect on the channel ATP-binding pocket than R27H, which may qualitatively explain why the ATP sensitivity of the R192H mutation is seven times less than R27H. The shape of the S116F117del channel may be compressed, which may explain why the mutated channel had no currents. Discontinuation of insulin and implementation of sulfonylureas for R27H or R192H carriers and continuation/switch to insulin therapy for S116F117del carriers resulted in good glycaemic control.Conclusions/interpretationOur results suggest that genetic diagnosis for the KCNJ11 mutations in familial early-onset type 2 diabetes mellitus may help in understanding the molecular aetiology and in providing more personalised treatment for these specific forms of diabetes in Chinese and other Asian patients.

Optical control of protein function provides excellent spatial-temporal resolution for studying proteins in situ. Although light-sensitive exogenous proteins and ligands have been used to manipulate neuronal activity, a method for optical control of neuronal proteins using unnatural amino acids (Uaa) in vivo is lacking. Here, we describe the genetic incorporation of a photoreactive Uaa into the pore of an inwardly rectifying potassium channel Kir2.1. The Uaa occluded the pore, rendering the channel nonconducting, and, on brief light illumination, was released to permit outward K(+) current. Expression of this photoinducible inwardly rectifying potassium (PIRK) channel in rat hippocampal neurons created a light-activatable PIRK switch for suppressing neuronal firing. We also expanded the genetic code of mammals to express PIRK channels in embryonic mouse neocortex in vivo and demonstrated a light-activated PIRK current in cortical neurons. These principles could be generally expanded to other proteins expressed in the brain to enable optical regulation.

Analysis of ion channel mutants is a widely used approach for dissecting ion channel function and for characterizing the mechanisms of action of channel-directed modulators. Expression of functional potassium channels in potassium-uptake-deficient yeast together with genetic selection approaches offers an unbiased, high-throughput, activity-based readout that can rapidly identify large numbers of active ion channel mutants. Because of the assumption-free nature of the method, detailed biophysical analysis of the functional mutants from such selections can provide new and unexpected insights into both ion channel gating and ion channel modulator mechanisms. Here, we present detailed protocols for generation and identification of functional mutations in potassium channels using yeast selections in the potassium-uptake-deficient strain SGY1528. This approach is applicable for the analysis of structure-function relationships of potassium channels from a wide range of sources including viruses, bacteria, plants, and mammals and can be used as a facile way to probe the interactions between ion channels and small-molecule modulators.

Mutations that disrupt function of the human inwardly rectifying potassium channel KIR2.1 are associated with the craniofacial and digital defects of Andersen-Tawil Syndrome, but the contribution of Kir channels to development is undefined. Deletion of mouse Kir2.1 also causes cleft palate and digital defects. These defects are strikingly similar to phenotypes that result from disrupted TGFβ/BMP signaling. We use Drosophila melanogaster to show that a Kir2.1 homolog, Irk2, affects development by disrupting BMP signaling. Phenotypes of irk2 deficient lines, a mutant irk2 allele, irk2 siRNA and expression of a dominant-negative Irk2 subunit (Irk2DN) all demonstrate that Irk2 function is necessary for development of the adult wing. Compromised Irk2 function causes wing-patterning defects similar to those found when signaling through a Drosophila BMP homolog, Decapentaplegic (Dpp), is disrupted. To determine whether Irk2 plays a role in the Dpp pathway, we generated flies in which both Irk2 and Dpp functions are reduced. Irk2DN phenotypes are enhanced by decreased Dpp signaling. In wild-type flies, Dpp signaling can be detected in stripes along the anterior/posterior boundary of the larval imaginal wing disc. Reducing function of Irk2 with siRNA, an irk2 deletion, or expression of Irk2DN reduces the Dpp signal in the wing disc. As Irk channels contribute to Dpp signaling in flies, a similar role for Kir2.1 in BMP signaling may explain the morphological defects of Andersen-Tawil Syndrome and the Kir2.1 knockout mouse.

ObjectiveType 2 diabetes is a common complex disorder with environmental and genetic components. We used a candidate gene-based approach to identify single nucleotide polymorphism (SNP) variants in 222 candidate genes that influence susceptibility to type 2 diabetes.Research design and methodsIn a case-control study of 1,161 type 2 diabetic subjects and 1,174 control Finns who are normal glucose tolerant, we genotyped 3,531 tagSNPs and annotation-based SNPs and imputed an additional 7,498 SNPs, providing 99.9% coverage of common HapMap variants in the 222 candidate genes. Selected SNPs were genotyped in an additional 1,211 type 2 diabetic case subjects and 1,259 control subjects who are normal glucose tolerant, also from Finland.ResultsUsing SNP- and gene-based analysis methods, we replicated previously reported SNP-type 2 diabetes associations in PPARG, KCNJ11, and SLC2A2; identified significant SNPs in genes with previously reported associations (ENPP1 [rs2021966, P = 0.00026] and NRF1 [rs1882095, P = 0.00096]); and implicated novel genes, including RAPGEF1 (rs4740283, P = 0.00013) and TP53 (rs1042522, Arg72Pro, P = 0.00086), in type 2 diabetes susceptibility.ConclusionsOur study provides an effective gene-based approach to association study design and analysis. One or more of the newly implicated genes may contribute to type 2 diabetes pathogenesis. Analysis of additional samples will be necessary to determine their effect on susceptibility.

Phosphatidylinosital-4,5-bisphosphate (PIP2) acts as an essential factor regulating the activity of all Kir channels. In most Kir members, the dependence on PIP2 is modulated by other factors, such as protein kinases (in Kir1), G(betagamma) (in Kir3), and the sulfonylurea receptor (in Kir6). So far, however, no regulator has been identified in Kir2 channels. Here we show that polyamines, which cause inward rectification by selectively blocking outward current, also regulate the interaction of PIP2 with Kir2.1 channels to maintain channel availability. Using spermine and diamines as polyamine analogs, we demonstrate that both spontaneous and PIP2 antibody-induced rundown of Kir2.1 channels in excised inside-out patches was markedly slowed by long polyamines; in contrast, polyamines with shorter chain length were ineffective. In K188Q mutant channels, which have a low PIP2 affinity, application PIP2 (10 microM) was unable to activate channel activity in the absence of polyamines, but markedly activated channels in the presence of long diamines. Using neomycin as a measure of PIP2 affinity, we found that long polyamines were capable of strengthening either the wild type or K188Q channels' interaction with PIP2. The negatively charged D172 residue inside the transmembrane pore region was critical for the shift of channel-PIP2 binding affinity by long polyamines. Sustained pore block by polyamines was neither sufficient nor necessary for this effect. We conclude that long polyamines serve a dual role as both blockers and coactivators (with PIP2) of Kir2.1 channels.

The Mg2+-inhibited cation (MIC) current, believed to represent activity of TRPM7 channels, is found in lymphocytes and mast cells, cardiac and smooth muscle, and several other eukaryotic cell types. MIC current is activated during whole-cell dialysis with divalent-free internal solutions. Millimolar concentrations of intracellular Mg2+ (or other divalent metal cations) inhibit the channels in a voltage-independent manner. The nature of divalent inhibition and the mechanism of channel activation in an intact cell remain unknown. We show that the polyamines (spermine, spermidine, and putrescine) inhibit the MIC current, also in a voltage-independent manner, with a potency that parallels the number of charges. Neomycin and poly-lysine also potently inhibited MIC current in the absence of Mg2+. These same positively charged ions inhibited IRK1 current in parallel with MIC current, suggesting that they probably act by screening the head group phosphates on PIP2 and other membrane phospholipids. In agreement with this hypothesis, internal protons also inhibited MIC current. By contrast, tetramethylammonium, tetraethylammonium, and hexamethonium produced voltage-dependent block but no inhibition. We show that inhibition by internal polyvalent cations can be relieved by alkalinizing the cytosol using externally applied ammonium or by increasing pH in inside-out patches. Furthermore, in perforated-patch and cell-attached recordings, when intracellular Mg2+ is not depleted, endogenous MIC or recombinant TRPM7 currents are activated by cytosolic alkalinization and inhibited by acidification; and they can be reactivated by PIP2 following rundown in inside-out patches. We propose that MIC (TRPM7) channels are regulated by a charge screening mechanism and may function as sensors of intracellular pH.

Animal and plant voltage-gated ion channels share a common architecture. They are made up of four subunits and the positive charges on helical S4 segments of the protein in animal K+ channels are the main voltage-sensing elements. The KAT1 channel cloned from Arabidopsis thaliana, despite its structural similarity to animal outward rectifier K+ channels is, however, an inward rectifier. Here we detected KAT1-gating currents due to the existence of an intrinsic voltage sensor in this channel. The measured gating currents evoked in response to hyperpolarizing voltage steps consist of a very fast (tau = 318 +/- 34 micros at -180 mV) and a slower component (4.5 +/- 0.5 ms at -180 mV) representing charge moved when most channels are closed. The observed gating currents precede in time the ionic currents and they are measurable at voltages (less than or equal to -60) at which the channel open probability is negligible ( approximately 10-4). These two observations, together with the fact that there is a delay in the onset of the ionic currents, indicate that gating charge transits between several closed states before the KAT1 channel opens. To gain insight into the molecular mechanisms that give rise to the gating currents and lead to channel opening, we probed external accessibility of S4 domain residues to methanethiosulfonate-ethyltrimethylammonium (MTSET) in both closed and open cysteine-substituted KAT1 channels. The results demonstrate that the putative voltage-sensing charges of S4 move inward when the KAT1 channels open.

In rat basophilic leukemia (RBL) cells and Jurkat T cells, Ca(2+) release-activated Ca(2+) (CRAC) channels open in response to passive Ca(2+) store depletion. Inwardly rectifying CRAC channels admit monovalent cations when external divalent ions are removed. Removal of internal Mg(2+) exposes an outwardly rectifying current (Mg(2+)-inhibited cation [MIC]) that also admits monovalent cations when external divalent ions are removed. Here we demonstrate that CRAC and MIC currents are separable by ion selectivity and rectification properties: by kinetics of activation and susceptibility to run-down and by pharmacological sensitivity to external Mg(2+), spermine, and SKF-96365. Importantly, selective run-down of MIC current allowed CRAC and MIC current to be characterized under identical ionic conditions with low internal Mg(2+). Removal of internal Mg(2+) induced MIC current despite widely varying Ca(2+) and EGTA levels, suggesting that Ca(2+)-store depletion is not involved in activation of MIC channels. Increasing internal Mg(2+) from submicromolar to millimolar levels decreased MIC currents without affecting rectification but did not alter CRAC current rectification or amplitudes. External Mg(2+) and Cs(+) carried current through MIC but not CRAC channels. SKF-96365 blocked CRAC current reversibly but inhibited MIC current irreversibly. At micromolar concentrations, both spermine and extracellular Mg(2+) blocked monovalent MIC current reversibly but not monovalent CRAC current. The biophysical characteristics of MIC current match well with cloned and expressed TRPM7 channels. Previous results are reevaluated in terms of separate CRAC and MIC channels.

K(ATP) channels, comprised of the pore-forming protein Kir6.x and the sulfonylurea receptor SURx, are regulated in an interdependent manner by adenine nucleotides, PIP2, and sulfonylureas. To gain insight into these interactions, we investigated the effects of mutating positively charged residues in Kir6.2, previously implicated in the response to PIP2, on channel regulation by adenine nucleotides and the sulfonylurea glyburide. Our data show that the Kir6.2 "PIP2-insensitive" mutants R176C and R177C are not reactivated by MgADP after ATP-induced inhibition and are also insensitive to glyburide. These results suggest that R176 and R177 are required for functional coupling to SUR1, which confers MgADP and sulfonylurea sensitivity to the K(ATP) channel. In contrast, the R301C and R314C mutants, which are also "PIP2-insensitive," remained sensitive to stimulation by MgADP in the absence of ATP and were inhibited by glyburide. Based on these findings, as well as previous data, we propose a model of the K(ATP) channel whereby in the presence of ATP, the R176 and R177 residues on Kir6.2 form a specific site that interacts with NBF1 bound to ATP on SUR1, promoting channel opening by counteracting the inhibition by ATP. This interaction is facilitated by binding of MgADP to NBF2 and blocked by binding of sulfonylureas to SUR1. In the absence of ATP, since K(ATP) channels are not blocked by ATP, they do not require the counteracting effect of NBF1 interacting with R176 and R177 to open. Nevertheless, channels in this state remain activated by MgADP. This effect may be explained by a direct stimulatory interaction of NBF2/MgADP moiety with another region of Kir6.2 (perhaps the NH2 terminus), or by NBF2/MgADP still promoting a weak interaction between NBF1 and Kir6.2 in the absence of ATP. The region delimited by R301 and R314 is not involved in the interaction with NBF1 or NBF2, but confers additional PIP2 sensitivity.

Kir6.2 channels linked to the green fluorescent protein (GFP) (Kir6. 2-GFP) have been expressed alone or with the sulfonylurea receptor SUR1 in HEK293 cells to study the regulation of K(ATP) channels by adenine nucleotides, phosphatidylinositol bisphosphate (PIP(2)), and phosphorylation. Upon excision of inside-out patches into a Ca(2+)- and MgATP-free solution, the activity of Kir6.2-GFP+SUR1 channels spontaneously ran down, first quickly within a minute, and then more slowly over tens of minutes. In contrast, under the same conditions, the activity of Kir6.2-GFP alone exhibited only slow rundown. Thus, fast rundown is specific to Kir6.2-GFP+SUR1 and involves SUR1, while slow rundown is a property of both Kir6.2-GFP and Kir6.2-GFP+SUR1 channels and is due, at least in part, to Kir6.2 alone. Kir6. 2-GFP+SUR1 fast phase of rundown was of variable amplitude and led to increased ATP sensitivity. Excising patches into a solution containing MgADP prevented this phenomenon, suggesting that fast rundown involves loss of MgADP-dependent stimulation conferred by SUR1. With both Kir6.2-GFP and Kir6.2-GFP+SUR1, the slow phase of rundown led to further increase in ATP sensitivity. Ca(2+) accelerated this process, suggesting a role for PIP(2) hydrolysis mediated by a Ca(2+)-dependent phospholipase C. PIP(2) could reactivate channel activity after a brief exposure to Ca(2+), but not after prolonged exposure. However, in both cases, PIP(2) reversed the increase in ATP sensitivity, indicating that PIP(2) lowers the ATP sensitivity by increasing P(o) as well as by decreasing the channel affinity for ATP. With Kir6.2-GFP+SUR1, slow rundown also caused loss of MgADP stimulation and sulfonylurea inhibition, suggesting functional uncoupling of SUR1 from Kir6.2-GFP. Ca(2+) facilitated the loss of sensitivity to MgADP, and thus uncoupling of the two subunits. The nonselective protein kinase inhibitor H-7 and the selective PKC inhibitor peptide 19-36 evoked, within 5-15 min, increased ATP sensitivity and loss of reactivation by PIP(2) and MgADP. Phosphorylation of Kir6.2 may thus be required for the channel to remain PIP(2) responsive, while phosphorylation of Kir6.2 and/or SUR1 is required for functional coupling. In summary, short-term regulation of Kir6.2+SUR1 channels involves MgADP, while long-term regulation requires PIP(2) and phosphorylation.

Inward rectifying K channels are essential for maintaining resting membrane potential and regulating excitability in many cell types. Previous studies have attributed the rectification properties of strong inward rectifiers such as Kir2.1 to voltage-dependent binding of intracellular polyamines or Mg to the pore (direct open channel block), thereby preventing outward passage of K ions. We have studied interactions between polyamines and the polyamine toxins philanthotoxin and argiotoxin on inward rectification in Kir2.1. We present evidence that high affinity polyamine block is not consistent with direct open channel block, but instead involves polyamines binding to another region of the channel (intrinsic gate) to form a blocking complex that occludes the pore. This interaction defines a novel mechanism of ion channel closure.

A K+ channel gene has been cloned from Drosophila melanogaster by complementation in Saccharomyces cerevisiae cells defective for K+ uptake. Naturally expressed in the neuromuscular tissues of adult flies, this gene confers K+ transport capacity on yeast cells when heterologously expressed. In Xenopus laevis oocytes, expression yields an ungated K(+)-selective current whose attributes resemble the "leak" conductance thought to mediate the resting potential of vertebrate myelinated neurons but whose molecular nature has long remained elusive. The predicted protein has two pore (P) domains and four membrane-spanning helices and is a member of a newly recognized K+ channel family. Expression of the channel in flies and yeast cells makes feasible studies of structure and in vivo function using genetic approaches that are not possible in higher animals.