Acid sensitive background potassium channels K2P3.1 and K2P9.1 undergo rapid dynamin-dependent endocytosis

Acid-sensitive, two-pore domain potassium channels, K_2P3.1 and K_2P9.1, are implicated in cardiac and nervous tissue responses to hormones, neurotransmitters and drugs. K_2P3.1 and K_2P9.1 leak potassium from the cell at rest and directly impact membrane potential. Hence altering channel number on the cell surface drives changes in cellular electrical properties. The rate of K_2P3.1 and K_2P9.1 delivery to and recovery from the plasma membrane determines both channel number at the cell surface and potassium leak from cells. This study examines the endocytosis of K_2P3.1 and K_2P9.1. Plasma membrane biotinylation was used to follow the fate of internalized GFP-tagged rat K_2P3.1 and K_2P9.1 transiently expressed in HeLa cells. Confocal fluorescence images were analyzed using Imaris software, which revealed that both channels are endocytosed by a dynamin-dependent mechanism and over the course of 60 min, move progressively toward the nucleus. Endogenous endocytosis of human K_2P3.1 and K_2P9.1 was examined in the lung carcinoma cell line, A549. Endogenous channels are endocytosed over a similar time-scale to the channels expressed transiently in HeLa cells. These findings both validate the use of recombinant systems and identify an endogenous model system in which K_2P3.1 and K_2P9.1 trafficking can be further studied.     

Altered Expression of Two-Pore Domain Potassium (K2P) Channels in Cancer

Potassium channels have become a focus in cancer biology as they play roles in cell behaviours associated with cancer progression, including proliferation, migration and apoptosis. Two-pore domain (K_2P) potassium channels are background channels which enable the leak of potassium ions from cells. As these channels are open at rest they have a profound effect on cellular membrane potential and subsequently the electrical activity and behaviour of cells in which they are expressed. The K_2P family of channels has 15 mammalian members and already 4 members of this family (K_2P2.1, K_2P3.1, K_2P9.1, K_2P5.1) have been implicated in cancer. Here we examine the expression of all 15 members of the K_2P family of channels in a range of cancer types. This was achieved using the online cancer microarray database, Oncomine (www.oncomine.org). Each gene was examined across 20 cancer types, comparing mRNA expression in cancer to normal tissue. This analysis revealed all but 3 K_2P family members (K_2P4.1, K_2P16.1, K_2P18.1) show altered expression in cancer. Overexpression of K_2P channels was observed in a range of cancers including breast, leukaemia and lung while more cancers (brain, colorectal, gastrointestinal, kidney, lung, melanoma, oesophageal) showed underexpression of one or more channels. K_2P1.1, K_2P3.1, K_2P12.1, were overexpressed in a range of cancers. While K_2P1.1, K_2P3.1, K_2P5.1, K_2P6.1, K_2P7.1 and K_2P10.1 showed significant underexpression across the cancer types examined. This analysis supports the view that specific K_2P channels may play a role in cancer biology. Their altered expression together with their ability to impact the function of other ion channels and their sensitivity to environmental stimuli (pO2, pH, glucose, stretch) makes understanding the role these channels play in cancer of key importance.     

Membrane Trafficking of Large Conductance Calcium-activated Potassium Channels Is Regulated by Alternative Splicing of a Transplantable, Acidic Trafficking Motif in the RCK1-RCK2 Linker

Trafficking of the pore-forming α-subunits of large conductance calcium- and voltage-activated potassium (BK) channels to the cell surface represents an important regulatory step in controlling BK channel function. Here, we identify multiple trafficking signals within the intracellular RCK1-RCK2 linker of the cytosolic C terminus of the channel that are required for efficient cell surface expression of the channel. In particular, an acidic cluster-like motif was essential for channel exit from the endoplasmic reticulum and subsequent cell surface expression. This motif could be transplanted onto a heterologous nonchannel protein to enhance cell surface expression by accelerating endoplasmic reticulum export. Importantly, we identified a human alternatively spliced BK channel variant, hSloΔ_579–664, in which these trafficking signals are excluded because of in-frame exon skipping. The hSloΔ_579–664 variant is expressed in multiple human tissues and cannot form functional channels at the cell surface even though it retains the putative RCK domains and downstream trafficking signals. Functionally, the hSloΔ_579–664 variant acts as a dominant negative subunit to suppress cell surface expression of BK channels. Thus alternative splicing of the intracellular RCK1-RCK2 linker plays a critical role in determining cell surface expression of BK channels by controlling the inclusion/exclusion of multiple trafficking motifs.     

Protein kinase A is central for forward transport of two-pore domain potassium channels K2P3.1 and K2P9.1

Acid-sensitive two-pore domain potassium channels (K2P3.1 and K2P9.1) play key roles in both physiological and pathophysiological mechanisms, the most fundamental of which is control of resting membrane potential of cells in which they are expressed. These background "leak" channels are constitutively active once expressed at the plasma membrane, and hence tight control of their targeting and surface expression is fundamental to the regulation of K(+) flux and cell excitability. The chaperone protein, 14-3-3, binds to a critical phosphorylated serine in the channel C termini of K2P3.1 and K2P9.1 (Ser(393) and Ser(373), respectively) and overcomes retention in the endoplasmic reticulum by βCOP. We sought to identify the kinase responsible for phosphorylation of the terminal serine in human and rat variants of K2P3.1 and K2P9.1. Adopting a bioinformatic approach, three candidate protein kinases were identified: cAMP-dependent protein kinase, ribosomal S6 kinase, and protein kinase C. In vitro phosphorylation assays were utilized to determine the ability of the candidate kinases to phosphorylate the channel C termini. Electrophysiological measurements of human K2P3.1 transiently expressed in HEK293 cells and cell surface assays of GFP-tagged K2P3.1 and K2P9.1 enabled the determination of the functional implications of phosphorylation by specific kinases. All of our findings support the conclusion that cAMP-dependent protein kinase is responsible for the phosphorylation of the terminal serine in both K2P3.1 and K2P9.1.     

Identification and functional characterization of zebrafish K2P10.1 (TREK2) two-pore-domain K+ channels

Two-pore-domain potassium (K_2P) channels mediate K⁺ background currents that stabilize the resting membrane potential and contribute to repolarization of action potentials in excitable cells. The functional significance of K_2P currents in cardiac electrophysiology remains poorly understood. Danio rerio (zebrafish) may be utilized to elucidate the role of cardiac K_2P channels in vivo. The aim of this work was to identify and functionally characterize a zebrafish otholog of the human K_2P10.1 channel. K_2P10.1 orthologs in the D. rerio genome were identified by database analysis, and the full zK_2P10.1 coding sequence was amplified from zebrafish cDNA. Human and zebrafish K_2P10.1 proteins share 61% identity. High degrees of conservation were observed in protein domains relevant for structural integrity and regulation. K_2P10.1 channels were heterologously expressed in Xenopus oocytes, and currents were recorded using two-electrode voltage clamp electrophysiology. Human and zebrafish channels mediated K⁺ selective background currents leading to membrane hyperpolarization. Arachidonic acid, an activator of hK_2P10.1, induced robust activation of zK_2P10.1. Activity of both channels was reduced by protein kinase C. Similar to its human counterpart, zK_2P10.1 was inhibited by the antiarrhythmic drug amiodarone. In summary, zebrafish harbor K_2P10.1 two-pore-domain K⁺ channels that exhibit structural and functional properties largely similar to human K_2P10.1. We conclude that the zebrafish represents a valid model to study K_2P10.1 function in vivo.     

Identification of endocrine cells of the stomach that express acid-sensitive background potassium (K<Subscript>2P</Subscript>9.1/TASK3) channels

The family of two-pore domain potassium (K_2P) channels is important in setting and controlling the background potassium current of excitable cells. This study examines the localisation of the acid-sensitive channel, K_2P9.1 (TASK3), in cells of the gastric mucosa. We observed K_2P9.1 immunoreactivity in endocrine cells of the mucosal glands of the guinea-pig, rat and mouse but the channels were not detected in parietal, chief, or mucous cells. K_2P9.1 channel immunoreactivity was consistently co-localised with histidine decarboxylase immunopositive enterochromaffin-like (ECL) cells, and with the majority of ghrelin immunoreactive X/A cells. Localisation in somatostatin immunoreactive D cells was rare in the guinea-pig, and did not occur in the stomach of rat, but, in the mouse, K_2P9.1 channels were observed in the majority of somatostatin-immunoreactive D cells. Conversely, sections taken from the guinea-pig and mouse stomachs, but not rat stomach, revealed K_2P9.1 in gastrin-containing G cells. These results demonstrate the presence of K_2P9.1 channels in the entero-endocrine ECL, G and D cell populations of the stomach that regulate acid secretion through the release of histamine, gastrin and somatostatin. K_2P9.1 channels were located in the ghrelin X/A cells that regulate food intake.     

Structural Insights into the Mechanisms and Pharmacology of K2P Potassium Channels

Leak currents, defined as voltage and time independent flows of ions across cell membranes, are central to cellular electrical excitability control. The K_2P (KCNK) potassium channel class comprises an ion channel family that produces potassium leak currents that oppose excitation and stabilize the resting membrane potential in cells in the brain, cardiovascular system, immune system, and sensory organs. Due to their widespread tissue distribution, K_2Ps contribute to many physiological and pathophysiological processes including anesthesia, pain, arrythmias, ischemia, hypertension, migraine, intraocular pressure regulation, and lung injury responses. Structural studies of six homomeric K_2Ps have established the basic architecture of this channel family, revealed key moving parts involved in K_2P function, uncovered the importance of asymmetric pinching and dilation motions in the K_2P selectivity filter (SF) C-type gate, and defined two K_2P structural classes based on the absence or presence of an intracellular gate. Further, a series of structures characterizing K_2P:modulator interactions have revealed a striking polysite pharmacology housed within a relatively modestly sized (~70 kDa) channel. Binding sites for small molecules or lipids that control channel function are found at every layer of the channel structure, starting from its extracellular side through the portion that interacts with the membrane bilayer inner leaflet. This framework provides the basis for understanding how gating cues sensed by different channel parts control function and how small molecules and lipids modulate K_2P activity. Such knowledge should catalyze development of new K_2P modulators to probe function and treat a wide range of disorders.     

Carbamazepine as a Novel Small Molecule Corrector of Trafficking-impaired ATP-sensitive Potassium Channels Identified in Congenital Hyperinsulinism

ATP-sensitive potassium (K_ATP) channels consisting of sulfonylurea receptor 1 (SUR1) and the potassium channel Kir6.2 play a key role in insulin secretion by coupling metabolic signals to β-cell membrane potential. Mutations in SUR1 and Kir6.2 that impair channel trafficking to the cell surface lead to loss of channel function and congenital hyperinsulinism. We report that carbamazepine, an anticonvulsant, corrects the trafficking defects of mutant K_ATP channels previously identified in congenital hyperinsulinism. Strikingly, of the 19 SUR1 mutations examined, only those located in the first transmembrane domain of SUR1 responded to the drug. We show that unlike that reported for several other protein misfolding diseases, carbamazepine did not correct K_ATP channel trafficking defects by activating autophagy; rather, it directly improved the biogenesis efficiency of mutant channels along the secretory pathway. In addition to its effect on channel trafficking, carbamazepine also inhibited K_ATP channel activity. Upon subsequent removal of carbamazepine, however, the function of rescued channels was recovered. Importantly, combination of the K_ATP channel opener diazoxide and carbamazepine led to enhanced mutant channel function without carbamazepine washout. The corrector effect of carbamazepine on mutant K_ATP channels was also demonstrated in rat and human β-cells with an accompanying increase in channel activity. Our findings identify carbamazepine as a novel small molecule corrector that may be used to restore K_ATP channel expression and function in a subset of congenital hyperinsulinism patients.     

Complex oligosaccharides are N-linked to Kv3 voltage-gated K channels in rat brain

Neuronal Kv3 voltage-gated K⁺ channels have two absolutely conserved N-glycosylation sites. Here, it is shown that Kv3.1, 3.3, and 3.4 channels are N-glycosylated in rat brain. Digestion of total brain membranes with peptide N glycosidase F (PNGase F) produced faster migrating immunobands than those of undigested membranes. Additionally, partial PNGase F digests showed that both sites are occupied by oligosaccharides. Neuraminidase treatment produced a smaller immunoband shift relative to PNGase F treatment. These results indicate that both sites are highly available and occupied by N-linked oligosaccharides for Kv3.1, 3.3, and 3.4 in rat brain, and furthermore that at least one oligosaccharide is of complex type. Additionally, these results point to an extracytoplasmic S1–S2 linker in Kv3 proteins expressed in native membranes. We suggest that N-glycosylation processing of Kv3 channels is critical for the expression of K⁺ currents at the surface of neurons, and perhaps contributes to the pathophysiology of congenital disorders of glycosylation.     

Constitutive Endocytic Recycling and Protein Kinase C-mediated Lysosomal Degradation Control KATP Channel Surface Density

Pancreatic ATP-sensitive potassium (K_ATP) channels control insulin secretion by coupling the excitability of the pancreatic β-cell to glucose metabolism. Little is currently known about how the plasma membrane density of these channels is regulated. We therefore set out to examine in detail the endocytosis and recycling of these channels and how these processes are regulated. To achieve this goal, we expressed K_ATP channels bearing an extracellular hemagglutinin epitope in human embryonic kidney cells and followed their fate along the endocytic pathway. Our results show that K_ATP channels undergo multiple rounds of endocytosis and recycling. Further, activation of protein kinase C (PKC) with phorbol 12-myristate 13-acetate significantly decreases K_ATP channel surface density by reducing channel recycling and diverting the channel to lysosomal degradation. These findings were recapitulated in the model pancreatic β-cell line INS1e, where activation of PKC leads to a decrease in the surface density of native K_ATP channels. Because sorting of internalized channels between lysosomal and recycling pathways could have opposite effects on the excitability of pancreatic β-cells, we propose that PKC-regulated K_ATP channel trafficking may play a role in the regulation of insulin secretion.     

Multiple modalities converge on a common gate to control K2P channel function

Members of the K_2P potassium channel family regulate neuronal excitability and are implicated in pain, anaesthetic responses, thermosensation, neuroprotection, and mood. Unlike other potassium channels, K_2Ps are gated by remarkably diverse stimuli that include chemical, thermal, and mechanical modalities. It has remained unclear whether the various gating inputs act through separate or common channel elements. Here, we show that protons, heat, and pressure affect activity of the prototypical, polymodal K_2P, K_2P2.1 (KCNK2/TREK‐1), at a common molecular gate that comprises elements of the pore‐forming segments and the N‐terminal end of the M4 transmembrane segment. We further demonstrate that the M4 gating element is conserved among K_2Ps and is employed regardless of whether the gating stimuli are inhibitory or activating. Our results define a unique gating mechanism shared by K_2P family members and suggest that their diverse sensory properties are achieved by coupling different molecular sensors to a conserved core gating apparatus.     

Ankyrin-B Regulates Kir6.2 Membrane Expression and Function in Heart

Ankyrin polypeptides are critical for normal membrane protein expression in diverse cell types, including neurons, myocytes, epithelia, and erythrocytes. Ankyrin dysfunction results in defects in membrane expression of ankyrin-binding partners (including ion channels, transporters, and cell adhesion molecules), resulting in aberrant cellular function and disease. Here, we identify a new role for ankyrin-B in cardiac cell biology. We demonstrate that cardiac sarcolemmal K_ATP channels directly associate with ankyrin-B in heart via the K_ATP channel α-subunit Kir6.2. We demonstrate that primary myocytes lacking ankyrin-B display defects in Kir6.2 protein expression, membrane expression, and function. Moreover, we demonstrate a secondary role for ankyrin-B in regulating K_ATP channel gating. Finally, we demonstrate that ankyrin-B forms a membrane complex with K_ATP channels and the cardiac Na/K-ATPase, a second key membrane transporter involved in the cardiac ischemia response. Collectively, our new findings define a new role for cardiac ankyrin polypeptides in regulation of ion channel membrane expression in heart.     

Functional Consequences of the Interactions among the Neural Cell Adhesion Molecule NCAM,the Receptor Tyrosine Kinase TrkB,and the Inwardly Rectifying K+ Channel KIR3.3

Cell adhesion molecules and neurotrophin receptors are crucial for the development and the function of the nervous system. Among downstream effectors of neurotrophin receptors and recognition molecules are ion channels. Here, we provide evidence that G protein-coupled inwardly rectifying K⁺ channel Kir3.3 directly binds to the neural cell adhesion molecule (NCAM) and neurotrophin receptor TrkB. We identified the binding sites for NCAM and TrkB at the C-terminal intracellular domain of Kir3.3. The interaction between NCAM, TrkB, and Kir3.3 was supported by immunocytochemical co-localization of Kir3.3, NCAM, and/or TrkB at the surface of hippocampal neurons. Co-expression of TrkB and Kir3.1/3.3 in Xenopus oocytes increased the K⁺ currents evoked by Kir3.1/3.3 channels. This current enhancement was reduced by the concomitant co-expression with NCAM. Both surface fluorescence measurements of microinjected oocytes and cell surface biotinylation of transfected CHO cells indicated that the cell membrane localization of Kir3.3 is regulated by TrkB and NCAM. Furthermore, the level of Kir3.3, but not of Kir3.2, at the plasma membranes was reduced in TrkB-deficient mice, supporting the notion that TrkB regulates the cell surface expression of Kir3.3. The premature expression of developmentally late appearing Kir3.1/3.3 in hippocampal neurons led to a reduction of NCAM-induced neurite outgrowth. Our observations indicate a decisive role for the neuronal K⁺ channel in regulating NCAM-dependent neurite outgrowth and attribute a physiologically meaningful role to the functional interplay of Kir3.3, NCAM, and TrkB in ontogeny.     

An Extracellular Ion Pathway Plays a Central Role in the Cooperative Gating of a K2P K+ Channel by Extracellular pH

Proton-gated TASK-3 K⁺ channel belongs to the K_2P family of proteins that underlie the K⁺ leak setting the membrane potential in all cells. TASK-3 is under cooperative gating control by extracellular [H⁺]. Use of recently solved K_2P structures allows us to explore the molecular mechanism of TASK-3 cooperative pH gating. Tunnel-like side portals define an extracellular ion pathway to the selectivity filter. We use a combination of molecular modeling and functional assays to show that pH-sensing histidine residues and K⁺ ions mutually interact electrostatically in the confines of the extracellular ion pathway. K⁺ ions modulate the pK_a of sensing histidine side chains whose charge states in turn determine the open/closed transition of the channel pore. Cooperativity, and therefore steep dependence of TASK-3 K⁺ channel activity on extracellular pH, is dependent on an effect of the permeant ion on the channel pH_o sensors.     

Gating the pore of potassium leak channels

A key feature of potassium channel function is the ability to switch between conducting and non-conducting states by undergoing conformational changes in response to cellular or extracellular signals. Such switching is facilitated by the mechanical coupling of gating domain movements to pore opening and closing. Two-pore domain potassium channels (K_2P) conduct leak or background potassium-selective currents that are mostly time- and voltage-independent. These channels play a significant role in setting the cell resting membrane potential and, therefore modulate cell responsiveness and excitability. Thus, K_2P channels are key players in numerous physiological processes and were recently shown to also be involved in human pathologies. It is well established that K_2P channel conductance, open probability and cell surface expression are significantly modulated by various physical and chemical stimuli. However, in understanding how such signals are translated into conformational changes that open or close the channels gate, there remain more open questions than answers. A growing line of evidence suggests that the outer pore area assumes a critical role in gating K_2P channels, in a manner reminiscent of C-type inactivation of voltage-gated potassium channels. In some K_2P channels, this gating mechanism is facilitated in response to external pH levels. Recently, it was suggested that K_2P channels also possess a lower activation gate that is positively coupled to the outer pore gate. The purpose of this review is to present an up-to-date summary of research describing the conformational changes and gating events that take place at the K_2P channel ion-conducting pathway during the channel regulation.     

Towards a TREK-1/2 (TWIK-Related K+ Channel 1 and 2) dual activator tool compound: Multi-dimensional optimization of BL-1249

This letter describes a focused, multi-dimensional optimization campaign around BL-1249, a fenamate class non-steroidal anti-inflammatory and a known activator of the K_2P potassium channels TREK-1 (K_2P2.1) and TREK-2 (K_2P10.1). While BL-1249 has been widely profiled in vitro as a dual TREK-1/2 activator, poor physicochemical and DMPK properties have precluded a deeper understanding of the therapeutic potential of these key K_2P channels across a broad spectrum of peripheral and central human disease. Here, we report multi-dimensional SAR that led to a novel TREK-1/2 dual activator chemotype, exemplified by ONO-2960632/VU6011992, with improved DMPK properties, representing a new lead for further optimization towards robust in vivo tool compounds.     

A structural model for K2P potassium channels based on 23 pairs of interacting sites and continuum electrostatics

K_2PØ, the two-pore domain potassium background channel that determines cardiac rhythm in Drosophila melanogaster, and its homologues that establish excitable membrane activity in mammals are of unknown structure. K_2P subunits have two pore domains flanked by transmembrane (TM) spans: TM1-P1-TM2-TM3-P2-TM4. To establish spatial relationships in K_2PØ, we identified pairs of sites that display electrostatic compensation. Channels silenced by the addition of a charge in pore loop 1 (P1) or P2 were restored to function by countercharges at specific second sites. A three-dimensional homology model was determined using the crystal structure of K_V1.2, effects of K_2PØ mutations to establish alignment, and compensatory charge–charge pairs. The model was refined and validated by continuum electrostatic free energy calculations and covalent linkage of introduced cysteines. K_2P channels use two subunits arranged so that the P1 and P2 loops contribute to one pore, identical P loops face each other diagonally across the pore, and the channel complex has bilateral symmetry with a fourfold symmetric selectivity filter.     

Cardiac expression and atrial fibrillation-associated remodeling of K2P2.1 (TREK-1) K channels in a porcine model

Aims

Effective management of atrial fibrillation (AF) often remains an unmet need. Cardiac two-pore-domain K⁺ (K_2P) channels are implicated in action potential regulation, and their inhibition has been proposed as a novel antiarrhythmic strategy. K_2P2.1 (TREK-1) channels are expressed in the human heart. This study was designed to identify and functionally express porcine K_2P2.1 channels. In addition, we sought to analyze cardiac expression and AF-associated K_2P2.1 remodeling in a clinically relevant porcine AF model.

Main methods

Three pK_2P2.1 isoforms were identified and amplified. Currents were recorded using voltage clamp electrophysiology in the Xenopus oocyte expression system. K_2P2.1 remodeling was studied by quantitative real time PCR and Western blot in domestic pigs during AF induced by atrial burst pacing.

Key findings

Human and porcine K_2P2.1 proteins share 99% identity. Residues involved in phosphorylation or glycosylation are conserved. Porcine K_2P2.1 channels carried outwardly rectifying K⁺ currents similar to their human counterparts. In pigs, K_2P2.1 was expressed ubiquitously in the heart with predominance in the atrial tissue. AF was associated with time-dependent reduction of K_2P2.1 protein in the RA by 70% (7 days of AF) and 80% (21 days of AF) compared to control animals in sinus rhythm. K_2P2.1 expression in the left atrium, AV node, and ventricles was not affected by AF.

Significance

Similarities between porcine and human K_2P2.1 channels indicate that the pig may represent a valid model for mechanistic and preclinical studies. AF-related atrial K_2P2.1 remodeling has potential implications for arrhythmia maintenance and antiarrhythmic therapy.     

Regulation of Cardiac ATP-sensitive Potassium Channel Surface Expression by Calcium/Calmodulin-dependent Protein Kinase II

Cardiac ATP-sensitive potassium (K_ATP) channels are key sensors and effectors of the metabolic status of cardiomyocytes. Alteration in their expression impacts their effectiveness in maintaining cellular energy homeostasis and resistance to injury. We sought to determine how activation of calcium/calmodulin-dependent protein kinase II (CaMKII), a central regulator of calcium signaling, translates into reduced membrane expression and current capacity of cardiac K_ATP channels. We used real-time monitoring of K_ATP channel current density, immunohistochemistry, and biotinylation studies in isolated hearts and cardiomyocytes from wild-type and transgenic mice as well as HEK cells expressing wild-type and mutant K_ATP channel subunits to track the dynamics of K_ATP channel surface expression. Results showed that activation of CaMKII triggered dynamin-dependent internalization of K_ATP channels. This process required phosphorylation of threonine at 180 and 224 and an intact ³³⁰YSKF³³³ endocytosis motif of the K_ATP channel Kir6.2 pore-forming subunit. A molecular model of the μ2 subunit of the endocytosis adaptor protein, AP2, complexed with Kir6.2 predicted that μ2 docks by interaction with ³³⁰YSKF³³³ and Thr-180 on one and Thr-224 on the adjacent Kir6.2 subunit. Phosphorylation of Thr-180 and Thr-224 would favor interactions with the corresponding arginine- and lysine-rich loops on μ2. We concluded that calcium-dependent activation of CaMKII results in phosphorylation of Kir6.2, which promotes endocytosis of cardiac K_ATP channel subunits. This mechanism couples the surface expression of cardiac K_ATP channels with calcium signaling and reveals new targets to improve cardiac energy efficiency and stress resistance.     

Acid-sensitive TWIK and TASK Two-pore Domain Potassium Channels Change Ion Selectivity and Become Permeable to Sodium in Extracellular Acidification

Two-pore domain K⁺ channels (K2P) mediate background K⁺ conductance and play a key role in a variety of cellular functions. Among the 15 mammalian K2P isoforms, TWIK-1, TASK-1, and TASK-3 K⁺ channels are sensitive to extracellular acidification. Lowered or acidic extracellular pH (pH_o) strongly inhibits outward currents through these K2P channels. However, the mechanism of how low pH_o affects these acid-sensitive K2P channels is not well understood. Here we show that in Na⁺-based bath solutions with physiological K⁺ gradients, lowered pH_o largely shifts the reversal potential of TWIK-1, TASK-1, and TASK-3 K⁺ channels, which are heterologously expressed in Chinese hamster ovary cells, into the depolarizing direction and significantly increases their Na⁺ to K⁺ relative permeability. Low pH_o-induced inhibitions in these acid-sensitive K2P channels are more profound in Na⁺-based bath solutions than in channel-impermeable N-methyl-d-glucamine-based bath solutions, consistent with increases in the Na⁺ to K⁺ relative permeability and decreases in electrochemical driving forces of outward K⁺ currents of the channels. These findings indicate that TWIK-1, TASK-1, and TASK-3 K⁺ channels change ion selectivity in response to lowered pH_o, provide insights on the understanding of how extracellular acidification modulates acid-sensitive K2P channels, and imply that these acid-sensitive K2P channels may regulate cellular function with dynamic changes in their ion selectivity.