Increased Channel Open Probability Research Articles

Allosteric Inhibition of the Epithelial Na+ Channel through Peptide Binding at Peripheral Finger and Thumb Domains

The epithelial Na(+) channel (ENaC) mediates the rate-limiting step in transepithelial Na(+) transport in the distal segments of the nephron and in the lung. ENaC subunits are cleaved by proteases, resulting in channel activation due to the release of inhibitory tracts. Peptides derived from these tracts inhibit channel activity. The mechanism by which these intrinsic inhibitory tracts reduce channel activity is unknown, as are the sites where these tracts interact with other residues within the channel. We performed site-directed mutagenesis in large portions of the predicted periphery of the extracellular region of the α subunit and measured the effect of mutations on an 8-residue inhibitory tract-derived peptide. Our data show that the inhibitory peptide likely binds to specific residues within the finger and thumb domains of ENaC. Pairwise interactions between the peptide and the channel were identified by double mutant cycle experiments. Our data suggest that the inhibitory peptide has a specific peptide orientation within its binding site. Extended to the intrinsic inhibitory tract, our data suggest that proteases activate ENaC by removing residues that bind at the finger-thumb domain interface.

Lithium acts as a potentiator of AMPAR currents in hippocampal CA1 cells by selectively increasing channel open probability

Recent evidence suggests that lithium, which is used in the treatment of bipolar disorders, may act by influencing AMPAR properties at central glutamatergic synapses. While it is clear that lithium potentiates recombinant AMPAR responses in a subunit specific way, the origin of this potentiation is not known. We examined the effects of lithium on native AMPAR channels in CA1 pyramidal cells in hippocampal slices where AMPARs are expected to be associated with auxiliary subunits. We found that lithium produced a selective increase in single-channel open probability (P(open)), with little effect on single-channel conductance or burst length. From the present and previous finding it is likely that lithium causes a reduction in the time to recovery from desensitization, resulting in the observed increase in P(open). This would be consistent with the view that lithium acts like certain other allosteric AMPAR modulators to reduce the time spent in the desensitized state, but differs from those that act by slowing dissociation of glutamate.

Green tea catechins are potent sensitizers of ryanodine receptor type 1 (RyR1)

Catechins, polyphenols extracted from green tea leaves, have a broad range of biological activities although the specific molecular mechanisms responsible are not known. At the high experimental concentrations typically used polyphenols bind to membrane phospholipid and also are easily auto-oxidized to generate superoxide anion and semiquinones, and can adduct to protein thiols. We report that the type 1 ryanodine receptor (RyR1) is a molecular target that responds to nanomolar (−)-epigallocatechin-3-gallate (EGCG) and (−)-epicatechin-3-gallate (ECG). Single channel analyses demonstrate EGCG (5–10 nM) increases channel open probability (Po) twofold, by lengthening open dwell time. The degree of channel activation is concentration-dependent and is rapidly and fully reversible. Four related catechins, EGCG, ECG, EGC ((−)-epigallocatechin) and EC ((−)-epicatechin) showed a rank order of activity toward RyR1 (EGCG > ECG ≫ EGC >>> EC). EGCG and ECG enhance the sensitivity of RyR1 to activation by ≤100 μM cytoplasmic Ca 2+ without altering inhibitory potency by >100 μM Ca 2+. EGCG as high as 10 μM in the extracellular medium potentiated Ca 2+ transient amplitudes evoked by electrical stimuli applied to intact myotubes and adult FDB fibers, without eliciting spontaneous Ca 2+ release or slowing Ca 2+ transient recovery. The results identify RyR1 as a sensitive target for the major tea catechins EGCG and ECG, and this interaction is likely to contribute to their observed biological activities.

A mutation of the epithelial sodium channel associated with atypical cystic fibrosis increases channel open probability and reduces Na+ self inhibition

Increased activity of the epithelial sodium channel (ENaC) in the respiratory airways contributes to the pathophysiology of cystic fibrosis (CF), a genetic disease caused by mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene. In some patients suffering from atypical CF a mutation can be identified in only one CFTR allele. We recently identified in this group of CF patients a heterozygous mutation (W493R) in the alpha-subunit of ENaC. Here, we investigate the functional effects of this mutation by expressing wild-type alpha beta gamma ENaC or mutant alpha(W493R)beta gamma ENaC in Xenopus oocytes. The alpha W493R mutation stimulated amiloride-sensitive whole-cell currents (Delta I(ami)) by approximately 4-fold without altering the single-channel conductance or surface expression of ENaC. As these data suggest that the open probability (P(o)) of the mutant channel is increased, we investigated the proteolytic activation of ENaC by chymotrypsin. Single-channel recordings revealed that chymotrypsin activated near-silent channels in outside-out membrane patches from oocytes expressing wild-type ENaC, but not in membrane patches from oocytes expressing the mutant channel. In addition, the alpha W493R mutation abolished Na(+) self inhibition of ENaC, which might also contribute to its gain-of-function effects. We conclude that the alpha W493R mutation promotes constitutive activation of ENaC by reducing the inhibitory effect of extracellular Na(+) and decreasing the pool of near-silent channels. The resulting gain-of-function phenotype of the mutant channel might contribute to the pathophysiology of CF in patients carrying this mutation.

Editorial [Hot Topic: Molecular Pharmacology of the GABAA Receptor (Guest Editor: Gustav Akk)

The GABAA receptor is a member of the Cys-loop family of transmitter-gated ion channels. GABAA receptors respond to synaptically-released or ambient transmitter with a conformational change, resulting in the opening of the gate that allows movement of small anions through the channel. Under most circumstances, GABAA receptor-mediated signaling leads to hyperpolarization in the mature brain. The normal functioning of the receptor is critical to cellular function, while errors in GABAA receptor activity can systemically manifest as seizures, anxiety disorders and others. The pharmacology of the GABAA receptor is remarkably rich. The receptor is a target for benzodiazepines, steroids, barbiturates, and a number of other classes of compounds, some of which are used clinically as anesthetics, anxiolytics, or anticonvulsants. The actions of the drugs are in many cases dependent on the subunit composition of the GABAA receptor. Thus, understanding how the existing therapeutics act can pave way to new drugs with improved specificity and reduced side effects. This Hot Topic Issue presents an overview of molecular pharmacology of the GABAA receptor focusing on three classes of positive modulators: general anesthetics, benzodiazepines, and neuroactive steroids. Paul Garcia, Scott Kolesky and Andrew Jenkins provide an introduction to GABAA receptors, and discuss the actions of major classes of volatile and intravenous anesthetics. The manuscript addresses the relationship between subunit composition of the receptor and modulation, and summarizes the data from work on mutated receptors on the location of the anesthetic binding cavity. Matt Bianchi presents a summary of the actions of benzodiazepines on the GABAA receptor. The major effect of benzodiazepines is to enhance the affinity of the receptor to the transmitter. The manuscript is aimed at comparing and contrasting the ability of benzodiazepines to modulate currents under conditions resembling synaptic conditions and tonic exposure to GABA. Finally, Gustav Akk and colleagues discuss the current knowledge about kinetic and structural features of GABAA receptor modulation by potentiating neuroactive steroids. This class of steroids acts by increasing channel open probability through a set of modifications in the channel open and closed time distributions. The review critically evaluates available experimental data discussing the possibility of a single vs. multiple interaction sites mediating the effects of potentiating steroids. This Hot Topics Issue provides an overview of the rapidly developing field of molecular pharmacology of the GABAA receptor. The manuscripts focus on several of the numerous positive modulators of the receptor summarizing the present state of knowledge and introducing new evidence and novel viewpoints about the kinetic and structural aspects of modulation. It is hoped that the presented material will provide an impetus for further research in this area.

Hydrogen sulfide increases calcium-activated potassium (BK) channel activity of rat pituitary tumor cells

Hydrogen sulfide (H(2)S) is the third gasotransmitter found to be produced endogenously in living cells to exert physiological functions. Large conductance (maxi) calcium-activated potassium channels (BK), which play an important role in the regulation of electrical activity in many cells, are targets of gasotransmitters. We examined the modulating action of H(2)S on BK channels from rat GH(3) pituitary tumor cells using patch clamp techniques. Application of sodium hydrogen sulfide as H(2)S donor to the bath solution in whole cell experiments caused an increase of calcium-activated potassium outward currents. In single channel recordings, H(2)S increased BK channel activity in a concentration-dependent manner. Hydrogen sulfide induced a reversible increase in channel open probability in a voltage-dependent, but calcium independent manner. The reducing agent, dithiothreitol, prevented the increase of open probability by H(2)S, whereas, the oxidizing agent thimerosal increased channel open probability in the presence of H(2)S. Our data show that H(2)S augments BK channel activity, and this effect can be linked to its reducing action on sulfhydryl groups of the channel protein.

Intracellular calcium strongly potentiates agonist-activated TRPC5 channels.

TRPC5 is a calcium (Ca2+)-permeable nonselective cation channel expressed in several brain regions, including the hippocampus, cerebellum, and amygdala. Although TRPC5 is activated by receptors coupled to phospholipase C, the precise signaling pathway and modulatory signals remain poorly defined. We find that during continuous agonist activation, heterologously expressed TRPC5 currents are potentiated in a voltage-dependent manner (∼5-fold at positive potentials and ∼25-fold at negative potentials). The reversal potential, doubly rectifying current–voltage relation, and permeability to large cations such as N-methyl-d-glucamine remain unchanged during this potentiation. The TRPC5 current potentiation depends on extracellular Ca2+: replacement by Ba2+ or Mg2+ abolishes it, whereas the addition of 10 mM Ca2+ accelerates it. The site of action for Ca2+ is intracellular, as simultaneous fura-2 imaging and patch clamp recordings indicate that potentiation is triggered at ∼1 µM [Ca2+]. This potentiation is prevented when intracellular Ca2+ is tightly buffered, but it is promoted when recording with internal solutions containing elevated [Ca2+]. In cell-attached and excised inside-out single-channel recordings, increases in internal [Ca2+] led to an ∼10–20-fold increase in channel open probability, whereas single-channel conductance was unchanged. Ca2+-dependent potentiation should result in TRPC5 channel activation preferentially during periods of repetitive firing or coincident neurotransmitter receptor activation.

Does the cytosolic amino ‐terminus of γ‐ENaC function as a nuclear signaling molecule?

Functional ENaC is in the apical plasma membrane of epithelial cells where it's activity is limiting for Na+ (re)absorption and thus, important for electrolyte balance. Enzymes that cleave the channel modify ENaC: including release of the N‐terminal portion of γ‐ENaC. This post‐translational modification increases ENaC activity by increasing channel open probability; however, the role of the section of ENaC cleaved from the core of the channel remains unclear. We previously showed that the amino‐terminal portion of γ‐ENaC when expressed alone targets to the nucleus rather than the plasma membrane. This leads us to wonder whether cleavage of ENaC resulting in release of the N‐terminus of γ‐ENaC results in both an acute activation of the channel and a secondary activation of a nuclear signaling event, similar to Notch signaling and release of the intracellular portion of polycystin. We find that the N‐terminal tail of γ‐ENaC targets to the nucleus decreasing activity of membrane resident channels in a dose‐dependent manner. This is in contrast to the carboxy‐terminal portion of this subunit that does not translocate to the nucleus or affect channel activity. These early results are consistent with the idea that post‐translation modification of ENaC by proteases has two effects: an acute activation of the channel and initiation of a negative‐feedback pathway involving nuclear signaling by the cleaved portion of ENaC.

Sulfhydryl Modification Of Cysteine Substitutions In The Second Hairpin Loop (HP2) Alters The Ion Permeation Properties Of The Glutamate Transporter, EAAT1

In the mammalian CNS, excitatory amino acid transporters (EAATs) limit glutamatergic signalling and regulate extracellular glutamate concentrations. EAATs have been shown to function not only as secondary active transporters, but also as anion-selective channels that regulate excitability. Under some conditions, EAATs have also been shown to mediate cation-selective conductances. The C-terminal portion of the protein contains two membrane spanning domains (TM7, TM8) and two hairpin loops (HP1, HP2), which are critical for substrate translocation, however, the anion permeation pathway has been linked to additional domains within the N-terminus of the protein (TM2). Application of bulky MTS-reagents to several cysteine substitution mutants in HP2 abolishes glutamate translocation, but preserves the anion conductance, suggesting that the anion permeation and substrate transport pathways are physically separated. Interestingly, MTS-modification of some of these mutants results in substantially greater macroscopic current amplitude. We have used two-electrode voltage clamp in Xenopus oocytes and whole-cell patch-clamp in mammalian cells to determine the basis for the change in ion permeation properties for a series of mutants that exhibit this phenotype. We examined the substrate-gated anion currents associated with one mutant, M451C EAAT1, in Xenopus oocytes and observed a profound time- and voltage-dependent deactivation of the macroscopic current at depolarizing potentials, suggesting that MTS-modification increases channel open probability. After modification, extracellular substitution with the impermeant anion gluconate, resulted in an outward current at positive potentials, suggesting that the current includes a major cation component. We are currently exploring the relationships between the substrate translocation pathway with this novel cation current and the enhanced anion current induced by modification, with the aim of better understanding the molecular determinants involved in EAAT-associated channel gating and permeation.

Menthol increases human glioblastoma intracellular Ca2+, BK channel activity and cell migration

This study examined the effect of menthol, an agonist for transient receptor potential melastatin 8 (TRPM8) ion channels, to increase intracellular Ca2+ concentration, [Ca2+]i, in human glioblastoma cells (DBTRG cells), which resulted in activation of the large-conductance Ca2+-activated K+ membrane ion channels (BK channels). Voltage ramps applied over 300 ms from -100 to 100 mV resulted in membrane currents with marked inwardly- and outwardly-rectifying components. Paxilline (2 μM) abolished the outwardly-rectifying current. Outwardly-rectifying on-cell patch currents were increased markedly by menthol (100 μM) added to the bath. The estimated on-cell conductance of these channels was 253 pS. Kinetic analysis showed that added menthol increased channel open probability and mean open frequency after 5 min. In a similar time course menthol increased [Ca2+]i, and this increase was abolished either by added paxilline, tetraethylammonium ion or by Ca2+-free external solution. Finally, menthol stimulated the rate of DBTRG cell migration into scratch wounds made in confluent cells, and this also was inhibited by paxilline or by tetraethylammonium ion. We conclude that menthol, a TRPM8 agonist, increases DBTRG cell [Ca2+]i that in turn activates membrane BK ion channels. Inhibition of BK channels by paxilline reverses menthol-stimulated increase of [Ca2+]i and of cell migration. Thus, BK channels function to maintain elevations in [Ca2+]i needed to sustain increases in DBTRG cell migration.

Amyloid-β protein impairs Ca2+ release and contractility in skeletal muscle

Inclusion body myositis (IBM), the most common muscle disorder in the elderly, is partly characterized by dysregulation of β-amyloid precursor protein (βAPP) expression and abnormal, intracellular accumulation of full-length βAPP and β-amyloid epitopes. The present study examined the effects of β-amyloid accumulation on force generation and Ca(2+) release in skeletal muscle from transgenic mice harboring human βAPP and assessed the consequence of Aβ(1-42) modulation of the ryanodine receptor Ca(2+) release channels (RyRs). β-Amyloid laden muscle produced less peak force and exhibited Ca(2+) transients with smaller amplitude. To determine whether modification of RyRs by β-amyloid underlie the effects observed in muscle, in vitro Ca(2+) release assays and RyR reconstituted in planar lipid bilayer experiments were conducted in the presence of Aβ(1-42). Application of Aβ(1-42) to RyRs in bilayers resulted in an increased channel open probability and changes in gating kinetics, while addition of Aβ(1-42) to the rabbit SR vesicles resulted in RyR-mediated Ca(2+) release. These data may relate altered βAPP metabolism in IBM to reductions in RyR-mediated Ca(2+) release and muscle contractility.

Interaction between STIM1 and TRPC1/TRPC4 complexes, a mechanism for store‐operated calcium entry in glomerular mesangial cells

Store‐operated Ca2+ entry (SOCE) participates in a diverse array of cell functions in a variety of cell types. Although recent evidence suggests that Orai proteins might be the molecular entity of store‐operated channels (SOC) in hematopoietic cells, multimers of canonical transient receptor potential (TRPC) proteins are still candidates of SOC in other types of cells. Our previous studies demonstrated that TRPC1 and TRPC4 are expressed and interact with each other in glomerular mesangial cells (MCs). The present study examined if complexes of TRPC1/TRPC4 proteins mediated SOCE in MCs and if activation of the channel complexes required STIM1, a Ca2+ sensor protein in the endoplasmic reticulum. Knockdown (KD) of TRPC1 or TRPC4 expression using RNAi inhibited an increase of channel open probability stimulated by store‐depletion with thapsigargin (TG), as measured by cell‐attached patch clamp. TRPC1 and TRPC4 interaction, as detected by co‐immunoprecipitation (co‐IP), was enhanced by TG treatment. KD of TRPC1 or TRPC4 also attenuated SOCE, measured by fluorescent Ca2+ imaging, while simultaneous KD of both subunits had a significantly greater effect. Furthermore, association of STIM1 with TRPC4 was also found by co‐IP, and KD of STIM1 inhibited TG‐induced SOCE. These data imply that TRPC1 and TRPC4 form a complex that is activated by store‐depletion. STIM1 also interacts with this complex and is required for SOCE.

Binding of Anionic Lipids to at Least Three Nonannular Sites on the Potassium Channel KcsA is Required for Channel Opening

In addition to the annular or boundary lipids that surround the transmembrane surface of the potassium channel KcsA from Streptomyces lividans, x-ray crystallographic studies have detected one anionic lipid molecule bound at each protein-protein interface in the homotetrameric structure, at sites referred to as nonannular sites. The binding constant for phosphatidylglycerol at the nonannular sites has been determined using fluorescence quenching methods with a mutant of KcsA lacking the normal three lipid-exposed Trp residues. Binding is weak, with a binding constant of 0.42 ± 0.06 in units of mol fraction, implying that the nonannular sites will only be ∼70% occupied in bilayers of 100% phosphatidylglycerol. However, the nonannular sites show high selectivity for anionic lipids over zwitterionic lipids, and it is suggested that a change in packing at the protein-protein interface leads to a closing of the nonannular binding site in the unbound state. Increasing the anionic lipid content of the membrane leads to a large increase in open channel probability, from ∼2.5% in the presence of 25 mol % phosphatidylglycerol to ∼62% in 100 mol % phosphatidylglycerol. The relationship between open channel probability and phosphatidylglycerol content shows cooperativity. The data are consistent with a model in which three or four of the four nonannular sites in the KcsA homotetramer have to be occupied by anionic lipid for the channel to open. The conductance of the open channel increases with increasing concentration of anionic lipid, an effect possibly due to effects of anionic lipid on the concentration of K + close to the membrane surface.

Regulation of ENaC by small G proteins

Small G proteins are recognized to regulate diverse cellular processes. We have shown previously that the epithelial Na+ channel (ENaC) is regulated by Ras and Rho GTPases. K‐Ras, RhoA and Rac1 markedly increase activity of ENaC reconstituted in CHO cells in a GTP‐dependent manner. However, their transductionpathways and mechanisms of activation are different; K‐Ras increases channel open probability via PI3‐kinase signaling, whereas RhoA increase channel membrane levels. Regulation of ENaC open probability by PI3‐kinaseis a direct consequence of the physical association of the product of PI3‐kinase, PI(3,4,5)P3with the channel. RhoA activates ENaC via Rho‐kinase and subsequent activation of PI(4)P5‐kinase with concomitant increases in PI(4,5)P2 levels promoting channel insertion into the plasma membrane. Recent studies reveal that multiple Rab isoforms physically interact with and/or modulate the activity of several ion channels. Rab11a is well‐established as a participant in the regulation of recycling endosomal trafficking. Rab11a is associated with vesicles in the apical portion of epithelial cells near the centrosome and beneath the apical plasma membrane. Co‐expression of Rab11a with ENaC results in a significant increase in channel activity. We conclude that Rab11a may be important in the trafficking of ENaC.Supported by AHA.

Potentiation of TRPC5 by Protons

Mammalian members of the classical transient receptor potential channel subfamily (TRPC) are Ca(2+)-permeable cation channels involved in receptor-mediated increases in intracellular Ca(2+). TRPC4 and TRPC5 form a group within the TRPC subfamily and are activated in a phospholipase C-dependent manner by an unidentified messenger. Unlike most other Ca(2+)-permeable channels, TRPC4 and -5 are potentiated by micromolar concentrations of La(3+) and Gd(3+). This effect results from an action of the cations at two glutamate residues accessible from the extracellular solution. Here, we show that TRPC4 and -5 respond to changes in extracellular pH. Lowering the pH increased both G protein-activated and spontaneous TRPC5 currents. Both effects were already observed with small reductions in pH (from 7.4 to 7.0) and increased up to pH 6.5. TRPC4 was also potentiated by decreases in pH, whereas TRPC6 was only inhibited, with a pIC(50) of 5.7. Mutation of the glutamate residues responsible for lanthanoid sensitivity of TRPC5 (E543Q and E595Q) modified the potentiation of TRPC5 by acid. Further evidence for a similarity in the actions of lanthanoids and H(+) on TRPC5 is the reduction in single channel conductance and dramatic increase in channel open probability in the presence of either H(+) or Gd(3+) that leads to larger integral currents. In conclusion, the high sensitivity of TRPC5 to H(+) indicates that, in addition to regulation by phospholipase C and other factors, the channel may act as a sensor of pH that links decreases in extracellular pH to Ca(2+) entry and depolarization.

Proteolytic cleavage of the voltage‐gated Ca2+ channel α2δ subunit: structural and functional features

By mediating depolarization-induced Ca(2+) influx, high-voltage-activated Ca(2+) channels control a variety of cellular events. These heteromultimeric proteins are composed of an ion-conducting (alpha(1)) and three auxiliary (alpha(2)delta, beta and gamma) subunits. The alpha(2)delta subunit enhances the trafficking of the channel complex to the cell surface and increases channel open probability. To exert these effects, alpha(2)delta must undergo important post-translational modifications, including a proteolytic cleavage that separates the extracellular alpha(2) from its transmembrane delta domain. After this proteolysis both domains remain linked by disulfide bonds. In spite of its central role in determining the final conformation of the fully mature alpha(2)delta, almost nothing is known about the physiological implications of this structural modification. In the current report, by using site-directed mutagenesis, the proteolytic site of alpha(2)delta was mapped to amino acid residues Arg-941 and Val-946. Substitution of these residues renders the protein insensitive to proteolytic cleavage as evidenced by the lack of molecular weight shift upon treatment with a disulfide-reducing agent. Interestingly, these mutations significantly decreased whole-cell patch-clamp currents without affecting the voltage dependence or kinetics of the channels, suggesting a reduction in the number of channels targeted to the plasma membrane.

Curcumin induces electrical activity in rat pancreatic β-cells by activating the volume-regulated anion channel

Curcumin, the principal active component of turmeric, is reported to exert a number of therapeutic actions, including a hypoglycaemic/antidiabetic action. The underlying mechanisms to this action are essentially unknown. We have investigated the hypothesis that a direct stimulatory action on the pancreatic β-cell could contribute towards the hypoglycaemic activity of this compound. Electrical and ion channel activity were recorded in rat β-cells using the patch–clamp technique. β-Cell volume was measured using a video-imaging technique. Insulin release was measured from intact islets by radioimmunoassay. Curcumin (2–10 μM) activated the volume-regulated anion channel in β-cells. Single channel studies indicated that activation was the result of increased channel open probability. This effect was accompanied by depolarisation of the cell membrane potential, the generation of electrical activity and enhanced insulin release. Curcumin also decreased β-cell volume, presumably reflecting loss of Cl − (and hence water) as a result of anion channel activation. These findings are consistent with the suggestion that Cl − fluxes play an important role in regulating β-cell function. The stimulation of β-cell function by curcumin could contribute to the hypoglycaemic actions of this compound, and these findings identify a novel potential therapeutic target for the treatment of type 2 diabetes mellitus.

Inhibition of MAPK stimulates the Ca 2+ -dependent big-conductance K channels in cortical collecting duct

The kidney plays a key role in maintaining potassium (K) homeostasis. K excretion is determined by the balance between K secretion and absorption in distal tubule segments such as the connecting tubule and cortical collecting duct. K secretion takes place by K entering principal cells (PC) from blood side through Na+, K+ -ATPase and being secreted into the lumen via both ROMK-like small-conductance K (SK) channels and Ca2+ -activated big-conductance K (BK) channels. K reabsorption occurs by stimulation of apical K/H-ATPase and inhibition of K recycling across the apical membrane in intercalated cells (IC). The role of ROMK channels in K secretion is well documented. However, the importance of BK channels in mediating K secretion is incompletely understood. It has been shown that their activity increases with high tubule flow rate and augmented K intake. However, BK channels have a low open probability and are mainly located in IC, which lack appropriate transporters for effective K secretion. Here we demonstrate that inhibition of ERK and P38 MAPKs stimulates BK channels in both PC and IC in the cortical collecting duct and that changes in K intake modulate their activity. Under control conditions, BK channel activity in PC was low but increased significantly by inhibition of both ERK and P38. Blocking MAPKs also increased channel open probability of BK in IC and thereby it may affect K backflux and net K absorption Thus, modulation of ERK and P38 MAPK activity is involved in controlling net K secretion in the distal nephron.

Oxidative modification of M-type K+ channels as a mechanism of cytoprotective neuronal silencing

Voltage-gated K(+) channels of the Kv7 family underlie the neuronal M current that regulates action potential firing. Suppression of M current increases excitability and its enhancement can silence neurons. We here show that three of five Kv7 channels undergo strong enhancement of their activity by oxidative modification induced by physiological concentrations of hydrogen peroxide. A triple cysteine pocket in the channel S2-S3 linker is critical for this effect. Oxidation-induced enhancement of M current produced a hyperpolarization and a dramatic reduction of action potential firing frequency in rat sympathetic neurons. As hydrogen peroxide is robustly produced during hypoxia-induced oxidative stress, we used an oxygen/glucose deprivation neurodegeneration model that showed neuronal death to be severely accelerated by M current blockade. Such blockade had no effect on survival of normoxic neurons. This work describes a novel pathway of M-channel regulation and suggests a role for M channels in protective neuronal silencing during oxidative stress.

RETRACTED: L-Type Ca2+ Channel Facilitation Mediated by Phosphorylation of the β Subunit by CaMKII

L-type Ca(2+) channels (LTCCs) are major entry points for Ca(2+) in many cells. Ca(2+)/calmodulin-dependent protein kinase II (CaMKII) is associated with cardiac LTCC complexes and increases channel open probability (P(O)) to dynamically increase Ca(2+) current (I(Ca)) and augment cellular Ca(2+) signaling by a process called facilitation. However, the critical molecular mechanisms for CaMKII localization to LTCCs and I(Ca) facilitation in cardiomyocytes have not been defined. We show CaMKII binds to the LTCC beta(2a) subunit and preferentially phosphorylates Thr498 in beta(2a). Mutation of Thr498 to Ala (T498A) in beta(2a) prevents CaMKII-mediated increases in the P(O) of recombinant LTCCs. Moreover, expression of beta(2a)(T498A) in adult cardiomyocytes ablates CaMKII-mediated I(Ca) facilitation, demonstrating that phosphorylation of beta(2a) at Thr498 modulates native calcium channels. These findings reveal a molecular mechanism for targeting CaMKII to LTCCs and facilitating I(Ca) that may modulate Ca(2+) entry in diverse cell types coexpressing CaMKII and the beta(2a) subunit.