Increased Channel Open Probability Research Articles

Mechanism and properties of positive allosteric modulation of N-methyl-d-aspartate receptors by 6-alkyl 2-naphthoic acid derivatives

The theory that N-methyl-d-aspartate receptor (NMDAR) hypofunction is responsible for the symptoms of schizophrenia is well supported by many pharmacological and genetic studies. Accordingly, positive allosteric modulators (PAMs) that augment NMDAR signaling may be useful for treating schizophrenia. Previously we have identified several NMDAR PAMs containing a carboxylic acid attached to naphthalene, phenanthrene, or coumarin ring systems. In this study, we describe several functional and mechanistic properties of UBP684, a 2-naphthoic acid derivative, which robustly potentiates agonist responses at each of the four GluN1a/GluN2 receptors and at neuronal NMDARs. UBP684 increases the maximal l-glutamate/glycine response while having minor subunit-specific effects on agonist potency. PAM binding is independent of agonist binding, and PAM activity is independent of membrane voltage, redox state, and the GluN1 exon 5 N-terminal insert. UBP684 activity is, however, markedly pH-dependent, with greater potentiation occurring at lower pHs and inhibitory activity at pH 8.4. UBP684 increases channel open probability (Po) and slows receptor deactivation time upon removal of l-glutamate, but not glycine. The structurally related PAM, UBP753, reproduced most of these findings, but did not prolong agonist removal deactivation time. Studies using cysteine mutants to lock the GluN1 and GluN2 ligand-binding domains (LBDs) in the agonist-bound states indicate that PAM potentiation requires GluN2 LBD conformational flexibility. Together, these findings suggest that UBP684 and UBP753 stabilize the GluN2 LBD in an active conformation and thereby increase Po. Thus, UBP684 and UBP753 may serve as lead compounds for developing agents to enhance NMDAR activity in disorders associated with NMDAR hypofunction.

Functional Evaluation of a De Novo GRIN2A Mutation Identified in a Patient with Profound Global Developmental Delay and Refractory Epilepsy.

The N-methyl-d-aspartate receptor (NMDAR), a ligand-gated ionotropic glutamate receptor, plays important roles in normal brain development and a wide range of neurologic disorders, including epilepsy. Here, we evaluate for the first time the functional properties of a de novo GRIN2A missense mutation (p.M817V) in the pre-M4 linker in a child with profound global developmental delay and refractory epilepsy. Electrophysiologic recordings revealed that the mutant GluN2A(M817V)-containing receptors showed enhanced agonist potency, reduced sensitivity to endogenous negative inhibitors (Mg2+, proton, and zinc), prolonged synaptic-like response time course, increased single-channel mean open time, and increased channel open probability. These results suggest that the gain-of-function M817V mutation causes overactivation of NMDAR and drives neuronal hyperexcitability, which may contribute to the patient's observed epileptic phenotype. Molecular modeling of the closed channel conformation reveals that this mutation weakens the interaction between GluN2 transmembrane helix M4 and two GluN1 transmembrane helices, and increases atomic fluctuation or movement of the pre-M1 region of GluN1 subunit, suggesting a mechanism by which channel function is enhanced. The functional changes of this mutation on agonist potency occur when the mutation is introduced into all other GluN2 subunits, suggesting a conserved role of this residue in control of NMDAR function through interactions of membrane spanning GluN2 and GluN1 helices. A number of NMDAR-targeted drugs including U.S. Food and Drug Association-approved NMDAR channel blockers were evaluated for their ability to inhibit receptors containing GluN2A(M817V) as a first step to exploring the potential for rescue pharmacology and personalized medicine.

Nitric Oxide Potentiates the Effect of Calcium/Calmodulin-Dependent Kinase II on Ca v 1.2 Protein

The L-type calcium channel (LTCC) is the major route for calcium influx into cardiac myocytes. Ca2+/calmodulin-dependent kinase II (CaMKII) facilitates calcium influx by binding to the C terminal domain of the Cav1.2 protein (main pore forming ion conducting subunit of the LTCC). Nitric oxide (NO) is proposed to bind to the LTCC and alter calcium influx. However, it is not known whether this occurs as a result of a direct effect of NO on the channel. The aim of this study was to clarify the effect of NO and CaMKII on Cav1.2 protein function. We expressed the human long N terminal (NT) isoform of Cav1.2 in HEK cells. The channel protein was purified by histidine tag purification and incorporated in liposomes for functional analysis by patch-clamp technique. Exposing the long NT isoform to the NO donor GSNO or sodium nitroprusside failed to increase channel open probability (relative Po 0.696 ± 0.153, n = 5 and 1.09 ± 0.02, n = 6 respectively). Application of CaMKII alone increased open probability (Po) of the long NT isoform 2.24 ± 0.16 fold (n = 6; p < 0.05). The effect was more pronounced when CaMKII was pre-exposed to the NO donor (2.98 ± 0.13 fold, n = 6; p < 0.05), and these conditions resulted in a significant increase in CaMKII activity as measured by our FRET reporter (relative activity 1.36 ± 0.08 vs. baseline, n = 6; p < 0.05). The results suggest that CaMKII, but not the channel itself, is sensitive to nitrosylation. We conclude that the function of Cav1.2 protein cannot be directly altered by NO, but the functionally relevant target of NO is CaMKII.

Synergistic interplay of G\u03b2\u03b3 and phosphatidylinositol 4,5-bisphosphate dictates Kv7.4 channel activity

Kv7.4 channels are key determinants of arterial contractility and cochlear mechanosensation that, like all Kv7 channels, have an obligatory requirement for phosphatidylinositol 4,5-bisphosphate (PIP2). βγ G proteins (Gβγ) have been identified as novel positive regulators of Kv7.4. The present study ascertained whether Gβγ increased Kv7.4 open probability through an increased sensitivity to PIP2. In HEK cells stably expressing Kv7.4, PIP2 or Gβγ increased open probability in a concentration dependent manner. Depleting PIP2 prevented any Gβγ-mediated stimulation whilst an array of Gβγ inhibitors prohibited any PIP2-induced current enhancement. A combination of PIP2 and Gβγ at sub-efficacious concentrations increased channel open probability considerably. The stimulatory effects of three Kv7.2-7.5 channel activators were also lost by PIP2 depletion or Gβγ inhibitors. This study alters substantially our understanding of the fundamental processes that dictate Kv7.4 activity, revealing a more complex and subtle paradigm where the reliance on local phosphoinositide is dictated by interaction with Gβγ.

An Extended Structure-Activity Relationship of Nondioxin-Like PCBs Evaluates and Supports Modeling Predictions and Identifies Picomolar Potency of PCB 202 Towards Ryanodine Receptors.

Nondioxin-like polychlorinated biphenyls (NDL PCBs) activate ryanodine-sensitive Ca2+ channels (RyRs) and this activation has been associated with neurotoxicity in exposed animals. RyR-active congeners follow a distinct structure-activity relationship and a quantitative structure-activity relationship (QSAR) predicts that a large number of PCBs likely activate the receptor, which requires validation. Additionally, previous structural based conclusions have been established using receptor ligand binding assays but the impact of varying PCB structures on ion channel gating behavior is not understood. We used [3H]Ryanodine ([3H]Ry) binding to assess the RyR-activity of 14 previously untested PCB congeners evaluating the predictability of the QSAR. Congeners determined to display widely varying potency were then assayed with single channel voltage clamp analysis to assess direct influences on channel gating kinetics. The RyR-activity of individual PCBs assessed in in vitro assays followed the general pattern predicted by the QSAR but binding and lipid bilayer experiments demonstrated higher potency than predicted. Of the 49 congeners tested to date, tetra-ortho PCB 202 was found to be the most potent RyR-active congener increasing channel open probability at 200 pM. Shifting meta-substitutions to the para-position resulted in a > 100-fold reduction in potency as seen with PCB 197. Non-ortho PCB 11 was found to lack activity at the receptor supporting a minimum mono-ortho substitution for PCB RyR activity. These findings expand and support previous SAR assessments; where out of the 49 congeners tested to date 42 activate the receptor demonstrating that the RyR is a sensitive and common target of PCBs.

GRIN2D Recurrent De Novo Dominant Mutation Causes a Severe Epileptic Encephalopathy Treatable with NMDA Receptor Channel Blockers

N-methyl-D-aspartate receptors (NMDARs) are ligand-gated cation channels that mediate excitatory synaptic transmission. Genetic mutations in multiple NMDAR subunits cause various childhood epilepsy syndromes. Here, we report a de novo recurrent heterozygous missense mutation-c.1999G>A (p.Val667Ile)-in a NMDAR gene previously unrecognized to harbor disease-causingmutations, GRIN2D, identified by exome and candidate panel sequencing in two unrelated children with epileptic encephalopathy. The resulting GluN2D p.Val667Ile exchange occurs in the M3 transmembrane domain involved in channel gating.This gain-of-function mutation increases glutamate and glycine potency by 2-fold, increases channel open probability by 6-fold, andreduces receptor sensitivity to endogenous negative modulators such as extracellular protons. Moreover, this mutation prolongs the deactivation time course after glutamate removal, which controls the synaptic time course. Transfection of cultured neurons with human GRIN2D cDNA harboring c.1999G>A leads to dendritic swelling and neuronal cell death, suggestive of excitotoxicity mediated by NMDAR over-activation. Because both individuals' seizures had proven refractory to conventional antiepileptic medications, the sensitivity of mutant NMDARs to FDA-approved NMDAR antagonists was evaluated. Based on these results, oral memantine was administered to both children, with resulting mild to moderate improvement in seizure burden and development. The older proband subsequently developed refractory status epilepticus, with dramatic electroclinical improvement upon treatment with ketamine and magnesium. Overall, these results suggest that NMDAR antagonists can be useful as adjuvant epilepsy therapy in individuals with GRIN2D gain-of-function mutations. This work further demonstrates the value of functionallyevaluating a mutation, enabling mechanistic understanding and therapeutic modeling to realize precision medicine for epilepsy.

Protein kinase A modulation of CaV1.4 calcium channels.

The regulation of L-type Ca2+ channels by protein kinase A (PKA) represents a crucial element within cardiac, skeletal muscle and neurological systems. Although much work has been done to understand this regulation in cardiac CaV1.2 Ca2+ channels, relatively little is known about the closely related CaV1.4 L-type Ca2+ channels, which feature prominently in the visual system. Here we find that CaV1.4 channels are indeed modulated by PKA phosphorylation within the inhibitor of Ca2+-dependent inactivation (ICDI) motif. Phosphorylation of this region promotes the occupancy of calmodulin on the channel, thus increasing channel open probability (PO) and Ca2+-dependent inactivation. Although this interaction seems specific to CaV1.4 channels, introduction of ICDI1.4 to CaV1.3 or CaV1.2 channels endows these channels with a form of PKA modulation, previously unobserved in heterologous systems. Thus, this mechanism may not only play an important role in the visual system but may be generalizable across the L-type channel family.

Diastolic Calcium Leak and the Role of Zinc

In heart failure, there is a significant accumulation of Zn2+ in left ventricular tissue (1). Dysregulation of Zn2+-homeostasis results in altered cardiac contractility and chronically raised cytosolic Ca2+ (2). We have recently shown that Zn2+ plays a key role in shaping intracellular Ca2+-dynamics through modulation of the cardiac ryanodine receptor (RyR2) (3). In healthy cardiac cells, RyR2 is inhibited by Mg2+, which acts to prevent inappropriate activation of RyR2 during diastole. We have investigated the relationship between cytosolic Ca2+, Mg2+ and Zn2+ in regulating RyR2 gating. Sheep RyR2 channels were incorporated into artificial membranes under voltage-clamp conditions, as previously described (3). At activating levels of Ca2+ (5µM) and in the presence of physiological concentrations of Mg2+ (1mM) the addition of 100pM cytosolic Zn2+ increased channel open probability (Po) from 0.11±0.02 to 0.34±0.12 (SD, n=3, p<0.05), suggesting that Zn2+ plays a role in enabling RyR2 to operate under conditions of systole. When [Ca2+] was lowered to diastolic levels (100nM), the addition of 1mM Mg2+ resulted in complete closure of the channel. Under these conditions, the addition of 100pM Zn2+ had no effect on channel Po. Elevating Zn2+ to pathophysiological levels (1nM) however, caused abnormal channel openings (Po=0.12; n=2). At activating levels of Ca2+ (5µM), in 38% of our bilayers, we observed currents consistent with those reported for Mitsugumin-23 (MG23) (4), with no contribution from RyR2. Interestingly, the addition of 1nM cytosolic Zn2+ increased these currents (mean average current increased from −0.56±0.05 to −0.98±0.1pA (SD, n=4, p<0.05)). These data suggest that dysregulated Zn2+-homeostasis leads to irregular gating of both RyR2 and MG23, which is likely to contribute to diastolic Ca2+ leak in heart failure.BHF Funded1. Hare et al.,(2009) The Biochemist;31:46-49.2. Kamalov et al.,(2010) J. Cardiovasc. Pharmacol;56:320-328.3. Woodier et al.,(2015) JBC;290:17599-17610.4. Venturi et al,.(2011) Biochem;50:2623-2632.

Low free drug concentration prevents inhibition of F508del CFTR functional expression by the potentiator VX-770 (ivacaftor).

The most common cystic fibrosis (CF) mutation F508del inhibits the gating and surface expression of CFTR, a plasma membrane anion channel. Optimal pharmacotherapies will probably require both a 'potentiator' to increase channel open probability and a 'corrector' that improves folding and trafficking of the mutant protein and its stability at the cell surface. Interaction between CF drugs has been reported but remains poorly understood. CF bronchial epithelial cells were exposed to the corrector VX-809 (lumacaftor) and potentiator VX-770 (ivacaftor) individually or in combination. Functional expression of CFTR was assayed as the forskolin-stimulated short-circuit current (Isc ) across airway epithelial monolayers expressing F508del CFTR. The potentiated Isc response during forskolin stimulation was increased sixfold after pretreatment with VX-809 alone and reached ~11% that measured across non-CF monolayers. VX-770 (100nM) and genistein (50μM) caused similar levels of potentiation, which were not additive and were abolished by the CFTR inhibitor CFTRinh -172. The unbound fraction of VX-770 in plasma was 0.13 ± 0.04%, which together with previous measurements in patients given 250mg p.o. twice daily, suggests a peak free plasma concentration of 1.5-8.5nM. Chronic exposure to high VX-770 concentrations (>1μM) inhibited functional correction by VX-809 but not in the presence of physiological protein levels (20-40mg·mL(-1) ). Chronic exposure to a low concentration of VX-770 (100nM) together with VX-809 (1μM) also did not reduce the forskolin-stimulated Isc , relative to cells chronically exposed to VX-809 alone, provided it was assayed acutely using the same, clinically relevant concentration of potentiator. Chronic exposure to clinically relevant concentrations of VX-770 did not reduce F508del CFTR function. Therapeutic benefit of VX-770 + VX-809 (Orkambi) is probably limited by the efficacy of VX-809 rather than by inhibition by VX-770.

Calcium homeostasis modulator (CALHM) ion channels.

Calcium homeostasis modulator 1 (CALHM1), formerly known as FAM26C, was recently identified as a physiologically important plasma membrane ion channel. CALHM1 and its Caenorhabditis elegans homolog, CLHM-1, are regulated by membrane voltage and extracellular Ca(2+) concentration ([Ca(2+)]o). In the presence of physiological [Ca(2+)]o (∼1.5 mM), CALHM1 and CLHM-1 are closed at resting membrane potentials but can be opened by strong depolarizations. Reducing [Ca(2+)]o increases channel open probability, enabling channel activation at negative membrane potentials. Together, voltage and Ca(2+) o allosterically regulate CALHM channel gating. Through convergent evolution, CALHM has structural features that are reminiscent of connexins and pannexins/innexins/LRRC8 (volume-regulated anion channel (VRAC)) gene families, including four transmembrane helices with cytoplasmic amino and carboxyl termini. A CALHM1 channel is a hexamer of CALHM1 monomers with a functional pore diameter of ∼14 Å. CALHM channels discriminate poorly among cations and anions, with signaling molecules including Ca(2+) and ATP able to permeate through its pore. CALHM1 is expressed in the brain where it plays an important role in cortical neuron excitability induced by low [Ca(2+)]o and in type II taste bud cells in the tongue that sense sweet, bitter, and umami tastes where it functions as an essential ATP release channel to mediate nonsynaptic neurotransmitter release. CLHM-1 is expressed in C. elegans sensory neurons and body wall muscles, and its genetic deletion causes locomotion defects. Thus, CALHM is a voltage- and Ca(2+) o-gated ion channel, permeable to large cations and anions, that plays important roles in physiology.

Functional Roles of Clusters of Hydrophobic and Polar Residues in the Epithelial Na+ Channel Knuckle Domain

The extracellular regions of epithelial Na(+) channel subunits are highly ordered structures composed of domains formed by α helices and β strands. Deletion of the peripheral knuckle domain of the α subunit in the αβγ trimer results in channel activation, reflecting an increase in channel open probability due to a loss of the inhibitory effect of external Na(+) (Na(+) self-inhibition). In contrast, deletion of either the β or γ subunit knuckle domain within the αβγ trimer dramatically reduces epithelial Na(+) channel function and surface expression, and impairs subunit maturation. We systematically mutated individual α subunit knuckle domain residues and assessed functional properties of these mutants. Cysteine substitutions at 14 of 28 residues significantly suppressed Na(+) self-inhibition. The side chains of a cluster of these residues are non-polar and are predicted to be directed toward the palm domain, whereas a group of polar residues are predicted to orient their side chains toward the space between the knuckle and finger domains. Among the mutants causing the greatest suppression of Na(+) self-inhibition were αP521C, αI529C, and αS534C. The introduction of Cys residues at homologous sites within either the β or γ subunit knuckle domain resulted in little or no change in Na(+) self-inhibition. Our results suggest that multiple residues in the α subunit knuckle domain contribute to the mechanism of Na(+) self-inhibition by interacting with palm and finger domain residues via two separate and chemically distinct motifs.

Phosphorylation-dependent Changes in Nucleotide Binding, Conformation, and Dynamics of the First Nucleotide Binding Domain (NBD1) of the Sulfonylurea Receptor 2B (SUR2B)

The sulfonylurea receptor 2B (SUR2B) forms the regulatory subunit of ATP-sensitive potassium (KATP) channels in vascular smooth muscle. Phosphorylation of the SUR2B nucleotide binding domains (NBD1 and NBD2) by protein kinase A results in increased channel open probability. Here, we investigate the effects of phosphorylation on the structure and nucleotide binding properties of NBD1. Phosphorylation sites in SUR2B NBD1 are located in an N-terminal tail that is disordered. Nuclear magnetic resonance (NMR) data indicate that phosphorylation of the N-terminal tail affects multiple residues in NBD1, including residues in the NBD2-binding site, and results in altered conformation and dynamics of NBD1. NMR spectra of NBD1 lacking the N-terminal tail, NBD1-ΔN, suggest that phosphorylation disrupts interactions of the N-terminal tail with the core of NBD1, a model supported by dynamic light scattering. Increased nucleotide binding of phosphorylated NBD1 and NBD1-ΔN, compared with non-phosphorylated NBD1, suggests that by disrupting the interaction of the NBD core with the N-terminal tail, phosphorylation also exposes the MgATP-binding site on NBD1. These data provide insights into the molecular basis by which phosphorylation of SUR2B NBD1 activates KATP channels.

A Conserved Residue Cluster That Governs Kinetics of ATP-dependent Gating of Kir6.2 Potassium Channels

ATP-sensitive potassium (KATP) channels are heteromultimeric complexes of an inwardly rectifying Kir channel (Kir6.x) and sulfonylurea receptors. Their regulation by intracellular ATP and ADP generates electrical signals in response to changes in cellular metabolism. We investigated channel elements that control the kinetics of ATP-dependent regulation of KATP (Kir6.2 + SUR1) channels using rapid concentration jumps. WT Kir6.2 channels re-open after rapid washout of ATP with a time constant of ∼60 ms. Extending similar kinetic measurements to numerous mutants revealed fairly modest effects on gating kinetics despite significant changes in ATP sensitivity and open probability. However, we identified a pair of highly conserved neighboring amino acids (Trp-68 and Lys-170) that control the rate of channel opening and inhibition in response to ATP. Paradoxically, mutations of Trp-68 or Lys-170 markedly slow the kinetics of channel opening (500 and 700 ms for W68L and K170N, respectively), while increasing channel open probability. Examining the functional effects of these residues using φ value analysis revealed a steep negative slope. This finding implies that these residues play a role in lowering the transition state energy barrier between open and closed channel states. Using unnatural amino acid incorporation, we demonstrate the requirement for a planar amino acid at Kir6.2 position 68 for normal channel gating, which is potentially necessary to localize the ϵ-amine of Lys-170 in the phosphatidylinositol 4,5-bisphosphate-binding site. Overall, our findings identify a discrete pair of highly conserved residues with an essential role for controlling gating kinetics of Kir channels.

Extracellular Chloride Regulates TMEM16A Gating

The gating mechanism of TMEM16A, an essential subunit of the Ca2+-activated Cl- channel, remains incompletely understood. Gating of TMEM16A channels is complex because elevation of intracellular calcium concentration ([Ca2+]i), strong depolarizations (Vm), and high extracellular Cl or highly permeant anions seem to contribute. In the present work we show that in TMEM16A no gating currents can be detected, however, external Cl ions might play a role in voltage sensing because the kinetics and magnitude of current activation were dependent on [Cl-]e. The onset and offset of Cl- currents produced by 0.5 s depolarisations of cells bathed in 137 mM [Cl-]e followed a mono-exponential behaviour; a subsequent reduction of [Cl-]e to 10 mM diminished the conductance ∼2-fold. These data suggest a single gating process regulated by extracellular Cl-. In contrast, when cells were exposed to 137 mM [Cl-]e the onset and offset currents elicited by 20s depolarisations exhibited fast and slow kinetics. Activation of the slow component resulted in large onset Cl- currents that did not reach steady-state at the end of the 20s pulse. This behaviour was abolished after activating TMEM16A with 5 µM [Ca2+]i or after deleting four residues (448EAVK451) located in the first intracellular loop. Deleting four additional residues (444EEEEEAVK451) resulted in Cl- currents that decayed at the end of the 20 s depolarization. To explain our data we developed a 12-state gating model assuming that TMEM16A is activated by a direct, Vm-dependent binding of two Ca2+ ions. In this model external Cl- increases channel open probability by promoting stable Vm-dependent Ca2+ binding. Our model reproduced the gating behaviour of the TMEM16A currents in response to voltage, Ca2+, and [Cl-]e. Thus, we conclude that external Cl stabilises Ca binding and promotes two gating modes in TMEM16A. Supported by grant 219949 from CONACyT.

Proteases, ENaCs and cystic fibrosis.

Proteases, ENaCs and cystic fibrosis.

Glutathionylation of the L-type Ca2+ channel in oxidative stress-induced pathology of the heart.

There is mounting evidence to suggest that protein glutathionylation is a key process contributing to the development of pathology. Glutathionylation occurs as a result of posttranslational modification of a protein and involves the addition of a glutathione moiety at cysteine residues. Such modification can occur on a number of proteins, and exerts a variety of functional consequences. The L-type Ca2+ channel has been identified as a glutathionylation target that participates in the development of cardiac pathology. Ca2+ influx via the L-type Ca2+ channel increases production of mitochondrial reactive oxygen species (ROS) in cardiomyocytes during periods of oxidative stress. This induces a persistent increase in channel open probability, and the resulting constitutive increase in Ca2+ influx amplifies the cross-talk between the mitochondria and the channel. Novel strategies utilising targeted peptide delivery to uncouple mitochondrial ROS and Ca2+ flux via the L-type Ca2+ channel following ischemia-reperfusion have delivered promising results, and have proven capable of restoring appropriate mitochondrial function in myocytes and in vivo.

Deletion of α-subunit exon 11 of the epithelial Na+ channel reveals a regulatory module

Epithelial Na(+) channel (ENaC) subunits (α, β, and γ) found in functional complexes are translated from mature mRNAs that are similarly processed by the inclusion of 13 canonical exons. We examined whether individual exons 3-12, encoding the large extracellular domain, are required for functional channel expression. Human ENaCs with an in-frame deletion of a single α-subunit exon were expressed in Xenopus oocytes, and their functional properties were examined by two-electrode voltage clamp. With the exception of exon 11, deletion of an individual exon eliminated channel activity. Channels lacking α-subunit exon 11 were hyperactive. Oocytes expressing this mutant exhibited fourfold greater amiloride-sensitive whole cell currents than cells expressing wild-type channels. A parallel fivefold increase in channel open probability was observed with channels lacking α-subunit exon 11. These mutant channels also exhibited a lost of Na(+) self-inhibition, whereas we found similar levels of surface expression of mutant and wild-type channels. In contrast, in-frame deletions of exon 11 from either the β- or γ-subunit led to a significant loss of channel activity, in association with a marked decrease in surface expression. Our results suggest that exon 11 within the three human ENaC genes encodes structurally homologous yet functionally diverse domains and that exon 11 in the α-subunit encodes a module that regulates channel gating.

An Intramolecular Interaction Controls a Rate-Limiting Step in ATP-Dependent Gating of Kir6.2 Channels

ATP-sensitive potassium (KATP) channels are complexes of inwardly-rectifying Kir channels (Kir6.x) and sulfonylurea receptors (SUR). These two subunits work together to encode sensitivity to intracellular adenine nucleotides (ATP and ADP) and other regulatory ligands including PIP2. We investigated the kinetics of ATP-dependent regulation of KATP (Kir6.2 + SUR1) channels using rapid concentration jumps. WT Kir6.2 channels rapidly re-open after washout of ATP, with a time constant of ∼60 ms. Extending similar kinetic measurements to mutants around the bundle crossing revealed modest effects on gating kinetics despite significant changes in ATP sensitivity. However, we identified a pair of highly conserved neighboring amino acids (Trp68, Lys170) that controls the rate of channel recovery and inhibition by ATP. Mutations of Trp68 or Lys170 have the paradoxical effect of dramatically slowing the kinetics of channel opening, while increasing channel open probability. Formalization of the transition state effects of these residues using phi-value analysis revealed a consistent steep negative slope. This finding implies that these residues play a specific role in lowering the transition state energy barrier between open and closed channel states. The demonstration of a negative phi-value is unique for an ion channel gating process, and diverges from interpretations that use phi-values to place specific residues in the spatial and temporal progression through the transition energy landscape. Using nonsense suppression for unnatural amino acid incorporation, we demonstrate the requirement for a planar amino acid at Kir6.2 position 68, potentially required to localize the e-amine of Lys170 in the PIP2 binding site. Our findings identify a discrete pair of residues with an essential role for controlling gating kinetics of Kir channels.

Chemical Uncoupling the DHPR-RyR1 Complex by Substituted Halogenated Biphenyls and Diphenylethers

Ca2+ release units (CRU) of skeletal muscle have two components necessary for excitation-contraction coupling (ECC); the DHPR and RyR1 that physically interact to engage conformational coupling that involves both orthograde and retrograde regulation of Ca2+ dynamics during ECC and SR Ca2+ leak when the muscle is at rest. We previously identified that 2,2',3,5',6-pentachlorobiphenyl (PCB95) promotes long-lived stability of the full open state of RyR1 channels (Samso et al 2009). Further investigations PCB95 and its 4- and 5-hydroxy (-OH) derivatives toward rabbit skeletal muscle ryanodine receptor (RyR1) were performed using [3H]ryanodine binding and SR Ca2+ flux analyses. 5-OH metabolites have comparable activity to their respective parent in both assays; however, 4-OH derivatives are unable to trigger Ca2+ release from SR microsomes in the presence of Ca2+-ATPase activity. PCB95 and derivatives are investigated using single channel voltage-clamp and primary murine embryonic muscle cells. Like PCB95, 5-OH-PCB95 quickly and persistently increases channel open probability (Po>0.9) by stabilizing the full-open channel state, whereas 4-OH-PCB95 transiently enhances Po. Ca2+ imaging of myotubes show acute exposure to PCB95 (5μM) potentiates ECC and caffeine responses and partially depletes SR Ca2+ stores. Exposure to 5-OH-PCB95 (5μM) increases cytoplasmic Ca2+, leading to ECC failure in 50% of myotubes with the remainder retaining negligible responses. 4-OH-PCB95 neither increases baseline Ca2+ nor causes ECC failure but depresses ECC and caffeine responses by 50%. With longer (3 h) exposure to 300nM PCB95, 5-OH-PCB95, or 4-OH-PCB95 decreases the number of ECC responsive myotubes by 22%, 81%, and 51% compared with control by depleting SR Ca2+ and/or uncoupling ECC. The position of hydroxylation of PCB95 and chemically related diphenylethers differentially influence RyR1 channel gating kinetics, SR Ca2+ leak, and the pattern of ECC impairment, including ECC uncoupling in intact muscle cells. P01 AR52354.

Estradiol activates epithelial sodium channels in rat alveolar cells through the G protein-coupled estrogen receptor

Female sex predisposes individuals to poorer outcomes during respiratory disorders like cystic fibrosis and influenza-associated pneumonia. A common link between these disorders is dysregulation of alveolar fluid clearance via disruption of epithelial sodium channel (ENaC) activity. Recent evidence suggests that female sex hormones directly regulate expression and activity of alveolar ENaC. In our study, we identified the mechanism by which estradiol (E2) or progesterone (P4) independently regulates alveolar ENaC. Using cell-attached patch clamp, we measured ENaC single-channel activity in a rat alveolar cell line (L2) in response to overnight exposure to either E2 or P4. In contrast to P4, E2 increased ENaC channel activity (NPo) through an increase in channel open probability (Po) and an increased number of patches with observable channel activity. Apical plasma membrane abundance of the ENaC α-subunit (αENaC) more than doubled in response to E2 as determined by cell surface biotinylation. αENaC membrane abundance was approximately threefold greater in lungs from female rats in proestrus, when serum E2 is greatest, compared with diestrus, when it is lowest. Our results also revealed a significant role for the G protein-coupled estrogen receptor (Gper) to mediate E2's effects on ENaC. Overall, our results demonstrate that E2 signaling through Gper selectively activates alveolar ENaC through an effect on channel gating and channel density, the latter via greater trafficking of channels to the plasma membrane. The results presented herein implicate E2-mediated regulation of alveolar sodium channels in the sex differences observed in the pathogenesis of several pulmonary diseases.