Inhibition Of Channel Activity Research Articles

The inhibition mechanism of the SUR2A-containing KATP channel by a regulatory helix

KATP channels are metabolic sensors for intracellular ATP/ADP ratios, play essential roles in many physiological processes, and are implicated in a spectrum of pathological conditions. SUR2A-containing KATP channels differ from other subtypes in their sensitivity to Mg-ADP activation. However, the underlying structural mechanism remains poorly understood. Here we present a series of cryo-EM structures of SUR2A in the presence of different combinations of Mg-nucleotides and the allosteric inhibitor repaglinide. These structures uncover regulatory helix (R helix) on the NBD1-TMD2 linker, which wedges between NBD1 and NBD2. R helix stabilizes SUR2A in the NBD-separated conformation to inhibit channel activation. The competitive binding of Mg-ADP with Mg-ATP to NBD2 mobilizes the R helix to relieve such inhibition, allowing channel activation. The structures of SUR2B in similar conditions suggest that the C-terminal 42 residues of SUR2B enhance the structural dynamics of NBD2 and facilitate the dissociation of the R helix and the binding of Mg-ADP to NBD2, promoting NBD dimerization and subsequent channel activation.

The Potential Antidepressant Compound Org 34167 Modulates HCN Channels Via a Novel Mode of Action.

Org 34167 is a small molecule hyperpolarization-activated cyclic nucleotide-gated (HCN) channel modulator that has been trialed in humans for its potential antidepressant activity. The precise action of Org 34167 is not fully understood. Here we use two-electrode voltage clamp recordings and an allosteric model to explore the interaction of Org 34167 with human HCN1 channels. The impact of Org 34167 on channel function included a hyperpolarizing shift in activation voltage dependence and a slowing of activation kinetics. Furthermore, a reduction in the maximum open probability at extreme hyperpolarization argued for an additional voltage-independent mechanism. Org 34167 had a similar impact on a truncated HCN1 channel lacking the C-terminal nucleotide binding domain, thus ruling out an interaction with this domain. Fitting a gating model, derived from a 10-state allosteric scheme, predicted that Org 34167 strongly reduced the equilibrium constant for the voltage-independent pore domain to favor a closed pore, as well as reducing the voltage sensing domain-pore domain coupling and shifting the zero voltage equilibrium constant of the voltage sensing domain to favor the inactive state. SIGNIFICANCE STATEMENT: The brain penetrant small molecule Org 34167 has been reported to have an antidepressant action by targeting HCN channels; however, its mode of action is unknown. We used heterologously expressed human HCN1 channels to show that Org 34167 inhibits channel activity by modulating kinetic parameters associated with the channel pore domain, voltage sensing domain, and interdomain coupling.

Untangling the inseparable life of an ABC transporter and a potassium channel: Towards understanding the structure-function relationship in the KATP channel complex.

KATP channels are unique protein complexes of a potassium channel, Kir6.1 or Kir6.2, and an ABC transporter, SUR1, SUR2A or SUR2B (splice variants of SUR2), in a 4:4 stoichiometry. Broadly expressed in many cell types and tissues, KATP channels couple changes in cellular metabolic activity with membrane excitability to control a wide range of physiological processes, including hormone secretion, control of vascular tone, learning and memory, and cardiac and neuronal protection against ischemic insults. In pancreatic β-cells KATP channels formed by Kir6.2 and SUR1 control insulin secretion, and gain- or loss-of-function mutations in these channels cause neonatal diabetes or congenital hyperinsulinism, respectively. Physiological activity of pancreatic KATP channels is regulated by intracellular ATP and ADP. Non-hydrolytic binding of ATP to Kir6.2 inhibits channel activity, while MgATP and MgADP binding to the nucleotide binding domains (NBDs) of SUR1 stimulates channel activity. Besides ATP and ADP, membrane phospholipids in particular PIP2 interacts with Kir6.2 and stimulates channel activity. In addition, pharmacological inhibitors and activators act on SUR1 to modulate channel activity. Although Kir6.2 and SUR1 harbor the primary binding sites for specific physiological and pharmacological ligands, both subunits participate in ligand regulation. How do the potassium channel and the silent ABC transporter work together to decipher signals from different ligands for gating has long been a major question in the KATP channel field. Here, I present structural and functional evidence that SUR1 contributes directly to ATP and PIP2 binding via its TMD0 and L0 linker to enhance Kir6.2 sensitivity to both ligands. Moreover, SUR1 regulates Kir6.2 activity allosterically via its dynamic interaction with the N-terminus of Kir6.2.

Phosphodiesterase delta governs the mechanical properties of erythrocytes infected with Plasmodium falciparum gametocytes

To persist in the blood circulation and to be available for mosquitoes, Plasmodium falciparum gametocytes modify the deformability and the permeability of their erythrocyte host via cyclic AMP (cAMP) signaling pathway. Cyclic nucleotide levels are tightly controlled by phosphodiesterases (PDE), however in Plasmodium these proteins are poorly characterized. Here, we characterize the P. falciparum phosphodiesterase delta (PfPDEδ) and we investigate its role in the cAMP signaling-mediated regulation of gametocyte-infected erythrocyte mechanical properties. Our results revealed that PfPDEδ is a dual-function enzyme capable of hydrolyzing both cAMP and cGMP, with a higher affinity for cAMP. We also show that PfPDEδ is the most expressed PDE in mature gametocytes and we propose that it is located in parasitophorous vacuole at the interface between the host cell and the parasite. We conclude that PfPDEδ is the master regulator of both the increase in deformability and the inhibition of channel activity in mature gametocyte stages, and may therefore play a crucial role in the persistence of mature gametocytes in the bloodstream.

Norfluoxetine inhibits TREK-2 K2P channels by multiple mechanisms including state-independent effects on the selectivity filter gate.

The TREK subfamily of two-pore domain K+ (K2P) channels are inhibited by fluoxetine and its metabolite, norfluoxetine (NFx). Although not the principal targets of this antidepressant, TREK channel inhibition by NFx has provided important insights into the conformational changes associated with channel gating and highlighted the role of the selectivity filter in this process. However, despite the availability of TREK-2 crystal structures with NFx bound, the precise mechanisms underlying NFx inhibition remain elusive. NFx has previously been proposed to be a state-dependent inhibitor, but its binding site suggests many possible ways in which this positively charged drug might inhibit channel activity. Here we show that NFx exerts multiple effects on single-channel behavior that influence both the open and closed states of the channel and that the channel can become highly activated by 2-APB while remaining in the down conformation. We also show that the inhibitory effects of NFx are unrelated to its positive charge but can be influenced by agonists which alter filter stability, such as ML335, as well as by an intrinsic voltage-dependent gating process within the filter. NFx therefore not only inhibits channel activity by altering the equilibrium between up and down conformations but also can directly influence filter gating. These results provide further insight into the complex allosteric mechanisms that modulate filter gating in TREK K2P channels and highlight the different ways in which filter gating can be regulated to permit polymodal regulation.

RyR2 disease mutations at the C-terminal domain intersubunit interface alter closed-state stability and channel activation

Ryanodine receptors (RyRs) are ion channels that mediate the release of Ca2+ from the sarcoplasmic reticulum/endoplasmic reticulum, mutations of which are implicated in a number of human diseases. The adjacent C-terminal domains (CTDs) of cardiac RyR (RyR2) interact with each other to form a ring-like tetrameric structure with the intersubunit interface undergoing dynamic changes during channel gating. This mobile CTD intersubunit interface harbors many disease-associated mutations. However, the mechanisms of action of these mutations and the role of CTD in channel function are not well understood. Here, we assessed the impact of CTD disease-associated mutations P4902S, P4902L, E4950K, and G4955E on Ca2+− and caffeine-mediated activation of RyR2. The G4955E mutation dramatically increased both the Ca2+-independent basal activity and Ca2+-dependent activation of [3H]ryanodine binding to RyR2. The P4902S and E4950K mutations also increased Ca2+ activation but had no effect on the basal activity of RyR2. All four disease mutations increased caffeine-mediated activation of RyR2 and reduced the threshold for activation and termination of spontaneous Ca2+ release. G4955D dramatically increased the basal activity of RyR2, whereas G4955K mutation markedly suppressed channel activity. Similarly, substitution of P4902 with a negatively charged residue (P4902D), but not a positively charged residue (P4902K), also dramatically increased the basal activity of RyR2. These data suggest that electrostatic interactions are involved in stabilizing the CTD intersubunit interface and that the G4955E disease mutation disrupts this interface, and thus the stability of the closed state. Our studies shed new insights into the mechanisms of action of RyR2 CTD disease mutations.

Molecular mechanism and structural basis of small-molecule modulation of the gating of acid-sensing ion channel 1

Acid-sensing ion channels (ASICs) are proton-gated cation channels critical for neuronal functions. Studies of ASIC1, a major ASIC isoform and proton sensor, have identified acidic pocket, an extracellular region enriched in acidic residues, as a key participant in channel gating. While binding to this region by the venom peptide psalmotoxin modulates channel gating, molecular and structural mechanisms of ASIC gating modulation by small molecules are poorly understood. Here, combining functional, crystallographic, computational and mutational approaches, we show that two structurally distinct small molecules potently and allosterically inhibit channel activation and desensitization by binding at the acidic pocket and stabilizing the closed state of rat/chicken ASIC1. Our work identifies a previously unidentified binding site, elucidates a molecular mechanism of small molecule modulation of ASIC gating, and demonstrates directly the structural basis of such modulation, providing mechanistic and structural insight into ASIC gating, modulation and therapeutic targeting.

Site-specific deacylation by ABHD17a controls BK channel splice variant activity

S-Acylation, the reversible post-translational lipid modification of proteins, is an important mechanism to control the properties and function of ion channels and other polytopic transmembrane proteins. However, although increasing evidence reveals the role of diverse acyl protein transferases (zDHHC) in controlling ion channel S-acylation, the acyl protein thioesterases that control ion channel deacylation are very poorly defined. Here we show that ABHD17a (α/β-hydrolase domain-containing protein 17a) deacylates the stress-regulated exon domain of large conductance voltage- and calcium-activated potassium (BK) channels inhibiting channel activity independently of effects on channel surface expression. Importantly, ABHD17a deacylates BK channels in a site-specific manner because it has no effect on the S-acylated S0–S1 domain conserved in all BK channels that controls membrane trafficking and is deacylated by the acyl protein thioesterase Lypla1. Thus, distinct S-acylated domains in the same polytopic transmembrane protein can be regulated by different acyl protein thioesterases revealing mechanisms for generating both specificity and diversity for these important enzymes to control the properties and functions of ion channels.

Voltage-dependent calcium channel β subunit-derived peptides reduce excitatory neurotransmission and arterial blood pressure

AimsVoltage-dependent calcium channels (VDCCs) play an important role in various physiological functions in the nervous system and the cardiovascular system. In L-, N-, P/Q-, and R-type VDCCs, β subunit assists the channels for membrane targeting and modulates channel properties. In this study, we investigated whether an inhibition of the β subunit binding to α subunit, the pore-forming main subunit of VDCCs, have any effect on channel activation and physiological functions. Main methodsPeptides derived from the specific regions of β subunit that bind to the α-interaction domain in I-II linker of α subunit were manufactured, presuming that the peptides interrupt α-β subunit interaction in the channel complex. Then, they were tested on voltage-activated Ca2+ currents recorded in acutely isolated trigeminal ganglion (TG) neurons, excitatory postsynaptic currents (EPSCs) in the spinal dorsal horn neurons, and arterial blood pressure (BP) recorded from the rat femoral artery. Key findingsWhen applied internally through patch pipettes, the peptides decreased the peak amplitudes of the voltage-activated Ca2+ currents. After fusing with HIV transactivator of transcription (TAT) sequence to penetrate cell membrane, the peptides significantly decreased the peak amplitudes of Ca2+ currents and the peak amplitudes of EPSCs upon the external application through bath solution. Furthermore, the TAT-fused peptides dose dependently reduced the rat BP when administered intravenously. SignificanceThese data suggest that an interruption of α-β subunit association in VDCC complex inhibits channel activation, thereby reducing VDCC-mediated physiological functions such as excitatory neurotransmission and arterial BP.

G551D mutation impairs PKA-dependent activation of CFTR channel that can be restored by novel GOF mutations.

G551D is a major disease-associated gating mutation in the cystic fibrosis transmembrane conductance regulator (CFTR) protein, an ATP- and phosphorylation-dependent chloride channel. G551D causes severe cystic fibrosis (CF) disease by disrupting ATP-dependent channel opening; however, whether G551D affects phosphorylation-dependent channel activation is unclear. Here, we use macropatch recording and Ussing chamber approaches to demonstrate that G551D impacts on phosphorylation-dependent activation of CFTR, and PKA-mediated phosphorylation regulates the interaction between the x-loop in nucleotide-binding domain 2 (NBD2) and cytosolic loop (CL) 1. We show that G551D not only disrupts ATP-dependent channel opening but also impairs phosphorylation-dependent channel activation by largely reducing PKA sensitivity consistent with the reciprocal relationship between channel opening/gating, ligand binding, and phosphorylation. Furthermore, we identified two novel GOF mutations: D1341R in the x-loop near the ATP-binding cassette signature motif in NBD2 and D173R in CL1, each of which strongly increased PKA sensitivity both in the wild-type (WT) background and when introduced into G551D-CFTR. When D1341R was combined with a second GOF mutation (e.g., K978C in CL3), we find that the double GOF mutation maximally increased G551D channel activity such that VX-770 had no further effect. We further show that a double charge-reversal mutation of D1341R/D173R-CFTR exhibited similar PKA sensitivity when compared with WT-CFTR. Together, our results suggest that charge repulsion between D173 and D1341 of WT-CFTR normally inhibits channel activation at low PKA activity by reducing PKA sensitivity, and negative allostery by the G551D is coupled to reduced PKA sensitivity of CFTR that can be restored by second GOF mutations.

SO040ROLE OF INTRACELLULAR CA2+ IN THE REGULATION OF THE RENAL CHLORIDE CHANNEL CLC-KA

Abstract Background and Aims It has been recognized that the physiological role of ClC-Ka and its mouse homolog ClC-K1 in the nephron is to confer high Cl- permeability in the thin Ascending Limb of loop of Henle (tAL), which in turn is essential for establishing the high osmolarity of the renal medulla in the countercurrent system. Accordingly ClC-K1 KO mice display a severe polyuria resembling a Nephrogenic Diabetes Insipidus (NDI) phenotype. Thus, ClC-Ka in human nephron could be an interesting target for diuretic and anti-hypertensive drugs. Unfortunately the regulation of ClC-Ka in and its electrophysiological properties are still not completely known. Method We analyzed, by whole-cell voltage-clamp, Cl- currents in HEK293 cells co-expressing GFP-ClC-Ka and the accessory m-Cherry-tagged barttin subunit, upon intracellular Ca2+ mobilization. The patch clamp protocol consists of a holding potential from 0 mV, which is stepped to various test potentials from -195 and +125 mV for 150 ms. Pulses ended with a tail pulse to -125 mV for 20 ms. Concomitantly we monitored by live confocal microscopy, the membrane expression of either GFP-ClC-Ka or barttin-mCherry. Results In control conditions we found a linear current/voltage relationship over a broad range of membrane voltages except of hyperpolarization below -100 mV, where the channel open probability was reduced, providing the well-known hook-shaped appearance of the I-V curve for ClC-Ka channel. Interestingly, both 100 µM ATP and 400 µg/ml of dandelion root extract (DRE), both increasing intracellular Ca2+ in HEK293, significantly inhibited Cl- currents in a time dependent way. Pre-treatment of cells with either the Ca2+ chelator BAPTA-AM or the PCK inhibitor Calphostin C, reverted the inhibitory effects of both ATP and DRE. Interestingly, 1µM of phorbol meristate acetate (PMA), a specific PKC activator, mimicked the inhibitory effect of ATP and DRE on Cl- currents. We finally demonstrated that 30 µM Heclin, an inhibitor of E3 ubiquitin ligase, did not revert the DRE- and ATP- induced Cl- currents inhibition, thus suggesting that the ClC-Ka inhibition is independent of channel ubiquitination. Accordingly, live confocal analysis showed that all the Ca2+ mobilizing compounds tested did not induced neither ClC-Ka nor Barttin internalization. We hypothesized that phosphorylation in the putative PKC consensus sites present in ClC-Ka amino acidic sequence may be involved in the inhibition of channel activity. Conclusion In conclusion, we demonstrated for the first time that the activity of ClC-Ka in renal cells could by inhibited by either receptor agonists or natural molecules able to activate an intracellular Ca2+/PKC pathway, thus opening venues for the development of new diuretic drugs.

New Insights into the Role of Intracellular Ca2+ in the Regulation of the Human Kidney Chloride Channel ClC‐Ka

It has been recognized that the physiological role of ClC‐Ka and its mouse homolog ClC‐K1 in the nephron is to confer high Cl− permeability in the thin Ascending Limb of Henle (tAL), which in turn is essential for establishing the high osmolarity of the renal medulla in the countercurrent system. Accordingly ClC‐K1 KO mice display a severe polyuria resembling a Nephrogenic Diabetes Insipidus (NDI) phenotype. Thus, ClC‐Ka in human nephron could be an interesting target for diuretic and anti‐hypertensive drugs. Unfortunately the regulation of ClC‐Ka in and its electrophysiological properties are still not completely known. We analyzed, by whole‐cell voltage‐clamp, Cl− currents in HEK293 cells expressing GFP‐ClC‐Ka and the accessory m‐Cherry Barttin subunit upon intracellular Ca2+mobilization. The patch clamp protocol consists of a holding potential from 0 mV, which is stepped to various test potentials from −195 and +125 mV for 150 ms. Pulses ended with a tail pulse to −125 mV for 20 ms. In control conditions we found a linear current/voltage relationship over a broad range of membrane voltages except of hyperpolarization below −100 mV, where the channel open probability was reduced, providing the well‐known hook‐shaped appearance of the I‐V curve for ClC‐Ka channel. Interestingly, both 100 μM ATP and 400 μg/ml of dandelion root extract (DRE), both increasing intracellular Ca2+ in HEK293, significantly inhibited Cl− currents in a time dependent way. Pretreatment of cells with either the Ca2+ chelator BAPTA‐AM or the PCK inhibitor Calphostin C, reverted the inhibitory effects of both ATP and DRE. Interestingly, 1μM of Phorbol Meristate Acetate (PMA), a specific PKC activator, mimicked the inhibitory effect of ATP and DRE on Cl− currents. We finally demonstrated that 30 μM Heclin, an inhibitor of E3 ubiquitin ligase, did not revert the DRE‐ and ATP‐ induced Cl− currents inhibition, thus suggesting that the ClC‐Ka inhibition is independent of channel ubiquitination. Accordingly, live confocal analysis showed that all the Ca2+mobilizing compounds tested did not induced neither ClC‐Ka nor Barttin internalization. We hypothesized that phosphorylation in the putative PKC consensus sites present in ClC‐Ka amino acidic sequence may be involved in the inhibition of channel activity. In conclusion, we demonstrated for the first time that the activity of ClC‐Ka in renal cells could by inhibited by either receptor agonists or natural molecules able to activate an intracellular Ca2+/PKC pathway, thus opening venues for the development of new diuretic drugs.Support or Funding InformationGrant n# ARS01_01081 to MC from The National Operational Program (PON) of the Ministry of Education, ITALYFigure 1

Modulation of a GIRK1 Active Mutant Subunit by Protein Kinase C Isoforms

G-protein gated inwardly rectifying potassium channels (GIRK)are activated by the Gᵦᵧ dimer of Gi/o-protein coupled receptors to inhibit excitability. There are four distinct subunits, GIRK1-GIRK4, which exist as heterotetramers or homotetramers throughout the body. Unlike GIRK1 and GIRK3 subunits that do not form functional homotetramers, GIRK2 and GIRK4 can each form homotetramers but can also assemble with the other subunits to form heterotetramers. These channels are activated by Na+, ethanol and PIP2, and are modulated by various factors like Protein kinase A and C (PKC). Phosphorylation of the channels by PKC has been widely reported to inhibit channel activity, however the effects of the various PKC isoforms on channel activity has yet to be fully defined. The 14 different isoforms of PKC are categorized into conventional, novel and atypical PKC's. In this study, we aimed to understand the various effects of two isoforms of PKC, the conventional PKCα and novel PKCε on GIRK1 through GIRK1(F137S), an active mutant of GIRK1 that can form functional homotetrameric channels. PKCε augmented GIRK1(F137S)activity, while PKCα inhibited GIRK1(F137S)activity. Due to the differential effect of these PKC isoforms on GIRK1, we sought to identify distinct phosphorylation sites on this subunit. Phosphorylation sites for both PKC isoforms on GIRK1(F137S) were predicted using the Group-based Prediction 3.0 software, and a site-directed mutagenesis approach is being employed to study alanine mutants of the channel in two-electrode voltage clamp experiments. Additionally, PIP2 dependence of PKC-mediated GIRK1 channel activity will be determined by depleting PIP2. This work aims to inform the elucidation of PKC modulation of GIRK1/2 and GIRK1/4 heterotetramers and the relevance of these effects on phosphorylation-mediated channelopathies.

Structural Determinants of Kv7.5 Potassium Channels That Confer Changes in Phosphatidylinositol 4,5-Bisphosphate (PIP2) Affinity and Signaling Sensitivities in Smooth Muscle Cells.

Smooth muscle cells express Kv7.4 and Kv7.5 voltage-dependent potassium channels, which have each been implicated as regulators of smooth muscle contractility, though they display different sensitivities to signaling via cAMP/protein kinase A (PKA) and protein kinase C (PKC). We expressed chimeric channels composed of different components of the Kv7.4 and Kv7.5 α-subunits in vascular smooth muscle cells to determine which components are essential for enhancement or inhibition of channel activity. Forskolin, an activator of the cAMP/PKA pathway, increased wild-type Kv7.5 but not wild-type Kv7.4 current amplitude. Replacing the amino terminus of Kv7.4 with the amino terminus of Kv7.5 conferred partial responsiveness to forskolin. In contrast, swapping carboxy-terminal phosphatidylinositol 4,5-bisphosphate (PIP2) binding domains, or the entire C terminus, was without effect on the forskolin response, but the latter conferred responsiveness to arginine-vasopressin (an inhibitory PKC-dependent response). Serine-to-alanine mutation at position 53 of the Kv7.5 amino terminus abrogated its ability to confer forskolin sensitivity to Kv7.4. Forskolin treatment reduced the sensitivity of Kv7.5 channels to Ciona intestinalis voltage-sensing phosphatase (Ci-VSP)-induced PIP2 depletion, whereas activation of PKC with phorbol-12-myristate-13-acetate potentiated the Ci-VSP-induced decline in Kv7.5 current amplitude. Our findings suggest that PKA-dependent phosphorylation of serine 53 on the amino terminus of Kv7.5 increases its affinity for PIP2, whereas PKC-dependent phosphorylation of the Kv7.5 carboxy terminus is associated with a reduction in PIP2 affinity; these changes in PIP2 affinity have corresponding effects on channel activity. Resting affinities for PIP2 differ for Kv7.4 and Kv7.5 based on differential responsiveness to Ci-VSP activation and different rates of current rundown in ruptured patch recordings. SIGNIFICANCE STATEMENT: Kv7.4 and Kv7.5 channels are known signal transduction intermediates and drug targets for regulation of smooth muscle tone. The present studies identify distinct functional domains that confer differential sensitivities of Kv7.4 and Kv7.5 to stimulatory and inhibitory signaling and reveal structural features of the channel subunits that determine their biophysical properties. These findings may improve our understanding of the roles of these channels in smooth muscle physiology and disease, particularly in conditions where Kv7.4 and Kv7.5 are differentially expressed.

A bivalent antihypertensive vaccine targeting L-type calcium channels and angiotensin AT1 receptors.

Hypertension has been the leading preventable cause of premature death worldwide. The aim of this study was to design a more efficient vaccine against novel targets for the treatment of hypertension. The epitope CE12, derived from the human L-type calcium channel (CaV 1.2), was designed and conjugated with Qβ bacteriophage virus-like particles to test the efficacy in hypertensive animals. Further, the hepatitis B core antigen (HBcAg)-CE12-CQ10 vaccine, a bivalent vaccine based on HBcAg virus-like particles and targeting both human angiotensin AT1 receptors and CaV 1.2 channels, was developed and evaluated in hypertensive rodents. The Qβ-CE12 vaccine effectively decreased the BP in hypertensive rodents. A monoclonal antibody against CE12 specifically bound to L-type calcium channels and inhibited channel activity. Injection with monoclonal antibody against CE12 effectively reduced the BP in angiotensin II-induced hypertensive mice. The HBcAg-CE12-CQ10 vaccine showed antihypertensive effects in hypertensive mice and relatively superior antihypertensive effects in spontaneously hypertensive rats and ameliorated L-NAME-induced renal injury. In addition, no obvious immune-mediated damage or electrophysiological adverse effects were detected. Immunotherapy against both AT1 receptors and CaV 1.2 channels decreased the BP in hypertensive rodents effectively and provided protection against hypertensive target organ damage without obvious feedback activation of renin-angiotensin system or induction of dominant antibodies against the carrier protein. Thus, the HBcAg-CE12-CQ10 vaccine may provide a novel and promising therapeutic approach for hypertension.

Membrane Lipid-KIR2.x Channel Interactions Enable Hemodynamic Sensing in Cerebral Arteries.

Objective- Inward rectifying K+ (KIR) channels are present in cerebral arterial smooth muscle and endothelial cells, a tandem arrangement suggestive of a dynamic yet undiscovered role for this channel. This study defined whether distinct pools of cerebral arterial KIR channels were uniquely modulated by membrane lipids and hemodynamic stimuli. Approach and Results- A Ba2+-sensitive KIR current was isolated in smooth muscle and endothelial cells of rat cerebral arteries; molecular analyses subsequently confirmed KIR2.1/KIR2.2 mRNA and protein expression in both cells. Patch-clamp electrophysiology next demonstrated that each population of KIR channels was sensitive to key membrane lipids and hemodynamic stimuli. In this regard, endothelial KIR was sensitive to phosphatidylinositol 4,5-bisphosphate content, with depletion impairing the ability of laminar shear stress to activate this channel pool. In contrast, smooth muscle KIR was sensitive to membrane cholesterol content, with sequestration blocking the ability of pressure to inhibit channel activity. The idea that membrane lipids help confer shear stress and pressure sensitivity of KIR channels was confirmed in intact arteries using myography. Virtual models integrating structural/electrical observations reconceptualized KIR as a dynamic regulator of membrane potential working in concert with other currents to set basal tone across a range of shear stresses and intravascular pressures. Conclusions- The data show for the first time that specific membrane lipid-KIR interactions enable unique channel populations to sense hemodynamic stimuli and drive vasomotor responses to set basal perfusion in the cerebral circulation.

Isomeric Carborane Neuromuscular Blocking Agents.

We synthesized a family of neuromuscular blocking agents (NMB) based on decamethonium, but containing a carborane cluster in the methylene chain between the two quaternary ammonium groups. The carborane cluster isomers o-NMB, m-NMB, and p-NMB were tested in animals for neuromuscular block and compared with agents used clinically: rocuronium and decamethonium. All three isomers caused reversible muscle weakness in mice as determined by grip strength and inverted screen tests, with a potency rank of p-NMB > rocuronium > decamethonium > m-NMB > o-NMB. The mechanism of action of the compounds was determined by using the in vitro rat phrenic nerve hemi-diaphragm preparation and electrophysiologic measurements in cells. Neostigmine reversed hemi-diaphragm weakness caused by the three isomers and rocuronium, but not succinylcholine. In electrophysiologic recordings of currents through acetylcholine receptor channels, the carborane compounds did not activate channel activity but did inhibit channel activation by acetylcholine. These results demonstrate that the carborane neuromuscular blocking agents are non-depolarizers in contrast to the depolarizing action of the parent compound.

Differential PI(4,5)P2 sensitivities of TRPC4, C5 homomeric and TRPC1/4, C1/5 heteromeric channels

Transient receptor potential canonical (TRPC) 4 and TRPC5 channels are modulated by the Gαq-PLC pathway. Since phosphatidylinositol 4,5-bisphosphate (PI(4,5)P2) maintains TRPC4 and TRPC5 channel function, the Gαq-PLC pathway inhibits channel activity by depleting PI(4,5)P2. Here we investigated the difference in PI(4,5)P2 sensitivity between homomeric and heteromeric TRPC channels. First, by using a Danio rerio voltage-sensing phosphatase (DrVSP), we show that PI(4,5)P2 dephosphorylation robustly inhibits TRPC4α, TRPC4β, and TRPC5 homotetramer currents and also TRPC1/4α, TRPC1/4β, and TRPC1/5 heterotetramer currents. Secondly, sensitivity of channels to PI(4,5)P2 dephosphorylation was suggested through the usage of FRET in combination with patch clamping. The sensitivity increased in the sequence TRPC4β < TRPC4α < TRPC5 in homotetramers, whereas when forming heterotetramers with TRPC1, the sensitivity was approximately equal between the channels. Thirdly, we determined putative PI(4,5)P2 binding sites based on a TRPC4 prediction model. By neutralization of basic residues, we identified putative PI(4,5)P2 binding sites because the mutations reduced FRET to a PI(4,5)P2 sensor and reduced the current amplitude. Therefore, one functional TRPC4 has 8 pockets with the two main binding regions; K419, K664/R511, K518, H630. We conclude that TRPC1 channel function as a regulator in setting PI(4,5)P2 affinity for TRPC4 and TRPC5 that changes PI(4,5)P2 sensitivity.

Single-Channel Resolution of the Interaction between C-Terminal CaV1.3 Isoforms and Calmodulin

Voltage-dependent calcium (CaV) 1.3 channels are involved in the control of cellular excitability and pacemaking in neuronal, cardiac, and sensory cells. Various proteins interact with the alternatively spliced channel C-terminus regulating gating of CaV1.3 channels. Binding of a regulatory calcium-binding protein calmodulin (CaM) to the proximal C-terminus leads to the boosting of channel activity and promotes calcium-dependent inactivation (CDI). The C-terminal modulator domain (CTM) of CaV1.3 channels can interfere with the CaM binding, thereby inhibiting channel activity and CDI. Here, we compared single-channel gating behavior of two natural CaV1.3 splice isoforms: the long CaV1.342 with the full-length CTM and the short CaV1.342A with the C-terminus truncated before the CTM. We found that CaM regulation of CaV1.3 channels is dynamic on a minute timescale. We observed that at equilibrium, single CaV1.342 channels occasionally switched from low to high open probability, which perhaps reflects occasional binding of CaM despite the presence of CTM. Similarly, when the amount of the available CaM in the cell was reduced, the short CaV1.342A isoform showed patterns of the low channel activity. CDI also underwent periodic changes with corresponding kinetics in both isoforms. Our results suggest that the competition between CTM and CaM is influenced by calcium, allowing further fine-tuning of CaV1.3 channel activity for particular cellular needs.

The epithelial Na+ channel γ subunit autoinhibitory tract suppresses channel activity by binding the γ subunit's finger–thumb domain interface

Epithelial Na+ channel (ENaC) maturation and activation require proteolysis of both the α and γ subunits. Cleavage at multiple sites in the finger domain of each subunit liberates their autoinhibitory tracts. Synthetic peptides derived from the proteolytically released fragments inhibit the channel, likely by reconstituting key interactions removed by the proteolysis. We previously showed that a peptide derived from the α subunit's autoinhibitory sequence (α-8) binds at the α subunit's finger-thumb domain interface. Despite low sequence similarity between the α and γ subunit finger domains, we hypothesized that a peptide derived from the γ subunit's autoinhibitory sequence (γ-11) inhibits the channel through an analogous mechanism. Using Xenopus oocytes, we found here that channels lacking a γ subunit thumb domain were no longer sensitive to γ-11, but remained sensitive to α-8. We identified finger domain sites in the γ subunit that dramatically reduced γ-11 inhibition. Using cysteines and sulfhydryl reactive cross-linkers introduced into both the peptide and the subunit, we also could cross-link γ-11 to both the finger domain and the thumb domain of the γ subunit. Our results suggest that α-8 and γ-11 occupy similar binding pockets within their respective subunits, and that proteolysis of the α and γ subunits activate the channel through analogous mechanisms.