Voltage-dependent Inhibition Research Articles

DHEA‐induced Voltage‐dependent Inhibition of ICa,L Is Augmented by Cholesterol Depletion and Down‐regulated by GPCR Blockade in Aortic Smooth Muscle Cells

Dehydroepiandrosterone (DHEA) is a steroid hormone synthesized from cholesterol and abundantly secreted as DHEA sulfate from the adrenal gland. DHEA produces relaxation of systemic and pulmonary arterial smooth muscles (ASM) and epiandrosterone, a metabolite of DHEA, causes voltage‐dependent inhibition (VDI) of ICa,L in ventricular myocytes. Here, we characterized DHEA‐induced VDI of ICa,L recorded with Ba2+ as a charge carrier by whole cell clamp and studied underlying mechanism and modulation by signal transduction in ASM cells (ASMCs) from bovine coronary artery and A7r5 cells from rat aorta. DHEA decreased peak amplitude of ICa,L rapidly, reversibly and dose‐dependently and accelerated time course of current decay decreasing the ratio of current amplitude at 500 ms (r500) to peak amplitude and shifted steady‐state inactivation curve (f∞‐V) to the left. DHEA‐induced inhibition of ICa,L became prominent by depolarization of holding potential; e.g. at −30 mV, the inhibition was 29% at 10 μM and 89% at 100 μM. Blockade of GPCR by dialysis of GDP βS shifted both f∞‐V and I–V relationships to the depolarizing direction, and decreased 10 μM‐DHEA‐induced VDI obtained at HP of −40 mV by 40%. Depletion of membrane cholesterol by methyl‐b‐cyclodextrin increased r500 and shifted f∞‐V to the right. Conversely, loading PEG‐cholesterol, a non‐ionic amphiphile, to the membrane decreased r500 and shifted f∞‐V to the left. In the cholesterol‐depleted cells, DHEA produced more potent decrease of r500 and larger negative shift of f∞‐V than in control. Therefore, DHEA‐induced VDI of ICa,L in ASMCs is due, at least partly, to cholesterol‐like action of DHEA on CaV1.2 in the membrane lipid bilayer. GPCR‐mediated signaling regulates voltage‐dependence of basal ICa,L and indirectly augments DHEA‐induced VDI of ICa,L in ASMCs.

Protein Kinase A-Dependent Phosphorylation Modulates the Affinity of Kv7.5 Potassium Channels to Phosphatidylinositol 4,5-Bisphosphate to Enhance Potassium Conductance

PKA-dependent phosphorylation of Kv7.5 potassium channels was previously found to enhance Kv7.5 currents, but the mechanism of the enhancement by PKA as well as putative PKA phosphorylation sites remained unknown. Activity of all known Kv7 channels critically depends on the presence of a minor membrane phospholipid, phosphatidylinositol 4,5-bisphosphate (PIP2). In the present study, we tested the hypothesis that PKA-dependent phosphorylation increases the affinity of Kv7.5 potassium channels to PIP2. Application of wortmannin (10 µM), an inhibitor of PI-4 kinase that decreases the level of membrane PIP2, caused time-dependent inhibition of the endogenous Kv7.5 current in A7r5 rat aortic smooth muscle cells. Activation of PKA by co-application of forskolin (activator of adenylyl cyclase, 1 µM) with IBMX (phosphodiesterase inhibitor, 500 µM) significantly attenuated the rate and total amount of wortmannin-induced inhibition of endogenous Kv7.5 current. Co-expression of ci-VSP, a voltage dependent phosphatase that dephosphorylates PIP2, with human Kv7.5 (hKv7.5) in A7r5 cells caused pronounced voltage and time-dependent inhibition of hKv7.5 current at membrane voltages positive to 0 mV. Enhancement of the hKv7.5 current observed in the presence of forskolin (10 µM) and IBMX (500 µM) occurred in parallel with the positive shift of the ci-VSP-induced voltage-dependent inhibition of the Kv7.5 current. In the presence of forskolin and IBMX, ci-VSP induced inhibition of the Kv7.5 current at voltages positive to +60 mV. To identify PKA phosphorylation sites we mutated predicted PKA phosphorylation sites S406 and S412 (located near the putative PIP2 binding domain on the C-terminus of the Kv7.5 channels) to alanine. Currents through both mutant channels Kv7.5 S406A and Kv7.5 S412A were enhanced by co-application of forskolin with IBMX to the same degree as wild type Kv7.5 current. Thus, phosphorylation of S406 and S412 cannot account for PKA-dependent Kv7.5 current enhancement. Overall, experimental results support our hypothesis that enhancement of Kv7.5 currents caused by PKA activation is associated with increased affinity of the Kv7.5 potassium channels to PIP2.

Antiarrhythmic effect of vernakalant in electrically remodeled goat atria is caused by slowing of conduction and prolongation of postrepolarization refractoriness

Vernakalant inhibits several potassium currents and causes a rate- and voltage-dependent inhibition of the sodium current. The aim of this study was to evaluate the antiarrhythmic mechanism of vernakalant in normal and electrically remodeled atria. Fourteen goats were instrumented with electrodes on both atria. Drug effects on refractory period (ERP), conduction velocity (CV), and atrial fibrillation cycle length (AFCL) were determined in normal goats (control) and after 2 (2dAF) or 11 (11dAF) days of pacing-induced atrial fibrillation (AF) in awake goats. To evaluate the contribution of changes in conduction and ERP, the same experiments were performed with flecainide and AVE0118. In a subset of goats, monophasic action potentials were recorded during anesthesia. Vernakalant dose-dependently prolonged ERP and decreased CV in CTL experiments. Both effects were maintained after 2dAF and 11dAF. After 11dAF, conduction slowed down by 8.2 ± 1.5 cm/s and AFCL increased by 55 ± 3 ms, leading to AF termination in 5 out of 9 goats. Monophasic action potential measurements revealed that ERP prolongation was due to enhanced postrepolarization refractoriness. During pacing, vernakalant had comparable effects on CV as flecainide, while effect on ERP was comparable to AVE0118. During AF, all compounds had comparable effects on median AFCL and ERP despite differences in their effects on CV during pacing. The antiarrhythmic effect of vernakalant in the goat, at clinically relevant plasma concentrations, is based on both conduction slowing and ERP prolongation due to postrepolarization refractoriness. These electrophysiological effects were not affected by long-term electrical remodeling of the atria.

Mechanism of inhibition by olanzapine of cloned hERG potassium channels

Olanzapine is widely used in the treatment of schizophrenia and related psychoses. We investigated the effects of olanzapine on human ether-a-go–go related gene (hERG) channels stably expressed in human embryonic kidney (HEK) cells using the whole-cell patch-clamp technique. Olanzapine inhibited hERG tail currents at −50mV in a concentration-dependent manner with an IC50 value of 8.0μM and a Hill coefficient of 0.9. The voltage-dependent inhibition of the hERG currents by olanzapine increased steeply in the voltage range of channel activation. Olanzapine also shifted the steady-state activation curve of the hERG currents in a hyperpolarizing direction. At more depolarized potentials where the channels were fully activated (between 0 and +50mV), the olanzapine inhibition was voltage-independent. The steady-state inactivation curve of the hERG currents was shifted in the hyperpolarizing direction in the presence of olanzapine. A fast application of olanzapine induced a reversible inhibition of the hERG tail currents during repolarization in a concentration-dependent manner with an IC50 value of 11.9μM, suggesting an open-channel block. Olanzapine also decreased the hERG current elicited by a 5s depolarizing pulse to +60mV to inactivate the hERG currents, suggesting an inhibition of the activated (open and/or inactivated) states of the channels. These results indicated that olanzapine inhibited the hERG current by preferentially interacting with the activated states of the channel.

Endoxifen, the active metabolite of tamoxifen, inhibits cloned hERG potassium channels

The effects of tamoxifen, and its active metabolite endoxifen (4-hydroxy-N-desmethyl-tamoxifen), on hERG currents stably expressed in HEK cells were investigated using the whole-cell patch-clamp technique and an immunoblot assay. Tamoxifen and endoxifen inhibited hERG tail currents at −50mV in a concentration-dependent manner with IC50 values of 1.2 and 1.6μM, respectively. The steady-state activation curve of the hERG currents was shifted to the hyperpolarizing direction in the presence of endoxifen. The voltage-dependent inhibition of hERG currents by endoxifen increased steeply in the voltage range of channel activation. The inhibition by endoxifen displayed a shallow voltage dependence (δ=0.18) in the full activation voltage range. A fast application of endoxifen induced a reversible block of hERG tail currents during repolarization in a concentration-dependent manner, which suggested an interaction with the open state of the channel. Endoxifen also decreased the hERG current elicited by a 5s depolarizing pulse to +60mV to inactivate the hERG currents, suggesting an interaction with the activated (open and/or inactivated) states of the channels. Tamoxifen and endoxifen inhibited the hERG channel protein trafficking to the plasma membrane in a concentration-dependent manner with endoxifen being more potent than tamoxifen. These results indicated that tamoxifen and endoxifen inhibited the hERG current by direct channel blockage and by the disruption of channel trafficking to the plasma membrane in a concentration-dependent manner. A therapeutic concentration of endoxifen inhibited the hERG current by preferentially interacting with the activated (open and/or inactivated) states of the channel.

Molecular basis for N-type voltage-gated Ca2+ channel modulation by Gq protein-coupled receptors

N-type voltage-gated Ca 2+ (Ca V 2.2) channels, which enable synaptic transmission by triggering neurotransmitter release, are tightly modulated by G protein-coupled receptors (GPCRs) via several downstream signaling messengers, such as Gbg, calmodulin, arachidonic acid and PIP 2 . However, the molecular mechanism by which G q/11 -coupled receptors (G q PCRs) suppress Ca V 2.2 currents remains unclear. In this research highlight, we review our recent finding that M 1 muscarinic receptors inhibit Ca V 2.2 channels through both Gbg-mediated voltage-dependent (VD) and Gα q/11 /PLC-mediated voltage-independent (VI) pathways. Our photometry results also demonstrate that Gbg-mediated VD inhibition of Ca V 2.2 channels initiates approximately 3s earlier than VI inhibition, and is strongly potentiated in cells expressing plasma membrane-localized Ca V b subunits. Our observations demonstrate a novel mechanism for Ca V 2.2 channel modulation by G q PCRs where the subcellular location of Ca V b subunits plays a critical role in determining the voltage-dependence of current suppression by M 1 receptors.

Pegylated Cholesterol and Methyl-Beta-Cyclodextrin are Modulators of L-Type Calcium Channel Current and Decrease Membrane Capacitance in Vascular Smooth Muscle Cells

Loading of pegylated cholesterol (PEG-cholesterol) by pretreatment of several hours decreases current density and augments voltage-dependent inactivation of L-type Ca channel current (ICa,L) and depletion of cholesterol by similar long pretreatment with methyl-beta-cyclodextrin (MbCD) increases the ICa,L density in A7r5 aortic smooth muscle cells. On the other hand, dehydroepiandrosterone (DHEA), a cholesterol-derived steroid hormone, rapidly induces voltage-dependent inhibition of ICa,L. If endocytosis and exocytosis of CaV1.2-containing membrane vesicles are involved in these modulations, they should induce a change in membrane capacitance (Cm). Cm could be affected also by a change of thickness and dielectric constant of lipid bilayer. Here we studied acute effects of PEG-cholesterol, MbCD and DHEA on Cm of isolated bovine coronary artery smooth muscle cells by whole-cell patch clamp technique. Ramp steps of 5 or 10 ms were applied repetitively before, during and after application of the modulators. Control Cm was 18.3±4.5 pF (mean±SD.). PEG-cholesterol and MbCD induced gradual and small decrease of Cm and their washout decelerated the rate of the decrease. Cm after 10 min of wash-in normalized by the initial value was: control without modulator, 1.01±0.02 (mean±SD.); 1 mM PEG-cholesterol, 0.96±0.02 (p<0.01, compared with control); 10 mM MbCD, 0.90±0.05 (p<0.0001); 0.1 mM DHEA, 1.00±0.03. Since the change of Cm is small, it little affects qualitative conclusion of the modulation of ICa,L density obtained by the pretreatment experiments with PEG-cholesterol and MbCD. The small but significant decrease of Cm reflects structural changes of the lipid bilayer which may be involved in the modulation of ICa,L by PEG-cholesterol and MbCD.

Somatostatin receptor subtype 4 modulates L-type calcium channels via Gβγ and PKC signaling in rat retinal ganglion cells

Somatostatin subtype-4 receptors (sst4) inhibit L-type calcium channel currents (ICa) in retinal ganglion cells (RGCs). Here we identify the signaling pathways involved in sst4 stimulation leading to suppression of ICa in RGCs. Whole cell patch clamp recordings were made on isolated immunopanned RGCs using barium as a charge carrier to isolate ICa. Application of the selective sst4 agonist, L-803 (10 nM), reduced ICa by 41.2%. Pretreatment of cells with pertussis toxin (Gi/o inhibitor) did not prevent the action of L-803, which reduced ICa by 34.7%. To determine the involvement of Gβγ subunits after sst4 activation, depolarizing pre-pulse facilitation paradigms were used to remove voltage-dependent inhibition of calcium channels. Pre-pulse facilitation did not reverse the inhibitory effects of L-803 on ICa (8.4 vs. 8.8% reductions, ctrl vs. L-803); however, pharmacologic inhibition of Gβγ reduced ICa suppression by L-803 (23.0%, P < 0.05). Inhibition of PKC (GF109203X; GFX) showed a concentration-dependent effect in preventing the action of L-803 on ICa (1 μM GFX, 34.3%; 5 μM GFX, 14.6%, P < 0.05). When both PKC and Gβγ were inhibited, the effects of L-803 on ICa were blocked (1.8%, P < 0.05). These results suggest that sst4 stimulation modulates RGC calcium channels via Gβγ and PKC activation. Since reducing intracellular Ca2+ is known to be neuroprotective in RGCs, modulating these sst4 signaling pathways may provide insights to the discovery of unique therapeutic targets to reduce intracellular Ca2+ levels in RGCs.

Voltage-dependent regulation of CaV2.2 channels by Gq-coupled receptor is facilitated by membrane-localized β subunit.

G protein-coupled receptors (GPCRs) signal through molecular messengers, such as Gβγ, Ca(2+), and phosphatidylinositol 4,5-bisphosphate (PIP2), to modulate N-type voltage-gated Ca(2+) (CaV2.2) channels, playing a crucial role in regulating synaptic transmission. However, the cellular pathways through which GqPCRs inhibit CaV2.2 channel current are not completely understood. Here, we report that the location of CaV β subunits is key to determining the voltage dependence of CaV2.2 channel modulation by GqPCRs. Application of the muscarinic agonist oxotremorine-M to tsA-201 cells expressing M1 receptors, together with CaV N-type α1B, α2δ1, and membrane-localized β2a subunits, shifted the current-voltage relationship for CaV2.2 activation 5 mV to the right and slowed current activation. Muscarinic suppression of CaV2.2 activity was relieved by strong depolarizing prepulses. Moreover, when the C terminus of β-adrenergic receptor kinase (which binds Gβγ) was coexpressed with N-type channels, inhibition of CaV2.2 current after M1 receptor activation was markedly reduced and delayed, whereas the delay between PIP2 hydrolysis and inhibition of CaV2.2 current was decreased. When the Gβγ-insensitive CaV2.2 α1C-1B chimera was expressed, voltage-dependent inhibition of calcium current was virtually abolished, suggesting that M1 receptors act through Gβγ to inhibit CaV2.2 channels bearing membrane-localized CaV β2a subunits. Expression of cytosolic β subunits such as β2b and β3, as well as the palmitoylation-negative mutant β2a(C3,4S), reduced the voltage dependence of M1 muscarinic inhibition of CaV2.2 channels, whereas it increased inhibition mediated by PIP2 depletion. Together, our results indicate that, with membrane-localized CaV β subunits, CaV2.2 channels are subject to Gβγ-mediated voltage-dependent inhibition, whereas cytosol-localized β subunits confer more effective PIP2-mediated voltage-independent regulation. Thus, the voltage dependence of GqPCR regulation of calcium channels can be determined by the location of isotype-specific CaV β subunits.

Different effects of lobeline on neuronal and muscle nicotinic receptors

Lobeline is a plant alkaloid known to interact with cholinergic system. The effect of lobeline on neuronal α3β4 receptors expressed in COS cells and muscle embryonic αβγδ receptors naturally expressed in TE671 cells was studied using a patch-clamp technique. Our results show that lobeline inhibited responses to acetylcholine in human embryonic muscle nicotinic receptor in a pseudo-noncompetitive manner. The responses of rat neuronal α3β4 receptors to a low concentration of acetylcholine were potentiated by a mixed occupation mechanism that corresponds to “competitive potentiation”. This potentiation turned into voltage-dependent inhibition for α3β4 receptors was activated by a high concentration of acetylcholine.

Binding of ArgTX-636 in the NMDA Receptor Ion Channel

The N-methyl-d-aspartate receptors (NMDARs) constitute an important class of ligand-gated cation channels that are involved in the majority of excitatory neurotransmission in the human brain. Compounds that bind in the NMDAR ion channel and act as blockers are use- and voltage-dependent inhibitors of NMDAR activity and have therapeutic potential for treatment of a variety of brain diseases or as pharmacological tools for studies of the neurobiological role of NMDARs. We have performed a kinetic analysis of the blocking mechanism of the prototypical polyamine toxin NMDAR ion channel blocker argiotoxin-636 (ArgTX-636) at recombinant GluN1/2A receptors to provide detailed information on the mechanism of block. The predicted binding site of ArgTX-636 is in the pore region of the NMDAR ion channel formed by residues in the transmembrane M3 and the M2 pore-loop segments of the GluN1 and GluN2A subunits. To assess the predicted binding mode in further detail, we performed an alanine- and glycine-scanning mutational analysis of this pore-loop segment to systematically probe the role of pore-lining M2 residues in GluN1 and GluN2A in the channel block by ArgTX-636. Comparison of M2 positions in GluN1 and GluN2A where mutation influences ArgTX-636 potency suggests differential contribution of the M2-loops of GluN1 and GluN2A to binding of ArgTX-636. The results of the mutational analysis are highly relevant for the future structure-based development of argiotoxin-derived NMDAR channel blockers.

Blockade of HERG Human K&lt;sup&gt;+&lt;/sup&gt; Channels by the Antidepressant Drug Paroxetine

The effects of paroxetine, a selective serotonin reuptake inhibitor, on human ether-a-go-go-related gene (HERG) channels were investigated using the whole-cell patch-clamp technique. The HERG channels were stably expressed in human embryonic kidney cells. Paroxetine inhibited the peak tail currents of the HERG channel in a concentration-dependent manner, with an IC50 value of 0.45 µM and a Hill coefficient of 0.85. These effects were reversible after wash-out of the drug. The paroxetine-induced inhibition of the HERG channels was voltage-dependent. There was a steep increase in inhibition over the voltage range of the channel opening. Also, a shallow voltage-dependent inhibition was detected over the voltage range in which the channels were fully activated. The fractional electrical distance was estimated to be 0.11. Paroxetine induced a leftward shift in the voltage-dependence of the steady-state activation of the HERG channels. Before and after application of the 1 µM paroxetine, the half-maximum activation was -14.21 mV and -27.04 mV, respectively, with no shift in the slope value. The HERG channel block was not use-dependent. The characteristics of the block were dependent on open and inactivated channel states rather than closed state. Paroxetine had no effect on activation and deactivation kinetics, steady-state inactivation. These results suggest that paroxetine blocks the HERG channels by binding to these channels in the open and inactivated states.

Aqueous cigarette smoke extract induces a voltage-dependent inhibition of CFTR expressed in Xenopus oocytes

The cystic fibrosis transmembrane conductance regulator (CFTR) chloride channel inhabits the apical membrane of airway epithelia, where its function is essential for mucus hydration, mucociliary clearance, and airway defense. Chronic obstructive pulmonary disease (COPD), most often a consequence of cigarette smoke (CS) exposure, affects 15 million persons in the US. Clinically, COPD is characterized by many of the salient features of cystic fibrosis lung disease, where CFTR is either absent or reduced in function. CS is an acidic aerosol (pH 5.3 to 6.3) reported to contain over 4,000 constituents. Acute CS exposure has been reported to decrease airway transepithelial voltage in vivo and short-circuit current in vitro; however, the mechanistic basis of these effects is uncertain. The goal of the studies described here was to develop a bioassay to characterize the effects of aqueous CS preparations on the channel function of CFTR. We studied aqueous CS extract (CSE) prepared in our laboratory, as well as commercial cigarette smoke condensate (CSC) in Xenopus oocytes expressing human CFTR. Application of CSE at pH 5.3 produced a reversible, voltage-dependent inhibition of CFTR conductance. CSE neutralized to pH 7.3 produced less inhibition of CFTR conductance. Serial dilution of CSE revealed a dose-dependent effect at acidic and neutral pH. In contrast, CSC did not inhibit CFTR conductance in oocytes. We conclude that one or more components of CSE inhibits CFTR in a manner similar to diphenylamine-2-carboxylate, a negatively charged, open-channel blocker.

β-Hydroxybutyrate Modulates N-Type Calcium Channels in Rat Sympathetic Neurons by Acting as an Agonist for the G-Protein-Coupled Receptor FFA3

Free fatty acids receptor 3 (FFA3, GPR41) and 2 (FFA2, GPR43), for which the short-chain fatty acids (SCFAs) acetate and propionate are agonist, have emerged as important G-protein-coupled receptors influenced by diet and gut flora composition. A recent study (Kimura et al., 2011) demonstrated functional expression of FFA3 in the rodent sympathetic nervous system (SNS) providing a potential link between nutritional status and autonomic function. However, little is known of the source of endogenous ligands, signaling pathways, or effectors in sympathetic neurons. In this study, we found that FFA3 and FFA2 are unevenly expressed in the rat SNS with higher transcript levels in prevertebral (e.g., celiac-superior mesenteric and major pelvic) versus paravertebral (e.g., superior cervical and stellate) ganglia. FFA3, whether heterologously or natively expressed, coupled via PTX-sensitive G-proteins to produce voltage-dependent inhibition of N-type Ca(2+) channels (Cav2.2) in sympathetic neurons. In addition to acetate and propionate, we show that β-hydroxybutyrate (BHB), a metabolite produced during ketogenic conditions, is also an FFA3 agonist. This contrasts with previous interpretations of BHB as an antagonist at FFA3. Together, these results indicate that endogenous BHB levels, especially when elevated under certain conditions, such as starvation, diabetic ketoacidosis, and ketogenic diets, play a potentially important role in regulating the activity of the SNS through FFA3.

Functional expression of a Kir2.1-like inwardly rectifying potassium channel in mouse mammary secretory cells

K(+) channels in mammary secretory (MS) cells are believed to play a role in transcellular electrolyte transport and thus determining ionic composition of the aqueous phase of milk. However, direct evidence for specific K(+) channel activity in native MS cells is lacking at the single-cell level. Here, we show for the first time that an inwardly rectifying K(+) (Kir) channel is functionally expressed in fully differentiated MS cells that were freshly isolated from the mammary gland of lactating mice. Using the standard whole cell patch-clamp technique, we found that mouse MS cells consistently displayed a K(+) current, whose electrophysiological properties are similar to those previously reported for Kir2.x channels, particularly Kir2.1: 1) current-voltage relationship with strong inward rectification, 2) slope conductance approximately proportional to the square root of external K(+) concentration, 3) voltage- and time-dependent and high-affinity block by external Ba(2+), and 4) voltage-dependent inhibition by external Cs(+). Accordingly, RT-PCR analysis revealed the gene expression of Kir2.1, but not Kir2.2, Kir2.3, and Kir2.4, in lactating mouse mammary gland, and immunohistochemical staining showed Kir2.1 protein expression in the secretory cells. Cell-attached patch recordings from MS cells revealed that a 31-pS K(+) channel with strong inward rectification was likely active at the resting membrane potential. Collectively, the present work demonstrates that a functional Kir2.1-like channel is expressed in lactating mouse MS cells. We propose that the channel might be involved, at least in part, in secretion and/or preservation of ionic components of milk stored into the lumen of these cells.

The dual effect of PNU-120596 on α7 nicotinic acetylcholine receptor channels

PNU-120596 (1-(5-chloro-2,4-dimethoxyphenyl)-3-(5-methylisoxazol-3-yl)urea), a Type-II positive allosteric modulator of α7 nicotinic acetylcholine receptors inhibits α7 desensitization and robustly prolongs openings of α7 channels. However, these effects may render α7 channels more accessible to positively charged molecules and thus, more susceptible to voltage-dependent open-channel-block-like inhibition. To test this hypothesis, choline chloride (i.e., choline), a selective endogenous α7 agonist, and bicuculline methochloride (i.e., bicuculline), a competitive α7 antagonist, were used as membrane voltage-sensitive probes in whole-cell voltage-clamp recordings from hippocampal CA1 interneurons in acute brain slices in the absence and presence of PNU-120596. PNU-120596 enhanced voltage-dependent inhibition of α7 responses by bicuculline and choline. In the presence of PNU-120596, α7 channels favored a burst-like kinetic modality in the presence, but not absence of bicuculline and bursts of α7 openings were voltage-dependent. These results suggest that PNU-120596 alters the pharmacology of α7 channels by making these channels more susceptible to voltage-dependent inhibitory interactions with positively charged drugs at concentrations that do not potently inhibit α7 channels without PNU-120596. This inhibition imitates α7 nicotinic receptor desensitization and compromises the potentiating anti-desensitization effects of PNU-120596 on α7 nicotinic receptors. This unexpected dual action of PNU-120596, and possibly other Type-II positive allosteric modulators of α7 nicotinic receptors, may lead to unanticipated α7 channel-drug interactions and misinterpretation of α7 single-channel data.

Structure and Function of Hainantoxin-III, a Selective Antagonist of Neuronal Tetrodotoxin-sensitive Voltage-gated Sodium Channels Isolated from the Chinese Bird Spider Ornithoctonus hainana

In the present study, we investigated the structure and function of hainantoxin-III (HNTX-III), a 33-residue polypeptide from the venom of the spider Ornithoctonus hainana. It is a selective antagonist of neuronal tetrodotoxin-sensitive voltage-gated sodium channels. HNTX-III suppressed Nav1.7 current amplitude without significantly altering the activation, inactivation, and repriming kinetics. Short extreme depolarizations partially activated the toxin-bound channel, indicating voltage-dependent inhibition of HNTX-III. HNTX-III increased the deactivation of the Nav1.7 current after extreme depolarizations. The HNTX-III·Nav1.7 complex was gradually dissociated upon prolonged strong depolarizations in a voltage-dependent manner, and the unbound toxin rebound to Nav1.7 after a long repolarization. Moreover, analysis of chimeric channels showed that the DIIS3-S4 linker was critical for HNTX-III binding to Nav1.7. These data are consistent with HNTX-III interacting with Nav1.7 site 4 and trapping the domain II voltage sensor in the closed state. The solution structure of HNTX-III was determined by two-dimensional NMR and shown to possess an inhibitor cystine knot motif. Structural analysis indicated that certain basic, hydrophobic, and aromatic residues mainly localized in the C terminus may constitute an amphiphilic surface potentially involved in HNTX-III binding to Nav1.7. Taken together, our results show that HNTX-III is distinct from β-scorpion toxins and other β-spider toxins in its mechanism of action and binding specificity and affinity. The present findings contribute to our understanding of the mechanism of toxin-sodium channel interaction and provide a useful tool for the investigation of the structure and function of sodium channel isoforms and for the development of analgesics.

Effect of mosapride on Kv4.3 potassium channels expressed in CHO cells

Mosapride and cisapride are gastroprokinetic agents with 5-hydroxytryptamine4 receptor agonist activity and have been widely used in the treatment of a variety of gastrointestinal disorders. The effects of mosapride and cisapride on cloned Kv4.3 channels stably expressed in Chinese hamster ovary cells were investigated using the whole-cell patch-clamp technique. Mosapride and cisapride inhibited Kv4.3 in a concentration-dependent manner with IC50 values of 15.2 and 9.8μM, respectively. Mosapride accelerated the rate of inactivation and activation of Kv4.3 in a concentration-dependent manner and thereby decreased the time to peak. The rate constants of association (k +1) and dissociation (k -1) for mosapride were 9.9μM(-1)s(-1) and 151.3s(-1), respectively. The K D (k -1/k +1) was 16.2μM, similar to the IC50 value calculated from the concentration-response curve. Voltage-dependent inhibition by mosapride was observed in the voltage range for channel opening but was not observed over a voltage range in which all Kv4.3 channels were open. Both the steady-state activation and inactivation curves of Kv4.3 were shifted in the hyperpolarizing direction in the presence of mosapride. Mosapride also caused a substantial acceleration in closed-state inactivation of Kv4.3. Mosapride produced use-dependent inhibition, which was consistent with a slow recovery from inactivation of Kv4.3. M1 and norcisapride, the major metabolites of mosapride and cisapride, respectively, had little or no effect on Kv4.3. These results indicate that mosapride inhibits Kv4.3 by both preferential binding to the open state of the channels during depolarization and acceleration of the closed-state inactivation at subthreshold potentials.

RGK Proteins are not Ras-Like Molecular Switches. A Prespective from Voltage-Dependent Calcium Channels Inhibition

The RGK family of small G-proteins, including Rad, Gem, Rem and Rem2, are inducibly expressed in various mammalian tissues and regulate voltage-dependent calcium channels (VDCC) and Rho kinase. In this study, two-electrode voltage-clamp measurements of CaV2.2 channels and their inhibition by various Gem constructs were conducted in Xenopus oocytes. Nucleotide identity had little or no effect on VDCC inhibition by Gem and Rad. Chimeras constructed from Ras G-domain or CaVβ fused to various Gem domain demonstrated that both the Gem G-domain and C-terminus have partial inhibitory effects. Gem sequences, beginning at the G5 loop through the C-terminus impart fully inhibition of the VDCC. This new data supports the hypothesis that RGK inhibition of VDCC activity does not conform to canonical G-protein modes of action and largely depends on the C-terminus, not the G-domain of the protein.

Cholestrol Depletion reduces Voltage-Dependent Inactivation of L-Type Calcium Channel and DHEA Augments the Inacivation in A7R5 Cells

Cholesterol (Chol), a structural component of the membrane, is involved also in the functional regulation of ion channels. The increase of membrane Chol is associated with the reduction of L-type Ca2+ channel current (ICa,L). The effect of Chol on the voltage-dependent inactivation is not known, however. Dehydroepiandrosterone (DHEA), a cholesterol-derived adrenal steroid hormone, produces a voltage-dependent inhibition of ICa,L at micro-molar concentrations. We studied the effect of Chol on the inactivation of ICa,L and on the DHEA-induced inhibition of ICa,L in A7r5, a cell line derived from fetal rat aorta. Chol was depleted with methyl-beta-cyclodextrin (CD) or loaded by cholesterol bound CD (chol-CD). Whole cell ICa,L was recorded with 10 mM Ba2+ as the charge carriers. T-type Ca2+ channel current was inactivated by 50 ms pre-pulse to −40 mV. The maximal density of ICa,L was decreased by 55% by loading Chol with 30 mM chol-CD, while it was not affected by the depletion of Chol by 10 and 30 mM CD. The depletion of Chol by 30 mM CD remarkably decreased the rate of inactivation of ICa,L, generating sustained current. It shifted V0.5 in the f∞-V curve to a depolarizing direction by 10 mV from control −30 mV, with appearance of 27% non-inactivating component at 0 mV. DHEA (30 micro M) inhibited the maximal amplitude of ICa,L by ∼30% irrespective of the manipulation of Chol. It shifted the V0.5 to a hyperpolarizing direction by 6 mV in control but 10 mV in the 30 mM CD treated cells. We conclude that membrane cholesterol is essential for the normal voltage-dependent inactivation of L-type Ca2+ channel. DHEA, at pharmacological concentrations, more potently augments the inactivation in the cholesterol depleted cells.