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Unlike other members of the voltage-gated ion channel superfamily, voltage-gated proton (Hv) channels are solely composed of voltage sensor domains without separate ion-conducting pores. Due to their unique dependence on both voltage and transmembrane pH gradients, Hv channels normally open to mediate proton efflux. Multiple cellular ligands were also found to regulate the function of Hv channels, including Zn 2+ , cholesterol, polyunsaturated arachidonic acid, and albumin. Our previous work showed that Zn 2+ and cholesterol inhibit the human voltage-gated proton channel (hHv1) by stabilizing its S4 segment at resting state conformations. Released from phospholipids by phospholipase A2 in cells upon infection or injury, arachidonic acid regulates the function of many ion channels, including hHv1. In the present work, we examined the effects of arachidonic acid on purified hHv1 channels using liposome flux assays and revealed underlying structural mechanisms using single-molecule FRET. Our data indicated that arachidonic acid strongly activates hHv1 channels by promoting transitions of the S4 segment toward opening or "preopening" conformations. Moreover, we found that arachidonic acid even activates hHv1 channels inhibited by Zn 2+ and cholesterol, providing a biophysical mechanism to activate hHv1 channels in nonexcitable cells upon infection or injury., Competing Interests: Conflict of interest The authors declare that they have no conflicts of interest with the contents of this article., (Copyright © 2023 The Authors. Published by Elsevier Inc. All rights reserved.)

Here, we report the identification and characterization of the first proton channels from fungi. The fungal proteins are related to animal voltage-gated Hv channels and are conserved in both higher and lower fungi. Channels from Basidiomycota and Ascomycota appear to be evolutionally and functionally distinct. Representatives from the two phyla share several features with their animal counterparts, including structural organization and strong proton selectivity, but they differ from each other and from animal Hvs in terms of voltage range of activation, pharmacology, and pH sensitivity. The activation gate of Hv channels is believed to be contained within the transmembrane core of the protein and little is known about contributions of peripheral regions to the activation mechanism. Using a chimeragenesis approach, we find that intra- and extracellular peripheral regions are main determinants of the voltage range of activation in fungal channels, highlighting the role of these overlooked components in channel gating.

Targeting receptor proteins, such as ligand-gated ion channels and G protein-coupled receptors, has directly enabled the discovery of most drugs developed to modulate receptor signalling. However, as the search for novel and improved drugs continues, an innovative approach - targeting receptor complexes - is emerging. Receptor complexes are composed of core receptor proteins and receptor-associated proteins, which have profound effects on the overall receptor structure, function and localization. Hence, targeting key protein-protein interactions within receptor complexes provides an opportunity to develop more selective drugs with fewer side effects. In this Review, we discuss our current understanding of ligand-gated ion channel and G protein-coupled receptor complexes and discuss strategies for their pharmacological modulation. Although such strategies are still in preclinical development for most receptor complexes, they exemplify how receptor complexes can be drugged, and lay the groundwork for this nascent area of research.

Epilepsy is characterised by spontaneous recurrent seizures that are caused by an imbalance between neuronal excitability and inhibition. Since ion channels play fundamental roles in the generation and propagation of action potentials as well as neurotransmitter release at a subset of excitatory and inhibitory synapses, their dysfunction has been linked to a wide variety of epilepsies. Indeed, these unique proteins are the major biological targets for antiepileptic drugs. Selective targeting of a specific ion channel subtype remains challenging for small molecules, due to the high level of homology among members of the same channel family. As a consequence, there is a growing trend to target ion channels with biologics. Venoms are the best known natural source of ion channel modulators, and venom peptides are increasingly recognised as potential therapeutics due to their high selectivity and potency gained through millions of years of evolutionary selection pressure. Here we describe the major ion channel families involved in the pathogenesis of various types of epilepsy, including voltage-gated Na + , K + , Ca 2+ channels, Cys-loop receptors, ionotropic glutamate receptors and P2X receptors, and currently available venom-derived peptides that target these channel proteins. Although only a small number of venom peptides have successfully progressed to the clinic, there is reason to be optimistic about their development as antiepileptic drugs, notwithstanding the challenges associated with development of any class of peptide drug., (Copyright © 2020 Elsevier Inc. All rights reserved.)

Ion channels are pore-forming protein complexes in membranes that play essential roles in a diverse array of biological activities. Ion channel activity is strictly regulated at multiple levels and by numerous cellular events to selectively activate downstream effectors involved in specific biological activities. For example, ions, binding proteins, nucleotides, phosphorylation, the redox state, channel subunit composition have all been shown to regulate channel activity and subsequently allow channels to participate in distinct cellular events. While these forms of modulation are well documented and have been extensively reviewed, in this article, we will first review and summarize channel proteolysis as a novel and quite widespread mechanism for altering channel activity. We will then highlight the recent findings demonstrating that proteolysis profoundly alters Inositol 1,4,5 trisphosphate receptor activity, and then discuss its potential functional ramifications in various developmental and pathological conditions., (Copyright © 2018 Elsevier B.V. All rights reserved.)

Immune cells express various voltage-gated and ligand-gated ion channels that mediate the influx and efflux of charged ions across the plasma membrane, thereby controlling the membrane potential and mediating intracellular signal transduction pathways. These channels thus present potential targets for experimental modulation of immune responses and for therapeutic interventions in immune disease. Small molecule drugs and natural toxins acting on ion channels have illustrated the potential therapeutic benefit of targeting ion channels on immune cells. Unwanted side effects and immunogenicity have however hampered the application of these molecules. Owing to their high specificity, low immunogenicity and beneficial pharmacodynamics, antibodies targeting membrane and secretory proteins have emerged as potent therapeutics in oncology and inflammation. Nanobodies-single domain fragments derived from heavy chain antibodies naturally occurring in camelids-offer additional benefits versus antibodies, including protrusion into cryptic epitopes and easy formatting of multi-specific reagents. Here we review recent progress in the development and application of antibodies and Nanobodies targeting ion channels on immune cells., (Copyright © 2018. Published by Elsevier Ltd.)

Chronic hepatitis C virus (HCV) infection causes severe liver disease and affects ca. 146 million individuals. Novel directly acting antivirals targeting HCV have revolutionized treatment. However, high costs limit access to therapy. Recently, several related drugs used in humans to treat allergies or as neuroleptics emerged as potent HCV cell entry inhibitors. Insights into their antiviral modes of action may increase opportunities for drug repurposing in hepatitis C and possibly other important human viral infections., (Copyright © 2017 American Society for Microbiology.)

GLIC receptor is a bacterial pentameric ligand-gated ion channel whose action is inhibited by xenon. Xenon has been used in clinical practice as a potent gaseous anaesthetic for decades, but the molecular mechanism of interactions with its integral membrane receptor targets remains poorly understood. Here we characterize by X-ray crystallography the xenon-binding sites within both the open and "locally-closed" (inactive) conformations of GLIC. Major binding sites of xenon, which differ between the two conformations, were identified in three distinct regions that all belong to the trans-membrane domain of GLIC: 1) in an intra-subunit cavity, 2) at the interface between adjacent subunits, and 3) in the pore. The pore site is unique to the locally-closed form where the binding of xenon effectively seals the channel. A putative mechanism of the inhibition of GLIC by xenon is proposed, which might be extended to other pentameric cationic ligand-gated ion channels.

Monoterpenes are a structurally diverse group of phytochemicals and a major constituent of plant-derived 'essential oils'. Monoterpenes such as menthol, carvacrol, and eugenol have been utilized for therapeutical purposes and food additives for centuries and have been reported to have anti-inflammatory, antioxidant and analgesic actions. In recent years there has been increasing interest in understanding the pharmacological actions of these molecules. There is evidence indicating that monoterpenes can modulate the functional properties of several types of voltage and ligand-gated ion channels, suggesting that some of their pharmacological actions may be mediated by modulations of ion channel function. In this report, we review the literature concerning the interaction of monoterpenes with various ion channels., (Copyright © 2015 Elsevier Inc. All rights reserved.)

In the last 2 decades biomedical research has provided great insights into the molecular signatures underlying painful conditions. However, chronic pain still imposes substantial challenges to researchers, clinicians and patients alike. Under pathological conditions, pain therapeutics often lack efficacy and exhibit only minimal safety profiles, which can be largely attributed to the targeting of molecules with key physiological functions throughout the body. In light of these difficulties, the identification of molecules and associated protein complexes specifically involved in chronic pain states is of paramount importance for designing selective interventions. Ion channels and receptors represent primary targets, as they critically shape nociceptive signaling from the periphery to the brain. Moreover, their function requires tight control, which is usually implemented by protein-protein interactions (PPIs). Indeed, manipulation of such PPIs entails the modulation of ion channel activity with widespread implications for influencing nociceptive signaling in a more specific way. In this review, we highlight recent advances in modulating ion channels and receptors via their PPI networks in the pursuit of relieving chronic pain. Moreover, we critically discuss the potential of targeting PPIs for developing novel pain therapies exhibiting higher efficacy and improved safety profiles.

The voltage sensitive domain (VSD) is a pivotal structure of voltage-gated ion channels (VGICs) and plays an essential role in the generation of electrochemical signals by neurons, striated muscle cells, and endocrine cells. The VSD is not unique to VGICs. Recent studies have shown that a VSD regulates a phosphatase. Similarly, Hv1, a voltage-sensitive protein that lacks an apparent pore domain, is a self-contained voltage sensor that operates as an H⁺ channel. VSDs are formed by four transmembrane helices (S1-S4). The S4 helix is positively charged due to the presence of arginine and lysine residues. It is surrounded by two water crevices that extend into the membrane from both the extracellular and intracellular milieus. A hydrophobic septum disrupts communication between these water crevices thus preventing the permeation of ions. The septum is maintained by interactions between the charged residues of the S4 segment and the gating charge transfer center. Mutating the charged residue of the S4 segment allows the water crevices to communicate and generate gating pore or omega pore. Gating pore currents have been reported to underlie several neuronal and striated muscle channelopathies. Depending on which charged residue on the S4 segment is mutated, gating pores are permeant either at depolarized or hyperpolarized voltages. Gating pores are cation selective and seem to converge toward Eisenmann's first or second selectivity sequences. Most gating pores are blocked by guanidine derivatives as well as trivalent and quadrivalent cations. Gating pores can be used to study the movement of the voltage sensor and could serve as targets for novel small therapeutic molecules.

Objectives: C-reactive protein (CRP), an inflammation marker, is a strong independent risk factor for cardiovascular disease. Vessels are able to express CRP; however, the molecular mechanism behind this expression is not clear., Methods: Reverse transcription PCR and ELISA were used to detect messenger RNA and proteins of CRP and nuclear factor κB (NF-κB) activity in vessel rings stretched with different mechanical strains., Results: Interleukin (IL)-6 treatment did not induce CRP expression in vessel rings of white rabbits in the absence of mechanical strain. In contrast, IL-6 augmented CRP expression in vessel rings stretched with mechanical strains of 3 and 5 g (CRP mRNA, IL-6: 11.367±1.68 and 12.78±0.76 vs vehicle: 7.27±0.88 and 8.3±0.91 folds, respectively; CRP, IL-6: 12.79±1.62 and 14.05±2.1 vs vehicle: 7.72±1.04 and 8.16±1.52 folds, respectively; p<0.05 vs 0 g group and vehicle control group; n=5), and this effect was completely blocked by treatment with gadolinium III chloride hexahydrate (GdCl3). Moreover, IL-6 treatment increased NF-κB activity in vessels stretched with a mechanical strain of 3 g, and this effect was blocked by stretch-activated channel inhibitors (streptomycin or GdCl3) and the NF-κB peptide inhibitor SN50, but not by the inactive SN50 analogue SN50M. We also performed similar experiments on human internal mammary arteries and obtained similar results., Conclusions: These results indicate that the inflammatory cytokine IL-6 alone does not induce CRP synthesis in vessels in the absence of mechanical strain; however, IL-6 augments mechanical strain-induced CRP synthesis in vessels via the stretch-activated channel-NF-κB pathway.

Voltage-gated sodium, potassium, and calcium channels are made of a pore domain (PD) controlled by four voltage-sensing domains (VSDs). The PD contains the ion permeation pathway and the activation gate located on the intracellular side of the membrane. A large number of small molecules are known to inhibit the PD by acting as open channel blockers. The voltage-gated proton channel Hv1 is made of two VSDs and lacks the PD. The location of the activation gate in the VSD is unknown and open channel blockers for VSDs have not yet been identified. Here, we describe a class of small molecules which act as open channel blockers on the Hv1 VSD and find that a highly conserved phenylalanine in the charge transfer center of the VSD plays a key role in blocker binding. We then use one of the blockers to show that Hv1 contains two intracellular and allosterically coupled gates., (Copyright © 2013 Elsevier Inc. All rights reserved.)

Background: Saturated free fatty acids (FFAs) have a dual action on pancreatic β-cells, consisting of an initial enhancement and subsequent suppression of glucose-induced electrical activity and insulin release. These stimulatory and inhibitory effects have been attributed, at least in part, to the activation and inhibition, respectively, of the volume-regulated anion channel (VRAC) by FFAs. Both effects were independent of their metabolism. We have now investigated the effects of related aliphatic compounds in order to further define the determinants of FFA interaction with VRAC., Methods: β-Cell VRAC and electrical activity were measured by conventional whole-cell and perforated patch recording, respectively. Cell volume was measured using a video-imaging technique., Results: In common with octanoic acid, addition of methyl octanoate or n-octanol resulted in a rapid, pronounced and reversible inhibition of VRAC activity. Addition of n-octane had no significant effect on VRAC activity. n-Octanol had a biphasic effect on β-cell membrane potential, namely a small transient depolarization followed by a marked hyperpolarization. n-Octanol was also found to prevent regulatory volume decrease in cells exposed to a hypotonic medium, consistent with VRAC inhibition., Conclusion: It is suggested that methyl octanoate and n-octanol can mimic the effects of FFAs on the pancreatic β-cell via modulation of VRAC activity. The structural requirements for this effect appear to be a medium or long chain aliphatic compound containing at least one oxygen atom.

The publication presents discussion of the modern vision of mechanisms of mechanoelectric feedback in heart as well as most recent findings regarding possible regulation of cardiomyocyte mechanically gated ion channels by endogenous compounds of immune origin--cytokines. Special attention is devoted to description of cytokine action on cardiac cells, in particular to nitrogen oxide effects on ionic currents, which contribute to generation of the action potential of the cardiomyocyte. We hypothesize that cytokines can potentially trigger such mechano-dependent cardiac pathologies as arrhythmias and fibrillation.

Crystal structures of Gloeobacter violaceus ligand-gated ion channel (GLIC), a proton-gated prokaryotic homologue of pentameric ligand-gated ion channel (LGIC) from G. violaceus, have provided high-resolution models of the channel architecture and its role in selective ion conduction and drug binding. However, it is still unclear which functional states of the LGIC gating scheme these crystal structures represent. Much of this uncertainty arises from a lack of thorough understanding of the functional properties of these prokaryotic channels. To elucidate the molecular events that constitute gating, we have carried out an extensive characterization of GLIC function and dynamics in reconstituted proteoliposomes by patch clamp measurements and EPR spectroscopy. We find that GLIC channels show rapid activation upon jumps to acidic pH followed by a time-dependent loss of conductance because of desensitization. GLIC desensitization is strongly coupled to activation and is modulated by voltage, permeant ions, pore-blocking drugs, and membrane cholesterol. Many of these properties are parallel to functions observed in members of eukaryotic LGIC. Conformational changes in loop C, measured by site-directed spin labeling and EPR spectroscopy, reveal immobilization during desensitization analogous to changes in LGIC and acetylcholine binding protein. Together, our studies suggest conservation of mechanistic aspects of desensitization among LGICs of prokaryotic and eukaryotic origin.

Dextromethorphan, an antitussive drug, has a neuroprotective property as evidenced by its inhibition of microglial production of pro-inflammatory cytokines and reactive oxygen species. The microglial activation requires NADPH oxidase activity, which is sustained by voltage-gated proton channels in microglia as they dissipate an intracellular acid buildup. In the present study, we examined the effect of dextromethorphan on proton currents in microglial BV2 cells. Dextromethorphan reversibly inhibited proton currents with an IC(50) value of 51.7 μM at an intracellular/extracellular pH gradient of 5.5/7.3. Dextromethorphan did not change the reversal potential or the voltage dependence of the gating. Dextrorphan and 3-hydroxymorphinan, major metabolites of dextromethorphan, and dextromethorphan methiodide were ineffective in inhibiting proton currents. The results indicate that dextromethorphan inhibition of proton currents would suppress NADPH oxidase activity and, eventually, microglial activation., (Copyright © 2012 Elsevier Ireland Ltd. All rights reserved.)

Microorganisms adapt to osmotic downshifts by releasing small osmolytes through mechanosensitive (MS) channels. We want to understand how the small mechanosensitive channel's (MscS) activation and inactivation, both driven by membrane tension, optimize survival in varying hypoosmotic shock situations. By measuring light scattering with a stopped-flow device, we estimate bacterial swelling time as 30-50 ms. A partial solute equilibration follows within 150-200 ms, during which optical responses from cells with WT MscS deviate from those lacking MS channels. MscS opening rates estimated in patch clamp show the channels readily respond to tensions below the lytic limit with a time course faster than 20 ms and close promptly upon tension release. To address the role of the tension-insensitive inactivated state in vivo, we applied short, long, and two-step osmotic shock protocols to WT, noninactivating G113A, and fast-inactivating D62N mutants. WT and G113A showed a comparable survival in short 1 min 800 mOsm downshock experiments, but G113A was at a disadvantage under a long 60 min shock. Preshocking cells carrying WT MscS for 15 s to 15 min with a 200 mOsm downshift did not sensitize them to the final 500 mOsm drop in osmolarity of the second step. However, these two-step shocks induced death in D62N more than just a one-step 700 mOsm downshift. We conclude MscS is able to activate and exude osmolytes faster than lytic pressure builds inside the cell under abrupt shock. During prolonged shocks, gradual inactivation prevents continuous channel activity and assists recovery. Slow kinetics of inactivation in WT MscS ensures that mild shocks do not inactivate the entire population, leaving some protection should conditions worsen.

Cells respond to a hyposmotic challenge by swelling and then returning toward the resting volume, a process known as the regulatory volume decrease or RVD. The sensors for this process have been proposed to include cationic mechanosensitive ion channels that are opened by membrane tension. We tested this hypothesis using a microfluidic device to measure cell volume and the peptide GsMTx4, a specific inhibitor of cationic mechanosensitive channels. GsMTx4 had no effect on RVD in primary rat astrocytes or Madin-Darby canine kidney (MDCK) cells but was able to completely inhibit RVD and the associated Ca(2+) uptake in normal rat kidney (NRK-49F) cells in a dose-dependent manner. Gadolinium (Gd(3+)), a nonspecific blocker of many mechanosensitive channels, inhibited RVD and Ca(2+) uptake in all three cell types, demonstrating the existence of at least two types of volume sensors. Single-channel stretch-activated currents are present in outside-out patches from NRK-49F, MDCK, and astrocytes, and they are reversibly inhibited by GsMTx4. While mechanosensitive channels are involved in volume regulation, their role for volume sensing is specialized. The NRK cells form a stable platform from which to screen drugs that affect volume regulation via mechanosensory channels and as a sensitive system to clone the channel.

Voltage-gated proton channels are strongly inhibited by Zn(2+), which binds to His residues. However, in a molecular model, the two externally accessible His are too far apart to coordinate Zn(2+). We hypothesize that high-affinity Zn(2+) binding occurs at the dimer interface between pairs of His residues from both monomers. Consistent with this idea, Zn(2+) effects were weaker in monomeric channels. Mutation of His(193) and His(140) in various combinations and in tandem dimers revealed that channel opening was slowed by Zn(2+) only when at least one His was present in each monomer, suggesting that in wild-type (WT) H(V)1, Zn(2+) binding between His of both monomers inhibits channel opening. In addition, monomeric channels opened exponentially, and dimeric channels opened sigmoidally. Monomeric channel gating had weaker temperature dependence than dimeric channels. Finally, monomeric channels opened 6.6 times faster than dimeric channels. Together, these observations suggest that in the proton channel dimer, the two monomers are closely apposed and interact during a cooperative gating process. Zn(2+) appears to slow opening by preventing movement of the monomers relative to each other that is prerequisite to opening. These data also suggest that the association of the monomers is tenuous and allows substantial freedom of movement. The data support the idea that native proton channels are dimeric. Finally, the idea that monomer-dimer interconversion occurs during activation of phagocytes appears to be ruled out.

This work completes previous findings and, using cysteine scanning mutagenesis (CSM) and biochemical methods, provides detailed analysis of conformational changes of the S6 domain and C-linker during gating of CNGA1 channels. Specific residues between Phe375 and Val424 were mutated to a cysteine in the CNGA1 and CNGA1(cys-free) background and the effect of intracellular Cd(2+) or cross-linkers of different length in the open and closed state was studied. In the closed state, Cd(2+) ions inhibited mutant channels A406C and Q409C and the longer cross-linker reagent M-4-M inhibited mutant channels A406C(cys-free) and Q409C(cys-free). Cd(2+) ions inhibited mutant channels D413C and Y418C in the open state, both constructed in a CNGA1 and CNGA1(cys-free) background. Our results suggest that, in the closed state, residues from Phe375 to approximately Ala406 form a helical bundle with a three-dimensional (3D) structure similar to those of the KcsA; furthermore, in the open state, residues from Ser399 to Gln409 in homologous subunits move far apart, as expected from the gating in K(+) channels; in contrast, residues from Asp413 to Tyr418 in homologous subunits become closer in the open state.

Previously, we have shown that murine HCN2 channels over-expressed in newborn and adult cardiac myocytes produce currents with different biophysical characteristics. To investigate the role of tyrosine kinase modulation in these age-dependent differences, we employed the broad spectrum tyrosine kinase inhibitor erbstatin. Our results demonstrated distinct and separable effects of erbstatin on channel gating and current amplitude and a marked age dependence to these effects. In newborn myocytes, erbstatin decreased current amplitude, shifted the activation relation negative, and slowed activation kinetics. The effect on activation voltage but not that on amplitude was absent when expressing a cAMP-insensitive mutant (HCN2R/E), while a C-terminal truncated form of HCN2 (HCN2DeltaCx) exhibited only the voltage dependent but not the amplitude effect of erbstatin. Thus, the action of erbstatin on the activation relation and current amplitude are distinct and separable in newborn myocytes, and the effect on activation voltage depends on the cAMP status of HCN2 channels. In contrast to newborn myocytes, erbstatin had no effect on HCN2 under control conditions in adult myocytes but induced a negative shift with no change in amplitude when saturated cAMP was added to the pipette solution. We conclude that erbstatin's effects on HCN2 current magnitude and voltage dependence are distinct and separable, and there are fundamental developmental differences in the heart that affect channel function and its modulation by the tyrosine kinase inhibitor erbstatin.

Objective: This study evaluated the ability of dihydrostreptomycin (DHSM) to go through the mechano-electric transduction (MET) channels in hair cells under physiological conditions., Materials and Methods: Tall hair cells were isolated from the chick basilar membrane (cochlea). Mechanical stimulation was applied by a glass rod attached to a piezoelectric bimorph, and MET currents were recorded with a whole-cell patch technique. The voltage-dependent block of DHSM to MET channel was estimated by calculating the relative conductances (the ratio of MET current in DHSM saline to DHSM-free saline) at various membrane potentials., Results and Conclusion: At membrane potentials between -100 and +50 mV, DHSM behaves as a voltage-dependent blocker according to a partial block model. At membrane potentials more negative than -100 mV, however, DHSM blocking decreased. This finding differed from the partial block model, but indicated that DHSM escaped through the channel pore into the cytoplasm by acting as a permeant channel blocker due to the large electrical driving force.

Although the accumulation of intracellular calcium ions ([Ca2+]i) is associated with muscle damage, little is known regarding the temporal profile of muscle [Ca2+]i under in vivo conditions, and, specifically, the effects of different contraction types [e.g., isometric (ISO); eccentric (ECC)] on [Ca2+]i remain to be determined. The following hypotheses were tested. 1) For 90 min at rest, an in vivo vs. in vitro preparation would better maintain initial [Ca2+]i. 2) Compared with ISO, ECC contractions (50 contractions, 10 sets, 5-min interval) would lead to a greater increase of [Ca2+]i. 3) Elevated [Ca2+]i during ECC would be reduced or prevented by the stretch-activated ion channel blockers streptomycin and gadolinium (Gd3+). Spinotrapezius muscles of Wistar rats were exteriorized (in vivo) or excised (in vitro). [Ca2+]i was evaluated by loading the muscle with fura 2-AM using fluorescence imaging. [Ca2+]i rose progressively beyond 40 min at rest under in vitro but not in vivo conditions during the 90-min protocol. In vivo [Ca2+]i increased more rapidly during ECC (first set) than ISO (fifth set) (P < 0.05 vs. precontraction values). The peak level of [Ca2+]i was increased by 21.5% (ISO) and 42.8% (ECC) after 10 sets (both P < 0.01). Streptomycin and Gd3+ abolished the majority of [Ca2+]i increase during ECC (69 and 86% reduction, respectively; P < 0.01 from peak [Ca2+]i of ECC). In conclusion, in vivo quantitative analyses demonstrated that ECC contractions elevate [Ca2+]i significantly more than ISO contractions and that stretch-activated channels may play a permissive role in this response.

Dielectrophoresis (DEP)--the motion of particles in non-uniform AC fields-has been used in the investigation of cell electrophysiology. The technique offers the advantages of rapid determination of the conductance and capacitance of membrane and cytoplasm. However, it is unable to directly determine the ionic strengths of individual cytoplasmic ions, which has potentially limited its application in assessing cell composition. In this paper, we demonstrate how dielectrophoresis can be used to investigate the cytoplasmic ion composition by using ion channel blocking agents. By blocking key ion transporters individually, it is possible to determine their overall contribution to the free ions in the cytoplasm. We use this technique to evaluate the relative contributions of chloride, potassium and calcium ions to the cytoplasmic conductivities of drug sensitive and resistant myelogenous leukaemic (K562) cells in order to determine the contributions of individual ion channel activity in mediating multi-drug resistance in cancer. Results indicate that whilst K(+) and Ca(2+) levels were extremely similar between sensitive and resistant lines, levels of Cl(-) were elevated by three times to that in the resistant line, implying increased chloride channel activity. This result is in line with current theories of MDR, and validates the use of ion channel blockers with DEP to investigate ion channel function.

Despite numerous reports implicating polyamines in plant salinity responses, the specific ionic mechanisms of polyamine-mediated adaptation to salt-stress remain elusive. In this work, we show that micromolar concentrations of polyamines are efficient in preventing NaCl-induced K(+) efflux from the leaf mesophyll, and that this effect can be attributed to the inhibition of non-selective cation channels in mesophyll. The inhibition by externally applied polyamines developed slowly over time, suggesting a cytosolic mode of action. Overall, we suggest that elevated levels of cellular polyamine may modulate the activity of plasma membrane ion channels, improving ionic relations and assisting in a plant's adaptation to salinity.

The 'funny' current, first described in cardiac pacemaker cells almost 30 years ago, is a key player in the generation of pacemaker activity and the autonomic modulation of heart rate. Because of these specific functions, a search for molecules able to interfere selectively with the 'funny' current was undertaken soon after its discovery, with the aim of developing tools for the pharmacological control of heart rate. This search has succeeded in generating a new class of drugs, the heart rate-reducing agents, which act through specific blockade of f-channels; one of these drugs, ivabradine, is presently marketed against stable angina. Because of their many functions in heart and other tissues, pharmacological utilization of "funny" channel properties is an exciting new frontier open to further developments.

Selective receptor antagonists are one of the most powerful resources in a pharmacologist's toolkit and are essential for the identification and classification of receptor subtypes and dissecting their roles in normal and abnormal body function. However, when the actions of antagonists are measured inappropriately and misleading results are reported, confusion and wrong interpretations ensue. This article gives a general overview of Schild analysis and the method of determining antagonist equilibrium constants. We demonstrate why this technique is preferable in the study of competitive receptor antagonism than the calculation of antagonist concentration that inhibit agonist-evoked responses by 50%. In addition we show how the use of Schild analysis can provide information on the outcome of single amino acid mutations in structure-function studies of receptors. Finally, we illustrate the need for caution when studying the effects of potent antagonists on synaptic transmission where the timescale of events under investigation is such that ligands and receptors never reach steady-state occupancy.

Human olfaction comprises the opposing actions of excitation and inhibition triggered by odorant molecules. In olfactory receptor neurons, odorant molecules not only trigger a G-protein-coupled signaling cascade but also generate various mechanisms to fine tune the odorant-induced current, including a low-selective odorant inhibition of the olfactory signal. This wide-range olfactory inhibition has been suggested to be at the level of ion channels, but definitive evidence is not available. Here, we report that the cyclic nucleotide-gated (CNG) cation channel, which is a key element that converts odorant stimuli into electrical signals, is inhibited by structurally unrelated odorants, consistent with the expression of wide-range olfactory inhibition. Interestingly, the inhibitory effect was small in the homo-oligomeric CNG channel composed only of the principal channel subunit, CNGA2, but became larger in channels consisting of multiple types of subunits. However, even in the channel containing all native subunits, the potency of the suppression on the cloned CNG channel appeared to be smaller than that previously shown in native olfactory neurons. Nonetheless, our results further showed that odorant suppressions are small in native neurons if the subsequent molecular steps mediated by Ca(2+) are removed. Thus, the present work also suggests that CNG channels switch on and off the olfactory signaling pathway, and that the on and off signals may both be amplified by the subsequent olfactory signaling steps.

Voltage changes across the cell membrane control the gating of many cation-selective ion channels. Conserved from bacteria to humans, the voltage-gated-ligand superfamily of ion channels are encoded as polypeptide chains of six transmembrane-spanning segments (S1-S6). S1-S4 functions as a self-contained voltage-sensing domain (VSD), in essence a positively charged lever that moves in response to voltage changes. The VSD 'ligand' transmits force via a linker to the S5-S6 pore domain 'receptor', thereby opening or closing the channel. The ascidian VSD protein Ci-VSP gates a phosphatase activity rather than a channel pore, indicating that VSDs function independently of ion channels. Here we describe a mammalian VSD protein (H(V)1) that lacks a discernible pore domain but is sufficient for expression of a voltage-sensitive proton-selective ion channel activity. H(v)1 currents are activated at depolarizing voltages, sensitive to the transmembrane pH gradient, H+-selective, and Zn2+-sensitive. Mutagenesis of H(v)1 identified three arginine residues in S4 that regulate channel gating and two histidine residues that are required for extracellular inhibition of H(v)1 by Zn2+. H(v)1 is expressed in immune tissues and manifests the characteristic properties of native proton conductances (G(vH+)). In phagocytic leukocytes, G(vH+) are required to support the oxidative burst that underlies microbial killing by the innate immune system. The data presented here identify H(v)1 as a long-sought voltage-gated H+ channel and establish H(v)1 as the founding member of a family of mammalian VSD proteins.

Beta sheet peptides (e.g., amyloid beta) are known to form ion channels in lipid bilayers possibly through aggregation, though the channel structure is not clear. We have recently reported that a short beta sheet peptide, (xSxG)(6), forms porin-like voltage-gated channels in lipid bilayers [Thundimadathil et al. (2005) Biochem. Biophys. Res. Commun. 330, 585-590]. To account for the porin-like activity, oligomerization of the peptide into a beta barrel-like structure was proposed. In this work, peptide aggregation in aqueous and membrane environments and a detailed study of channel properties were performed to gain insight into the mechanism of channel formation. The complex nature of the channel was revealed by kinetic analysis and the occurrence of interconverting multiple conductance states. Ion channels were inhibited by Congo red, suggesting that the peptide aggregates are the active channel species. Peptide aggregation and fibril formation in water were confirmed by electron microscopy (EM) and Congo red binding studies. Furthermore, oligomeric structures in association with lipid bilayers were detected. Circular dichroism of peptide-incorporated liposomes and peptide-lipid binding studies using EM suggest a lipid-induced beta sheet aggregation. Gel electrophoresis of peptide-incorporated liposomes showed dimeric and multimeric structures. Taken together, this work indicates insertion of (xSxG)(6) as oligomers into the lipid bilayer, followed by rearrangement into a beta barrel-like pore structure. A large peptide pore comprising several individual beta sheets or smaller beta sheet aggregates is expected to have a complex behavior in membranes. A dyad repeat sequence and the presence of glycine, serine, and hydrophobic residues in a repeated pattern in this peptide may be providing a favorable condition for the formation of a beta barrel-like structure in lipid bilayers.

Based on the analysis of kinetic scheme of blocking of open channels at any number of blocker binding sites, the dependence of current on blocker concentration was found. A variant of this dependence for a trapping blocker was also found. The restrictions of the applicability of the Hill equation and the necessity of taking into account the dependence of the concentration of demi-maximal blocker action (IC50) on the concentration of agonist were shown.

Voltage-gated proton channels are expressed highly in rat alveolar epithelial cells. Here we investigated whether these channels contribute to pH regulation. The intracellular pH (pH(i)) was monitored using BCECF in cultured alveolar epithelial cell monolayers and found to be 7.13 in nominally HCO(3)(-)-free solutions [at external pH (pH(o)) 7.4]. Cells were acid-loaded by the NH(4)(+) prepulse technique, and the recovery was observed. Under conditions designed to eliminate the contribution of other transporters that alter pH, addition of 10 microM ZnCl(2), a proton channel inhibitor, slowed recovery about twofold. In addition, the pH(i) minimum was lower, and the time to nadir was increased. Slowing of recovery by ZnCl(2) was observed at pH(o) 7.4 and pH(o) 8.0 and in normal and high-K(+) Ringer solutions. The observed rate of Zn(2+)-sensitive pH(i) recovery required activation of a small fraction of the available proton conductance. We conclude that proton channels contribute to pH(i) recovery after an acid load in rat alveolar epithelial cells. Addition of ZnCl(2) had no effect on pH(i) in unchallenged cells, consistent with the expectation that proton channels are not open in resting cells. After inhibition of all known pH regulators, slow pH(i) recovery persisted, suggesting the existence of a yet-undefined acid extrusion mechanism in these cells.

Although widely regarded as attractive drug targets, less than a tenth of known ion channels are currently commercially exploited as therapeutic targets. Historically, drug discovery efforts on ion channel targets have been encumbered by a lack of molecular and structural information, sub-optimal screening technologies and a paucity of discriminating pharmacological tools. Although challenges remain, recent scientific and technological advances in the area of ion channel research and screening offer the exciting prospect of a new, more-predictive era of ion channel drug discovery. In this article, focusing primarily on non voltage gated cation channels, we describe the continuing evolution of approaches to ion channel drug discovery, highlight recent developments in the ion channel field and consider their potential impact on discovering and ascribing function to ion channel targets. We discuss the renaissance of known ion channel targets, such as nicotinic acetylcholine receptors and calcium-activated potassium channels, as well as the emergence of the transient receptor potential (TRP) channels as a gene family of cation channels with broad therapeutic potential.

The influence of an NO donor, nitroglycerine (NG) on Ca2+ accumulation in rat heart and liver mitochondria was studied. A rapid dose-dependent increase in Ca(2+)-uptake was observed within 5 min after NG injection in vivo, with a peak in NG concentration of 0.5-1.0 mg/kg body weight. There is a correlation between a dose-dependent increase in Ca(2+)-accumulating capacity of mitochondria and sharp dose-dependent rise in stable NO metabolites level (NO2-, NO3- and nitrosothiols) and reactive oxygen species (H2O2 and hydroxyl radical, *OH) as well as lipid oxidation products (diene conjugates), the markers of the oxidative damage of the organelles in mitochondria in comparison to a whole tissue (liver) where only moderate increase of ROS and NO metabolites was detected. A 20-30-fold increase of NO3- level from 32. +/- 2.6 to 736.4 +/- 96.5 nmol/mg protein (p < 0.05) and from 45.4 +/- 6.2 to 1395.0 +/- 121.1 nmol/mg protein (p < 0.05) in liver and heart mitochondria respectively also provide a strong evidence for nitrosative stress. The results obtained suggest that calcium elimination from cytosol and its increased accumulation in mitochondria (likely dependent on permeability transition pore blockade by NO) could account for the cardioprotective action of NG although resulted in the development of oxidative stress with following long-term alterations of the energy supply and metabolism of subcellular structures.

Although widely regarded as attractive drug targets, less than a tenth of known ion channels are currently commercially exploited as therapeutic targets. Historically, drug discovery efforts on ion channel targets have been encumbered by a lack of molecular and structural information, sub-optimal screening technologies and a paucity of discriminating pharmacological tools. Although challenges remain, recent scientific and technological advances in the area of ion channel research and screening offer the exciting prospect of a new, more-predictive era of ion channel drug discovery. In this article, focusing primarily on non-voltage-gated cation channels, we describe the continuing evolution of approaches to ion channel drug discovery, highlight recent developments in the ion channel field and consider their potential impact on discovering and ascribing function to ion channel targets. We discuss the renaissance of known ion channel targets, such as nicotinic acetylcholine receptors and calcium-activated potassium channels, as well as the emergence of the transient receptor potential (TRP) channels as a gene family of cation channels with broad therapeutic potential.

The peptide GsMTx4, isolated from the venom of the tarantula Grammostola spatulata, is a selective inhibitor of stretch-activated cation channels (SACs). The mechanism of inhibition remains unknown; but both GsMTx4 and its enantiomer, enGsMTx4, modify the gating of SACs, thus violating a trademark of the traditional lock-and-key model of ligand-protein interactions. Suspecting a bilayer-dependent mechanism, we examined the effect of GsMTx4 and enGsMTx4 on gramicidin A (gA) channel gating. Both peptides are active, and the effect increases with the degree of hydrophobic mismatch between bilayer thickness and channel length, meaning that GsMTx4 decreases the energy required to deform the boundary lipids adjacent to the channel. GsMTx4 decreases inward SAC single-channel currents but has no effect on outward currents, suggesting it is located within a Debye length of the outer vestibule of the SACs, but significantly farther from the inner vestibule. Likewise, GsMTx4 decreases gA single-channel currents. Our results suggest that modulation of membrane proteins by amphipathic peptides--mechanopharmacology--involves not only the protein itself but also the surrounding lipids. The surprising efficacy of the d form of GsMTx4 peptide has important therapeutic implications, because d peptides are not hydrolysed by endogenous proteases and may be administered orally.

Ion channels and transporter proteins are prerequisites for formation and conduction of cardiac electrical impulses. Acting in concert, these proteins maintain cellular Na(+) and Ca(2+) homeostasis. Since intracellular Ca(2+) concentration determines contractile activation, we expect the majority of agents that modulate activity of ion channels and transporters not only to influence cellular action potentials but also contractile force. Drugs which block ion channels usually possess antiarrhythmic properties, those inhibiting the Na(+) pump have predominantly inotropic effects and those affecting Na(+),Ca(2+)- or Na(+),H(+)-exchanger protect against ischaemic cell damage. However, irrespective of their primary indication, all compounds targeted against ion channels and transporter proteins possess potential proarrhythmic activity.

Rod vision begins when 11-cis-retinal absorbs a photon and isomerizes to all-trans-retinal (ATR) within the photopigment, rhodopsin. Photoactivated rhodopsin triggers an enzyme cascade that lowers the concentration of cGMP, thereby closing cyclic nucleotide-gated (CNG) ion channels. After isomerization, ATR dissociates from rhodopsin, and after a bright light, this release is expected to produce a large surge of ATR near the CNG channels. Using excised patches from Xenopus oocytes, we recently showed that ATR shuts down cloned rod CNG channels, and that this inhibition occurs in the nanomolar range (aqueous concentration) at near-physiological concentrations of cGMP. Here we further characterize the ATR effect and present mechanistic information. ATR was found to decrease the apparent cGMP affinity, as well as the maximum current at saturating cGMP. When ATR was applied to outside-out patches, inhibition was much slower and less effective than when it was applied to inside-out patches, suggesting that ATR requires access to the intracellular surface of the channel or membrane. The apparent ATR affinity and maximal inhibition of heteromeric (CNGA1/CNGB1) channels was similar to that of homomeric (CNGA1) channels. Single-channel and multichannel data suggest that channel inhibition by ATR is reversible. Inhibition by ATR was not voltage dependent, and the form of its dose-response relation suggested multiple ATR molecules interacting per channel. Modeling of the data obtained with cAMP and cGMP suggests that ATR acts by interfering with the allosteric opening transition of the channel and that it prefers closed, unliganded channels. It remains to be determined whether ATR acts directly on the channel protein or instead alters channel-bilayer interactions.

Hyperpolarization-activated HCN channels are modulated by direct binding of cyclic nucleotides. For HCN2 channels, cAMP shifts the voltage dependence for activation, with relatively little change in the maximal conductance. By contrast, in spHCN channels, cAMP relieves a rapid inactivation process and produces a large increase in maximum conductance. Our results suggest that these two effects of cAMP represent the same underlying process. We also find that spHCN inactivation occurs not by closure of a specialized inactivation gate, as for other voltage-dependent channels, but by reclosure of the same intracellular gate opened upon activation. Effectively, the activation gate exhibits a "desensitization to voltage," perhaps by slippage of the coupling between the voltage sensors and the gate. Differences in the initial coupling efficiency could allow cAMP to produce either the inactivation or the shift phenotype by strengthening effective coupling: a shift would naturally occur if coupling is already strong in the absence of cAMP.

Cyclic nucleotide-gated (CNG) ion channels are nonselective cation channels with a high permeability for Ca(2+). Not surprisingly, they are blocked by a number of Ca(2+) channel blockers including tetracaine, pimozide, and diltiazem. We studied the effects of dequalinium, an extracellular blocker of the small conductance Ca(2+)-activated K(+) channel. We previously noted that dequalinium is a high-affinity blocker of CNGA1 channels from the intracellular side, with little or no state dependence at 0 mV. Here we examined block by dequalinium at a broad range of voltages in both CNGA1 and CNGA2 channels. We found that dequalinium block was mildly state dependent for both channels, with the affinity for closed channels 3-5 times higher than that for open channels. Mutations in the S4-S5 linker did not alter the affinity of open channels for dequalinium, but increased the affinity of closed channels by 10-20-fold. The state-specific effect of these mutations raises the question of whether/how the S4-S5 linker alters the binding of a blocker within the ion permeation pathway.

Felbamate (FBM) is a potent nonsedative anticonvulsant whose clinical effect may be related to the inhibition of N-methyl-D-aspartate (NMDA) currents, but the exact molecular action remains unclear. Using whole-cell patch-clamp recording in rat hippocampal neurons, we found that submillimolar FBM effectively modifies the gating process of NMDA channels. During a single high-concentration (1 mM) NMDA pulse, FBM significantly inhibits the late sustained current but not the early peak current. However, if the 1 mM NMDA pulse is preceded by a low-concentration (10 microM) NMDA prepulse, then FBM significantly inhibits both the peak and the sustained currents in the 1 mM pulse. In sharp contrast, the NMDA currents elicited by micromolar NMDA are only negligibly inhibited or even enhanced by FBM. These findings indicate that the inhibitory effect of FBM on NMDA currents is stronger with both higher NMDA concentration and longer NMDA exposure, and is thus "use-dependent". FBM also slows recovery of the desensitized NMDA channel, and quantitative analyses of FBM effects on the activation kinetics and the desensitization curve of the NMDA currents further disclose dissociation constants of approximately 200, approximately 110, and approximately 55 microM for FBM binding to the resting, activated, and desensitized NMDA channels, respectively. We conclude that therapeutic concentrations (50-300 microM) of FBM could bind to and modify a significant proportion of the resting NMDA channel even when NMDA or other glutamatergic ligand is not present and then decrease the NMDA currents at subsequent NMDA pulses by stabilization of the desensitized channels. Because the inhibitory effect is apparent only when there is excessive NMDA exposure, FBM may effectively inhibit many seizure discharges but preserve most normal neuronal firings.

Excitotoxicity in brain ischemia triggers neuronal death and neurological disability, and yet these are not prevented by antiexcitotoxic therapy (AET) in humans. Here, we show that in neurons subjected to prolonged oxygen glucose deprivation (OGD), AET unmasks a dominant death mechanism perpetuated by a Ca2+-permeable nonselective cation conductance (IOGD). IOGD was activated by reactive oxygen/nitrogen species (ROS), and permitted neuronal Ca2+ overload and further ROS production despite AET. IOGD currents corresponded to those evoked in HEK-293 cells expressing the nonselective cation conductance TRPM7. In cortical neurons, blocking IOGD or suppressing TRPM7 expression blocked TRPM7 currents, anoxic 45Ca2+ uptake, ROS production, and anoxic death. TRPM7 suppression eliminated the need for AET to rescue anoxic neurons and permitted the survival of neurons previously destined to die from prolonged anoxia. Thus, excitotoxicity is a subset of a greater overall anoxic cell death mechanism, in which TRPM7 channels play a key role.

Peptide toxins are invaluable tools for studying the structure and physiology of ion channels. Pseudechetoxin (PsTx) is the first known peptide toxin that targets cyclic nucleotide-gated (CNG) ion channels, which play a critical role in sensory transduction in the visual and olfactory systems. PsTx inhibited channel currents at low nM concentrations when applied to the extracellular face of membrane patches expressing olfactory CNGA2 subunits. Surprisingly, 500 nM PsTx did not inhibit currents through channels formed by the CNGA3 subunit from cone photoreceptors. We have exploited this difference to identify the PsTx-binding site on the extracellular face of CNG channels. Studies using chimeric channels revealed that transplantation of the pore domain from CNGA2 was sufficient to confer high affinity PsTx binding upon a CNGA3 background. To further define the binding site, reciprocal mutations were made at 10 nonidentical amino acid residues in this region. We found that two residues in CNGA2, D316 and Y321, were essential for high-affinity inhibition by PsTx. Furthermore, replacement of both residues was required to confer high-affinity PsTx inhibition upon CNGA3. Several other residues, including E325, also form favorable interactions with PsTx. In the CNGA2-E325K mutant, PsTx affinity was reduced by approximately 5-fold to 120 nM. An electrostatic interaction with D316 does not appear to be the primary determinant of PsTx affinity, as modification of the D316C mutant with a negatively charged methanethiosulfonate reagent did not restore high affinity inhibition. The residues involved in PsTx binding are found within the pore turret and helix, in similar positions to residues that form the receptor for pore-blocking toxins in voltage-gated potassium channels. Furthermore, biophysical properties of PsTx block, including an unfavorable interaction with permeant ions, also suggest that it acts as a pore blocker. In summary, PsTx seems to occlude the entrance to the pore by forming high-affinity contacts with the pore turret, which may be larger than that found in the KcsA structure.

Small molecules can activate membrane bound receptors to elicit cellular responses and approximately 80% of all such responses are mediated through membranes. Solid state nuclear magnetic resonance (NMR) methods have been developed for the resolution of specifically labelled ligands, designed to report back structural, dynamic and electronic details of the ligand whilst at its site of action. In particular, ion channel blockers, drugs, inhibitors, prosthetic groups and solutes have been studied when at their target site in functionally competent membranes. A blocker of the channel in the anion transporter of erythrocytes (AE1) has been studied to show that considerable motional freedom within the channel site occurs. The activating agonist of the ligand gated ion channel, the nicotinic acetylcholine receptor, is constrained at its site of action and binds through a cation-pi interaction as revealed in the NMR spectra. The footprint for the inhibitor binding site of the digitalis receptor, the Na(+)/K(+)-ATPase has been resolved as well as the molecular conformation of digitalis showing a bent structure whilst at its site of action. For the gastric proton pump, however, a planar conformation is observed for a reversible substituted imidazopyridine. In all cases not only the structure, but also some indications about the electronic interactions of ligand and target have been resolved using these approaches.

In 1999, we purified pseudechetoxin (PsTx), the first peptide toxin known to block cyclic nucleotide-gated (CNG) ion channels, from the venom of Pseudechis australis [Brown, R. L., Haley, T. L., West, K. A., and Crabb, J. W. (1999) Proc. Natl. Acad. Sci. U.S.A. 96, 754-759]. Here we report the cloning of the cDNA encoding PsTx, as well as the discovery and cloning of pseudecin, a homologous toxin from the venom of Pseudechis porphyriacus. The mature proteins are 211 and 210 amino acids in length, and the amino acid sequences are 96.7% identical, differing in only seven residues. The purified toxins were applied to outside-out patches excised from Xenopus oocytes expressing CNG channels composed of the rod CNGA1 or olfactory CNGA2 channel subunits. Surprisingly, these patch-clamp studies revealed a 30-fold difference in affinity between PsTx and pseudecin for channels composed of CNGA2 subunits. The apparent K(i) of PsTx was 15 nM, while the affinity of pseudecin was 460 nM. The difference in affinities for the CNGA1 subunit from rod photoreceptors was less pronounced, but the affinity of PsTx was 70 nM, compared with 1000 nM for pseudecin. This difference in affinity may be instructive as we attempt to identify the regions of the toxins that contact CNG channels. As the only known protein blockers of CNG channels, these toxins promise to be valuable tools to study the structure of the external face of these channels.

Rod cyclic nucleotide-gated (CNG) channels are heterotetramers comprised of both CNGA1 and CNGB1 subunits. Calcium/calmodulin (Ca(2+)/CaM) binds to a site in the N-terminal region of CNGB1 subunits and inhibits the opening conformational change in CNGA1/CNGB1 channels. Here, we show that polypeptides derived from an N-terminal region of CNGB1 form a specific interaction with polypeptides derived from a C-terminal region of CNGA1 that is distal to the cyclic nucleotide-binding domain. Deletion of the Ca(2+)/CaM-binding site from the N-terminal region of CNGB1 eliminated both Ca(2+)/CaM modulation of the channel and the intersubunit interaction. Furthermore, the interaction was disrupted by the presence of Ca(2+)/CaM. These results suggest that Ca(2+)/CaM-dependent inhibition of rod channels is caused by the direct binding of Ca(2+)/CaM to a site in the N-terminal region in CNGB1, which disrupts the interaction between this region and a distal C-terminal region of CNGA1. The mechanism underlying Ca(2+)/CaM modulation of rod channels is distinct from that in olfactory (CNGA2) CNG channels.

We report here that NO(3)(-) in the xylem exerts positive feedback on its loading into the xylem through a change in the voltage dependence of the Quickly Activating Anion Conductance, X-QUAC. Properties of this conductance were investigated on xylem-parenchyma protoplasts prepared from roots of Hordeum vulgare by applying the patch-clamp technique. Chord conductances were minimal around -40 mV and increased with plasma membrane depolarisation as well as with hyperpolarisation. Two gates with opposite voltage dependences were postulated. When 30 mM Cl- in the bath was replaced by NO(3)(-), a shift in the midpoint potential of the depolarisation-activated gate by about -60 mV from 43 to -16 mV occurred (K(m) = 3.4 mM). No such effect was seen when chloride was replaced by malate. Addition of 10 mM NO(3)(-)to the pipette solution and reduction of [Cl-] from 124 to 4 mM (to simulate cytoplasmic concentrations) did not interfere with the voltage dependence of X-QUAC activation, nor was it affected by changes in external [K+]. If only the NO(3)(-) effect on gating was considered, an increase of the NO(3)(-) concentration in the xylem sap to 5 mM would result in an enhancement of NO(3)(-) efflux by about 30%. Although the driving force for NO(3)(-) efflux would be reduced simultaneously, NO(3)(-) efflux into the xylem through X-QUAC would be maintained with high NO(3)(-) concentrations in the xylem sap; a situation which occurs for instance during the night.