Inhibition Of Channel Opening Research Articles

Pharmacologic Characterization of LTGO-33, a Selective Small Molecule Inhibitor of the Voltage-Gated Sodium Channel NaV1.8 with a Unique Mechanism of Action.

Discovery and development of new molecules directed against validated pain targets is required to advance the treatment of pain disorders. Voltage-gated sodium channels (NaVs) are responsible for action potential initiation and transmission of pain signals. NaV1.8 is specifically expressed in peripheral nociceptors and has been genetically and pharmacologically validated as a human pain target. Selective inhibition of NaV1.8 can ameliorate pain while minimizing effects on other NaV isoforms essential for cardiac, respiratory, and central nervous system physiology. Here we present the pharmacology, interaction site, and mechanism of action of LTGO-33, a novel NaV1.8 small molecule inhibitor. LTGO-33 inhibited NaV1.8 in the nM potency range and exhibited over 600-fold selectivity against human NaV1.1-NaV1.7 and NaV1.9. Unlike prior reported NaV1.8 inhibitors that preferentially interacted with an inactivated state via the pore region, LTGO-33 was state-independent with similar potencies against closed and inactivated channels. LTGO-33 displayed species specificity for primate NaV1.8 over dog and rodent NaV1.8 and inhibited action potential firing in human dorsal root ganglia neurons. Using chimeras combined with mutagenesis, the extracellular cleft of the second voltage-sensing domain was identified as the key site required for channel inhibition. Biophysical mechanism of action studies demonstrated that LTGO-33 inhibition was relieved by membrane depolarization, suggesting the molecule stabilized the deactivated state to prevent channel opening. LTGO-33 equally inhibited wild-type and multiple NaV1.8 variants associated with human pain disorders. These collective results illustrate LTGO-33 inhibition via both a novel interaction site and mechanism of action previously undescribed in NaV1.8 small molecule pharmacologic space. SIGNIFICANCE STATEMENT: NaV1.8 sodium channels primarily expressed in peripheral pain-sensing neurons represent a validated target for the development of novel analgesics. Here we present the selective small molecule NaV1.8 inhibitor LTGO-33 that interdicts a distinct site in a voltage-sensor domain to inhibit channel opening. These results inform the development of new analgesics for pain disorders.

Resolvin D1 and D2 Inhibit Transient Receptor Potential Vanilloid 1 and Ankyrin 1 Ion Channel Activation on Sensory Neurons via Lipid Raft Modification

Transient Receptor Potential Vanilloid 1 and Ankyrin 1 (TRPV1, TRPA1) cation channels are expressed in nociceptive primary sensory neurons and regulate nociceptor and inflammatory functions. Resolvins are endogenous lipid mediators. Resolvin D1 (RvD1) is described as a selective inhibitor of TRPA1-related postoperative and inflammatory pain in mice acting on the G protein-coupled receptor DRV1/GPR32. Resolvin D2 (RvD2) is a very potent TRPV1 and TRPA1 inhibitor in DRG neurons, and decreases inflammatory pain in mice acting on the GPR18 receptor, via TRPV1/TRPA1-independent mechanisms. We provided evidence that resolvins inhibited neuropeptide release from the stimulated sensory nerve terminals by TRPV1 and TRPA1 activators capsaicin (CAPS) and allyl-isothiocyanate (AITC), respectively. We showed that RvD1 and RvD2 in nanomolar concentrations significantly decreased TRPV1 and TRPA1 activation on sensory neurons by fluorescent calcium imaging and inhibited the CAPS- and AITC-evoked 45Ca-uptake on TRPV1- and TRPA1-expressing CHO cells. Since CHO cells are unlikely to express resolvin receptors, resolvins are suggested to inhibit channel opening through surrounding lipid raft disruption. Here, we proved the ability of resolvins to alter the membrane polarity related to cholesterol composition by fluorescence spectroscopy. It is concluded that targeting lipid raft integrity can open novel peripheral analgesic opportunities by decreasing the activation of nociceptors.

Structure of the receptor-activated human TRPC6 and TRPC3 ion channels.

TRPC6 and TRPC3 are receptor-activated nonselective cation channels that belong to the family of canonical transient receptor potential (TRPC) channels. They are activated by diacylglycerol, a lipid second messenger. TRPC6 and TRPC3 are involved in many physiological processes and implicated in human genetic diseases. Here we present the structure of human TRPC6 homotetramer in complex with a newly identified high-affinity inhibitor BTDM solved by single-particle cryo-electron microscopy to 3.8 Å resolution. We also present the structure of human TRPC3 at 4.4 Å resolution. These structures show two-layer architectures in which the bell-shaped cytosolic layer holds the transmembrane layer. Extensive inter-subunit interactions of cytosolic domains, including the N-terminal ankyrin repeats and the C-terminal coiled-coil, contribute to the tetramer assembly. The high-affinity inhibitor BTDM wedges between the S5-S6 pore domain and voltage sensor-like domain to inhibit channel opening. Our structures uncover the molecular architecture of TRPC channels and provide a structural basis for understanding the mechanism of these channels.

Structure Inhibition and Regulation of a Two-Pore Channel TPC1

Membrane transport serves vital functions in the cell, providing nutrients for growth, relaying electrical signals, evading pathogens, and maintaining homeostasis. Voltage- and ligand-gated ion channels propagate cellular electrical signals by coupling changes in membrane potential and the binding of molecules to channel opening and selective passage of ions through the membrane barrier. Two-pore channels (TPCs) are intracellular ion channels that integrate changes in membrane potential, second messengers, and phosphorylation to control endolysosomal trafficking, autophagy, cellular ion and amino acid homeostasis, and ultra-long action potential-like signals. They broadly impact human diseases related to trafficking including filoviral infections, Parkinson's disease, obesity, fatty liver disease, and Alzheimer's disease. The response of TPCs to multiple cellular inputs suggests a multi-state gating mechanism. Nevertheless, the mechanisms that govern cycles of activation and deactivation or ‘gating’ in the channel remain poorly understood. To understand the bases for ion permeation, channel activation, the location of voltage-sensing domains and regulatory ion-binding sites, and phosphoregulation, we determined the crystal structure of TPC1 from Arabidopsis thaliana to 2.87A resolution. This reveals for the first time how TPC channels assemble as ‘quasi-tetramers’ from two non-equivalent tandem pore-forming subunits. We determined sites of phosphorylation in the N-terminal and C-terminal domains that are positioned to allosterically modulate channel activation by cytoplasmic calcium. One of the two voltage sensing domains (VSD2) encodes voltage sensitivity and inhibition by luminal calcium locks VSD2 in a ‘resting’ conformation, distinct from the activated VSDs observed in structures of other voltage-gated ion channels. The structure shows how potent pharmacophore trans-Ned-19 allosterically acts to inhibit channel opening. In animals, trans-Ned-19 prevents infection by Ebola virus and Filoviruses by blocking fusion of the viral and endolysosomal membranes, thereby preventing delivery of their RNA into the host cytoplasm. The structure of TPC1 paves the way for understanding the complex function of these channels and may aid the development of antiviral compounds.

Apocalmodulin Itself Promotes Ion Channel Opening and Ca2+ Regulation

The Ca(2+)-free form of calmodulin (apoCaM) often appears inert, modulating target molecules only upon conversion to its Ca(2+)-bound form. This schema has appeared to govern voltage-gated Ca(2+) channels, where apoCaM has been considered a dormant Ca(2+) sensor, associated with channels but awaiting the binding of Ca(2+) ions before inhibiting channel opening to provide vital feedback inhibition. Using single-molecule measurements of channels and chemical dimerization to elevate apoCaM, we find that apoCaM binding on its own markedly upregulates opening, rivaling the strongest forms of modulation. Upon Ca(2+) binding to this CaM, inhibition may simply reverse the initial upregulation. As RNA-edited and -spliced channel variants show different affinities for apoCaM, the apoCaM-dependent control mechanisms may underlie the functional diversity of these variants and explain an elongation ofneuronal action potentials by apoCaM. More broadly, voltage-gated Na channels adopt this same modulatory principle. ApoCaM thus imparts potent and pervasive ion-channel regulation. PAPERCLIP:

State-Dependent Crosslinking in IKS Demonstrates a Closed-State Interaction between KCNE1 at F57 and KCNQ1 that Inhibits Channel Opening

The slow delayed rectifier current (IKs) is a key repolarizing potassium current in the cardiac action potential. IKs is composed of KCNQ1 which forms the tetrameric voltage gated pore subunit and KCNE1, a single transmembrane domain accessory subunit, proposed to reside in the channel's exterior cleft. KCNE1 imposes a dramatic regulation on KCNQ1, significantly delaying opening compared to the unchaperoned channel. Here, we have investigated this interaction using the UV-crosslinking unnatural amino acid, p-benzoyl-L-phenyl alanine (pBpa). pBpa was genetically incorporated into KCNE1 at residue F57 in the transmembrane domain using the amber stop codon (TAG) suppression system. Successful incorporation into KCNE1-F57TAG was confirmed by a lack of functional IKs current in transfected cells not supplemented with pBpa. Characterization of the pBpa-incorporated channel complex revealed a right shifted V0.5 of activation compared to wild-type (+27 mV vs. +14 mV). Channels were UV-irradiated in the closed state by applying a 300 ms light pulse at −90 mV followed by a 4s activation step (+60 mV). A diary plot of the peak current vs. UV-exposure with repeated exposure revealed a rapid decrease in available current compared to UV-treated wild-type channels. This indicates the permanent closure of channels by crosslinking. Application of UV at the end of a 4s activation step (+60 mV) produced an immediate downward deflection in current and resulted in a slower rate of channel crosslinking compared to the closed-state UV treatment. This suggests that KCNE1 can revisit a closed-state orientation during channel activation and is then trapped in the closed-state by the covalent crosslink. These findings provide new insight into the interactions that regulate the IKs channel complex.

Scorpion β-toxin interference with NaV channel voltage sensor gives rise to excitatory and depressant modes

Scorpion β toxins, peptides of ∼70 residues, specifically target voltage-gated sodium (NaV) channels to cause use-dependent subthreshold channel openings via a voltage–sensor trapping mechanism. This excitatory action is often overlaid by a not yet understood depressant mode in which NaV channel activity is inhibited. Here, we analyzed these two modes of gating modification by β-toxin Tz1 from Tityus zulianus on heterologously expressed NaV1.4 and NaV1.5 channels using the whole cell patch-clamp method. Tz1 facilitated the opening of NaV1.4 in a use-dependent manner and inhibited channel opening with a reversed use dependence. In contrast, the opening of NaV1.5 was exclusively inhibited without noticeable use dependence. Using chimeras of NaV1.4 and NaV1.5 channels, we demonstrated that gating modification by Tz1 depends on the specific structure of the voltage sensor in domain 2. Although residue G658 in NaV1.4 promotes the use-dependent transitions between Tz1 modification phenotypes, the equivalent residue in NaV1.5, N803, abolishes them. Gating charge neutralizations in the NaV1.4 domain 2 voltage sensor identified arginine residues at positions 663 and 669 as crucial for the outward and inward movement of this sensor, respectively. Our data support a model in which Tz1 can stabilize two conformations of the domain 2 voltage sensor: a preactivated outward position leading to NaV channels that open at subthreshold potentials, and a deactivated inward position preventing channels from opening. The results are best explained by a two-state voltage–sensor trapping model in that bound scorpion β toxin slows the activation as well as the deactivation kinetics of the voltage sensor in domain 2.

TRIP8B Allosterically Regulates the Ability of cAMP to Enhance the HCN2 Channel Opening

The opening of hyperpolarization-activated cyclic nucleotide-gated cation (HCN) channels is enhanced by the binding of cAMP to a cytoplasmic cyclic nucleotide binding domain (CNBD). This action results from an allosteric mechanism in which cAMP binds more tightly to the open versus closed state of the channel, thereby shifting channel opening to more depolarized voltages (quantified by the shift in the midpoint voltage of activation, ΔV1/2). Recently, the brain-specific cytoplasmic protein TRIP8b has been shown to form an auxiliary subunit of HCN channels. In heterologous systems, TRIP8b both alters channel membrane trafficking and inhibits channel opening by shifting the V1/2 to more negative potentials. Previous results indicate that this inhibitory action of TRIP8b results from an antagonism of the ability of cAMP to enhance channel opening. Moreover, the effect of TRIP8b on HCN gating was found to result from the interaction of a central core region of TRIP8b with the CNBD of the channel. Here we ask: Does the effect of TRIP8b involve a direct competition with cAMP for the CNBD, or does it reflect an allosteric action? By creating TRIP8b-HCN2 fusion proteins, we demonstrate that TRIP8b shifts the relation between cAMP concentration and ΔV1/2 to higher cAMP concentrations. Furthermore, we find that direct application of TRIP8bmini, an 80 amino-acid domain in the conserved central core of TRIP8b, to inside-out patches is sufficient to antagonize the action of cAMP similar to that seen with full-length TRIP8b. Moreover, we find that TRIP8bmini reduces the ΔV1/2 observed with saturating levels of cAMP (i.e., decreases ΔVmax), suggesting that TRIP8b does not directly compete with cAMP but rather regulates HCN2 channels through an allosteric mechanism, decreasing both the efficacy of cAMP and the sensitivity of the channel to this ligand.

TRIP8b Regulates HCN1 Channel Trafficking and Gating through Two Distinct C-Terminal Interaction Sites

Hyperpolarization-activated cyclic nucleotide-regulated (HCN) channels in the brain associate with their auxiliary subunit TRIP8b (also known as PEX5R), a cytoplasmic protein expressed as a family of alternatively spliced isoforms. Recent in vitro and in vivo studies have shown that association of TRIP8b with HCN subunits both inhibits channel opening and alters channel membrane trafficking, with some splice variants increasing and others decreasing channel surface expression. Here, we address the structural bases of the regulatory interactions between mouse TRIP8b and HCN1. We find that HCN1 and TRIP8b interact at two distinct sites: an upstream site where the C-linker/cyclic nucleotide-binding domain of HCN1 interacts with an 80 aa domain in the conserved central core of TRIP8b; and a downstream site where the C-terminal SNL (Ser-Asn-Leu) tripeptide of the channel interacts with the tetratricopeptide repeat domain of TRIP8b. These two interaction sites play distinct functional roles in the effects of TRIP8b on HCN1 trafficking and gating. Binding at the upstream site is both necessary and sufficient for TRIP8b to inhibit channel opening. It is also sufficient to mediate the trafficking effects of those TRIP8b isoforms that downregulate channel surface expression, in combination with the trafficking motifs present in the N-terminal region of TRIP8b. In contrast, binding at the downstream interaction site serves to stabilize the C-terminal domain of TRIP8b, allowing for optimal interaction between HCN1 and TRIP8b as well as for proper assembly of the molecular complexes that mediate the effects of TRIP8b on HCN1 channel trafficking.

Dual Regulation of Intracellular CFTR Gating by the R Domain

Most ATP-binding cassette (ABC) transporters have two intracellular nucleotide-binding domains (NBDs) and two membrane-spanning domains (MSDs). Each MSD consists of six helical segments probably extended to four gating-controled cytoplasmic loops (CLs). However, the cyctic fibrosis transmembrane conductance regulator (CFTR) has an additional regulatory (R) domain (645-838). It has fourteen PKA phosphorylation sites exerting stimulatory or inhibitory effects on channel activity. My previous studies suggested that S768, a well-known inhibitory PKA phosphorylation site, may prevent channel opening by a putative Fe3+ bridge or an H-bond between the R domain and CL3. Here an electrostatic attraction between the R domain and CL1 or CL3 was reported as a new approach to regulating channel gating dually. On the one hand, diamide-induced disulfide bond crosslinking of K946C to D836C, confirmed by SDS-PAGE mobility, inhibited channel opening, and inhibition was reversed by DTT. However, both K946A and D835A or D836A clearly increased the sensitivity of channel activity to PKA phosphorylation while K951A failed to. Thus, an electrostatic attraction between K946 and D835 or D836 may stop channel opening primarily by preventing an interaction of CL3 with CL1. On the other hand, diamide-induced disulfide bond crosslinking of S700C or S795C to K162C or K163C, which was confirmed by SDS-PAGE mobility upon pretreatment of CFTR-expressing HEK-293T cells with forskolin, increased a channel open probability, and the effect was reversed by DTT. However, S700A, S795A and K162A enhanced PKA dependence of channel activity while K163A did not. Therefore, an electrostatic attraction between K162 and phosphorylated S700 or S795 may promote channel opening possibly by stabilizing the interaction between CL1 and CL3.

Zinc inhibition of monomeric and dimeric proton channels suggests cooperative gating

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.

Structural Determinants of the High Affinity Extracellular Zinc Binding Site on Cav3.2 T-type Calcium Channels

Ca(v)3.2 T-type channels contain a high affinity metal binding site for trace metals such as copper and zinc. This site is occupied at physiologically relevant concentrations of these metals, leading to decreased channel activity and pain transmission. A histidine at position 191 was recently identified as a critical determinant for both trace metal block of Ca(v)3.2 and modulation by redox agents. His(191) is found on the extracellular face of the Ca(v)3.2 channel on the IS3-S4 linker and is not conserved in other Ca(v)3 channels. Mutation of the corresponding residue in Ca(v)3.1 to histidine, Gln(172), significantly enhances trace metal inhibition, but not to the level observed in wild-type Ca(v)3.2, implying that other residues also contribute to the metal binding site. The goal of the present study is to identify these other residues using a series of chimeric channels. The key findings of the study are that the metal binding site is composed of a Asp-Gly-His motif in IS3-S4 and a second aspartate residue in IS2. These results suggest that metal binding stabilizes the closed conformation of the voltage-sensor paddle in repeat I, and thereby inhibits channel opening. These studies provide insight into the structure of T-type channels, and identify an extracellular motif that could be targeted for drug development.

TRIP8b Splice Variants Form a Family of Auxiliary Subunits that Regulate Gating and Trafficking of HCN Channels in the Brain

Hyperpolarization-activated cyclic nucleotide-regulated (HCN) channels, which generate the I(h) current, mediate a number of important brain functions. The HCN1 isoform regulates dendritic integration in cortical pyramidal neurons and provides an inhibitory constraint on both working memory in prefrontal cortex and spatial learning and memory in the hippocampus. Altered expression of HCN1 following seizures may contribute to the development of temporal lobe epilepsy. Yet the regulatory networks and pathways governing HCN channel expression and function in the brain are largely unknown. Here, we report the presence of nine alternative N-terminal splice forms of the brain-specific cytoplasmic protein TRIP8b and demonstrate the differential effects of six isoforms to downregulate or upregulate HCN1 surface expression. Furthermore, we find that all TRIP8b isoforms inhibit channel opening by shifting activation to more negative potentials. TRIP8b thus functions as an auxiliary subunit that provides a mechanism for the dynamic regulation of HCN1 channel expression and function.

C-terminal Movement during Gating in Cyclic Nucleotide-modulated Channels

Activation of cyclic nucleotide-modulated channels such as CNG and HCN channels is promoted by ligand-induced conformational changes in their C-terminal regions. The primary intersubunit interface of these C termini includes two salt bridges per subunit, formed between three residues (one positively charged and two negatively charged amino acids) that we term the SB triad. We previously hypothesized that the SB triad is formed in the closed channel and breaks when the channel opens. Here we tested this hypothesis by dynamically manipulating the SB triad in functioning CNGA1 channels. Reversing the charge at positions Arg-431 and Glu-462, two of the SB triad residues, by either mutation or application of charged reagents increased the favorability of channel opening. To determine how a charge reversal mutation in the SB triad structurally affects the channel, we solved the crystal structure of the HCN2 C-terminal region with the equivalent E462R mutation. The backbone structure of this mutant was very similar to that of wild type, but the SB triad was rearranged such that both salt bridges did not always form simultaneously, suggesting a mechanism for the increased ease of opening of the mutant channels. To prevent movement in the SB triad, we tethered two components of the SB triad region together with cysteine-reactive cross-linkers. Preventing normal movement of the SB triad region with short cross-linkers inhibited channel opening, whereas longer cross-linkers did not. These results support our hypothesis that the SB triad forms in the closed channel and indicate that this region expands as the channel opens.

Inhibition Mechanism of the Acetylcholine Receptor by α-Neurotoxins as Revealed by Normal-Mode Dynamics

The nicotinic acetylcholine receptor (AChR) is the prototype of ligand-gated ion channels. Here, we calculate the dynamics of the muscle AChR using normal modes. The calculations reveal a twist-like gating motion responsible for channel opening. The ion channel diameter is shown to increase with this twist motion. Strikingly, the twist motion and the increase in channel diameter are not observed for the AChR in complex with two alpha-bungarotoxin (alphaBTX) molecules. The toxins seems to lock together neighboring receptor subunits, thereby inhibiting channel opening. Interestingly, one alphaBTX molecule suffices to prevent the twist motion. These results shed light on the gating mechanism of the AChR and present a complementary inhibition mechanism by snake-venom-derived alpha-neurotoxins.

Tricyclic antidepressants inhibit homomeric Cys-loop receptors by acting at different conformational states

Tricyclic antidepressants not only inhibit monoamine reuptake but also modulate Cys-loop receptors. However, it is not understood how this modulation is involved in their therapeutic effects. We analyzed the mechanisms of inhibition of homomeric 5-HT 3A and α7-5HT 3A receptors by tricyclic antidepressants at the single-channel and macroscopic current levels. These drugs reduce agonist-evoked currents in a noncompetitive and concentration-dependent manner. When they act on the open state, the reduction is similar for both receptors and it is voltage-dependent, thus suggesting an open-channel block process in which the blocked channel can either close or remain stabilized. By acting on the resting state, tricyclic antidepressants reduce the peak current in a voltage-independent manner, with a potency 6-fold higher for 5-HT 3A than for α7-5HT 3A (IC 50: 6 μM and 1 μM for α7-5HT 3A and 5-HT 3A, respectively). Thus, tricyclic antidepressants may act on closed channels at the unshared extracellular domain from where they inhibit channel opening. Single α7-5HT 3A channels in the continued presence of tricyclic antidepressants show: i) reduced open durations, compatible with open-channel block; ii) reduced burst durations, compatible with closing of blocked channels; and iii) reduced frequency of opening events, compatible with both impaired opening and stabilization of a closed state. In summary, our study reveals that tricyclic antidepressants inhibit homomeric Cys-loop receptors by acting through different mechanisms at open and closed conformational states and probably at two different domains, namely, the pore in the open state and the extracellular domain in the closed state.

The Block of CFTR by Scorpion Venom is State-Dependent

Cystic fibrosis transmembrane conductance regulator (CFTR) adenosine triphosphate-dependent chloride channels are expressed in epithelial cells and are associated with a number of genetic disorders, including cystic fibrosis. Venom of the scorpion Leirus quinquestriatus hebraeus reversibly inhibits CFTR when applied to its cytoplasmic surface. To examine the state-dependence of inhibition we recorded wild-type and mutant CFTR channel currents using inside-out membrane patches from Xenopus oocytes. Application of either venom or diphenylamine-2-carboxylate to channels that were either activated (open) or resting (closed) indicate primarily closed state-dependent inhibition of CFTR by venom, whereas diphenylamine-2-carboxylate showed no state-dependence of block. Efficacy of venom-mediated macroscopic current inhibition was inversely related to channel activity. Analysis of single-channel and macropatch data indicated that venom could either inhibit channel opening, if it binds during an interburst closed state or in the absence of cytosolic adenosine triphosphate, or introduce new intraburst closed states, if it binds during an open event. The on-rate of venom binding for intraburst block could be modulated by changing CFTR activity with vanadate or adenylyl-imidodiphosphate, or by introducing the Walker A mutation K1250A. These findings represent the first description of state-dependent inhibition of CFTR and suggest that the active toxin could be used as a tool to study the conformational changes that occur during CFTR gating.

Inhibitory regulation of constitutive transient receptor potential-like cation channels in rabbit ear artery myocytes.

In the present study we have investigated an inhibitory pathway regulating a constitutively active Ca(2+)-permeable non-selective cation conductance (I(cat)) in rabbit ear artery smooth muscle cells. Constitutive single channel activity of I(cat) was recorded in cell-attached and inside-out patches with similar unitary conductance values. In inside-out patches with relatively high constitutive activity the G-protein activator GTPgammaS inhibited channel activity which was reversed by the protein kinase C (PKC) inhibitor chelerythrine indicating a G-protein pathway inhibits channel activity via PKC. Spontaneous channel activity was also suppressed by the G-protein inhibitor GDPbetaS suggesting a G-protein is also involved in initiation of constitutive channel activity. Bath application of antibodies to G(alphaq)/G(alpha11) enhanced channel activity whereas anti-G(alpha1-3)/G(alphao) antibodies decreased basal channel activity which suggests that G(alphaq)/G(alpha11) and G(alphaiota)/G(alphao) proteins initiate, respectively, the inhibitory and excitatory cascades. The phospholipase C (PLC) inhibitor U73122 increased spontaneous activity which implies a role for PLC in the inhibitory pathway. Bath application of the diacylycerol (DAG) analogue 1-oeoyl-2-acetyl-sn-glycerol (OAG) decreased the probability of channel opening (NP(o)) and this was reversed by chelerythrine. Application of the PKC activator phorbol 12, 13-dibutyrate (PDBu) and chelerythrine, respectively, decreased and increased NP(o). These data indicate that spontaneously active cation channels are inhibited by a tonic inhibitory pathway involving G(alphaq)/G(alpha11)-mediated stimulation of PLC to generate DAG which activates PKC to inhibit channel opening. There were some patches with relatively low NP(o) and it was evident that the inhibitory pathway was particularly marked in these cases. Moreover in the latter patches GTPgammaS and OAG caused marked increases in NP(o). Together with inhibitory effects of GDPbetaS and anti-G(alpha1-3)/G(alphao) antibodies the results suggest that there is constitutive G(alphai)/G(alphao) protein activity leading to channel opening via a DAG-mediated but PKC-independent mechanism. Finally, with whole-cell recording it is shown that noradrenaline increases I(cat) and the noradrenaline-evoked response is markedly potentiated by PKC inhibition. This latter observation shows that PKC also limits agonist-evoked I(cat) in these arterial myocytes.

Multimerization of the Ligand Binding Domains of Cyclic Nucleotide-Gated Channels

Cyclic nucleotide-gated (CNG) channels comprise four subunits and are activated by the direct binding of cyclic nucleotide to an intracellular domain on each subunit. This ligand binding domain is thought to contain a β roll followed by two α helices, designated the B and C helices. To examine the quaternary structure of CNG channels and how it changes during ion channel gating, we introduced single cysteines along the C helix of each subunit in an otherwise cysteineless channel. We found that cysteines on the C helices could form intersubunit disulfide bonds, even between diagonal subunits. Disulfide bond formation occurred primarily in closed channels and inhibited channel opening. These data suggest that the C helices from all four channel subunits are in close proximity in the closed state and move apart during channel opening.

Enhanced inhibition of a mutant neuronal nicotinic acetylcholine receptor by agonists: protection of function by (E)-N-methyl-4-(3-pyridinyl)-3-butene-1-amine (TC-2403).

Inhibition of neuronal nicotinic receptors can be regulated by sequence in the beta subunit second transmembrane domain (TM2). The incorporation of a beta4(6'F10'T) subunit, which contains sequence from the muscle beta subunit at the TM2 6' and 10' positions of the neuronal beta4 subunit, increases the loss of receptor responsiveness after the application of acetylcholine (ACh), nicotine, or 3-(2,4-dimethoxybenzylidene)-anabaseine (DMXB), an alpha7-selective partial agonist. Inhibition of receptor responsiveness following agonist exposure may occur through either an enhancement of desensitization, increased channel block by an agonist, or alternatively via allosteric modulation. Although DMXB produces very little activation of either alpha3beta4 or alpha3beta4(6'F10'T) receptors, DMXB shows an enhanced use-and voltage-dependent inhibition of alpha3beta4(6'F10'T) receptors compared with wild-type. In contrast, the alpha4beta2-selective agonist (E)-N-methyl-4-(3-pyridinyl)-3-butene-1-amine (TC-2403, previously identified as RJR-2403) shows increased activation of alpha3beta4(6'F10'T) receptors compared with alpha3beta4 receptors (as related to ACh activation) but with no significant increase in antagonist activity. The interaction between the binding of local anesthetics and the functional inhibition produced by these agonists was evaluated. The binding of the local anesthetics to their inhibitory sites does not affect inhibitory effects of DMXB and nicotine. However, TC-2403 can protect receptor function from the inhibitory effects of other agonists, suggesting that TC-2403, as well as agonists that cause inhibition, may be binding to an allosteric site, either promoting or inhibiting channel opening. The ability of TC-2403 to protect receptor function from agonist-induced inhibition may point toward valuable new combination drug therapies.