Phenylthiophenecarboxamide Antagonists of the Olfactory Receptor Co-Receptor Subunit from a Mosquito

Insects detect environmental chemicals using chemosensory receptors, such as the ORs, a family of odorant-gated ion channels. Insect ORs are multimeric complexes of unknown stoichiometry, formed by a common subunit (the odorant receptor co-receptor subunit, Orco) and one of many variable subunits that confer odorant specificity. The recent discovery of Orco directed ligands, including both agonists and antagonists, suggests Orco as a promising target for chemical control of insects. In addition to competitively inhibiting OR activation by Orco agonists, several Orco antagonists have been shown to act through a non-competitive mechanism to inhibit OR activation by odorants. We previously identified a series of Orco antagonists, including N-(4-ethylphenyl)-2-thiophenecarboxamide (OX1a, previously referred to as OLC20). Here, we explore the chemical space around the OX1a structure to identify more potent Orco antagonists. Cqui\Orco+Cqui\Or21, an OR from Culex quinquefasciatus (the Southern House Mosquito) that responds to 3-methylindole (skatole) and is thought to mediate oviposition behavior, was expressed in Xenopus oocytes and receptor function assayed by two-electrode voltage clamp electrophysiology. 22 structural analogs of OX1a were screened for antagonism of OR activation by an Orco agonist. By varying the moieties decorating the phenyl and thiophene rings, and altering the distance between the rings, we were able to identify antagonists with improved potency. Detailed examination of three of these compounds (N-mesityl-2-thiophenecarboxamide, N-(4-methylbenzyl)-2-thiophenecarboxamide and N-(2-ethylphenyl)-3-(2-thienyl)-2-propenamide) demonstrated competitive inhibition of receptor activation by an Orco agonist and non-competitive inhibition of receptor activation by an odorant. The ability to inhibit OR activation by odorants may be a general property of this class of Orco antagonist, suggesting that odorant mediated behaviors can be manipulated through Orco antagonism. The high conservation of Orco across insect species and previous demonstrations that various Orco ligands are active at ORs derived from several different insect orders suggests that Orco antagonists may have broad applicability.     

The effects of extracellular calmodulin on initiation of Hippeastrum rutilum pollen germination and tube growth

The effects of anti-calmodulin (CaM) serum, the CaM antagonist W7-agarose, the Ca²⁺ chelator ethyleneglycol-bis-(β-aminoethyl)-N,N,N′,N′-tetraacetic acid (EGTA) and exogenous pure CaM on pollen germination and tube growth of Hippeastrum rutilum Herb were studied. Pollen germination and tube growth were inhibited or completely stopped by anti-CaM serum in a dose-dependent manner, while the same amount of preimmune serum had no effect on either process. Pollen germination and tube growth were also inhibited or completely stopped by the CaM antagonist W7-agarose and the Ca²⁺ chelator EGTA. The addition of exogenous pure CaM enhanced pollen germination and tube growth, whereas the same amount of bovine serum albumin had no effect. The inhibitory effects caused by anti-CaM serum, W7-agarose and EGTA-washing could be reversed completely by the addition of exogenous pure CaM. These results indicate that extracellular CaM initiates pollen germination and tube growth, whereas exogenous CaM enhances the above processes, and may provide a novel view for understanding the control of pollen germination and tube growth. Received: 12 December 1996 / Accepted: 15 January 1997     

Insights into subunit interactions within the insect olfactory receptor complex using FRET

Insect olfactory receptors (ORs) are a novel family of ligand-gated cation channels that can respond to volatile organic compounds at low concentrations. They are involved in the detection of odorants associated with mate recognition, food localisation and predator avoidance. These receptors form a complex that is currently thought to contain at least two subunit members: the non-canonical Orco ion channel subunit and a ligand-binding receptor subunit. The integral membrane proteins SNMP1 and 2 are also associated with olfactory function, with SNMP1 required for cis-vaccinyl acetate reception in Drosophila melanogaster. In order to investigate protein–protein interactions among these membrane proteins we measured intermolecular Förster/Fluorescence Resonance Energy Transfer (FRET) in live insect cells by acceptor photobleaching. Fusion proteins containing Cyan Fluorescent Protein or Yellow Fluorescent Protein were produced using baculovirus-mediated expression in High Five? cells. The majority of the recombinant products were of the expected size for the fusion proteins and located within intracellular membranes. We were able to show FRET efficiencies providing evidence for homomeric and heteromeric interactions of the ligand-binding OR, Or22a, and Orco (Or22a–Or22a, Or22a–Orco, Orco–Orco). There was no evidence for an interaction between SNMP1 and Orco or between SNMP2 and Orco or Or22a. However, fusion proteins of SNMP1 and Or22a did show an interaction by FRET, suggesting SNMP1 may interact with at least some insect olfactory receptor complexes. In summary, this study supports previously observed homomeric and heteromeric interactions between Orco and the ligand-binding OR, Or22a, and identifies a novel interaction between Or22a and SNMP1.     

Evidence for calmodulin inter-domain compaction in solution induced by W-7 binding

Small-angle X-ray scattering and nuclear magnetic resonance were used to investigate the structural change of calcium-bound calmodulin (Ca²⁺/CaM) in solution upon binding to its antagonist, N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide (W-7). The radius of gyration was 17.4±0.3 Å for Ca²⁺/CaM-W-7 with a molar ratio of 1:5 and 20.3±0.7 Å for Ca²⁺/CaM. Comparison of the radius of gyration and the pair distance distribution function of the Ca²⁺/CaM-W-7 complex with those of other complexes indicates that binding of two W-7 molecules induces a globular shape for Ca²⁺/CaM, probably caused by an inter-domain compaction. The results suggest a tendency for Ca²⁺/CaM to form a globular structure in solution, which is inducible by a small compound like W-7.     

Inhibition of Anopheles gambiae Odorant Receptor Function by Mosquito Repellents

The identification of molecular targets of insect repellents has been a challenging task, with their effects on odorant receptors (ORs) remaining a debatable issue. Here, we describe a study on the effects of selected mosquito repellents, including the widely used repellent N,N-diethyl-meta-toluamide (DEET), on the function of specific ORs of the African malaria vector Anopheles gambiae. This study, which has been based on quantitative measurements of a Ca²⁺-activated photoprotein biosensor of recombinant OR function in an insect cell-based expression platform and a sequential compound addition protocol, revealed that heteromeric OR (ORx/Orco) function was susceptible to strong inhibition by all tested mosquito repellents except DEET. Moreover, our results demonstrated that the observed inhibition was due to efficient blocking of Orco (olfactory receptor coreceptor) function. This mechanism of repellent action, which is reported for the first time, is distinct from the mode of action of other characterized insect repellents including DEET.     

Role of calcium and calmodulin in the regulation of the rabbit ileal brush-border membrane Na+/H+ antiporter

Summary In rabbit ileum, Ca²⁺/calmodulin (CaM) appears to be involved in physiologically inhibiting the linked NaCl absorptive process, since inhibitors of Ca²⁺/CaM stimulate linked Na⁺ and Cl^– absorption. The role of Ca²⁺/CaM-dependent phosphorylation in regulation of the brush-border Na⁺/H⁺ antiporter, which is believed to be part of the neutral linked NaCl absorptive process, was studied using purified brush-border membrane vesicles, which contain both the Na⁺/H⁺ antiporter and Ca²⁺/CaM-dependent protein kinase(s) and its phosphoprotein substrates. Rabbit ileal villus cell brush-border membrane vesicles were prepared by Mg precipitation and depleted of ATP. Using a freezethaw technique, the ATP-depleted vesicles were loaded with Ca²⁺, CaM, ATP and an ATP-regenerating system consisting of creatine kinase and creatine phosphate. The combination of Ca²⁺/CaM and ATP inhibited Na⁺/H⁺ exchange by 45±13%. This effect was specific since Ca²⁺/CaM and ATP did not alter diffusive Na⁺ uptake, Na⁺-dependent glucose entry, or Na⁺ or glucose equilibrium volumes. The inhibition of the Na⁺/H⁺ exchanger by Ca²⁺/CaM/ATP was due to an effect on theV _max and not on theK _m for Na⁺. In the presence of CaM and ATP, Ca²⁺ caused a concentration-dependent inhibition of Na⁺ uptake, with an effect 50% of maximum occurring at 120nm. This Ca²⁺ concentration dependence was similar to the Ca²⁺ concentration dependence of Ca²⁺/CaM-dependent phosphorylation of specific proteins in the vesicles. The Ca²⁺/CaM/ATP-inhibition of Na⁺/H⁺ exchange was reversed by W₁₃, a Ca²⁺/CaM antagonist, but not by a hydrophobic control, W₁₂, or by H-7, a protein kinase C antagonist. we conclude that Ca²⁺, acting through CaM, regulates ileal brush-border Na⁺/H⁺ exchange, and that this may be involved in the regulation of neutral linked NaCl absorption.     

Calmodulin stimulation of unidirectional calcium uptake by the endoplasmic reticulum of barley aleurone

The role of calmodulin (CaM) in gibberellic acid (GA₃)-stimulated Ca²⁺ uptake was investigated in endomembranes isolated from aleurone cells of barley (Hordeum vulgare L.). Unidirectional Ca²⁺ -uptake activity of endoplasmic reticulum (ER) was higher in membranes isolated from aleurone layers treated for 16 h with GA₃ and Ca²⁺ compared with those isolated from layers incubated in Ca²⁺ alone. However, the level of uptake from Ca²⁺-treated tissue could be stimulated to that of the GA₃-treated cells by applying exogenous CaM which increased the V _max of the Ca²⁺ transporter approximately threefold. Calcium uptake in ER from GA₃-treated tissue was inhibited by the CaM antagonist W7 in 50% of experiments, whereas the activity in membranes from non-GA₃-treated tissue was unaffected. Treatment with GA₃ also led to a twofold increase in CaM levels in aleurone layers within 4–6 h, paralleling the time course of the stimulation of Ca²⁺ uptake and preceding the stimulation of α-amylase secretion. We propose that the elevation of Ca²⁺ uptake into the ER induced by GA₃ may be coordinated and regulated by elevated levels of membrane-associated CaM and this may regulate Ca²⁺-dependent α-amylase synthesis in the lumen of the ER.     

Pharmacological evidence for the implication of both cyclic GMP-dependent and -independent transduction pathways within auxin-induced stomatal opening in Commelina communis (L.)

It has been previously suggested that auxin-induced stomatal opening results from at least two transduction pathways, one of which involves cyclic GMP (cGMP) as the mediator within a Ca²⁺ signalling cascade. This hypothesis was investigated further in epidermal peels of Commelina communis by comparing the effects of potential inhibitors of plant Ca²⁺-dependent enzymes on the stomatal opening responses to the auxin indolyl-3-butyric acid (IBA) and to the cGMP membrane-permeable derivative 8-bromoguanosine 3′,5′-cyclic monophosphate (8-Br-cGMP). In the 30–50 μM range, the potential plant calmodulin (CaM) antagonist N-(aminohexyl)-5-chloro-1-naphthalenesulphonamide (W-7) positively interacted with IBA but not with 8-Br-cGMP to open the stomata. The CaM antagonists W-7 (in the 10–20 μM range) and N-(aminohexyl)-1-naphthalenesulphonamide (40 μM), the potential inhibitors of plant protein kinases 1-(5-isoquinolinylsulfonyl)-2-methylpiperazine (20 and 200 μM) and K-252a (0.6 μM), and cyclosporine A and FK506, potential inhibitors of plant homologs of Ca²⁺–CaM complex (Ca²⁺/CaM)-dependent protein phosphatase 2B, prevented the IBA and 8-Br-cGMP responses by about 70% and 100%, respectively. Together, these results provide indirect pharmacological evidence that, in addition to the cGMP-dependent pathway, the auxin signal is transduced through at least one cGMP-independent pathway.     

Calcium/Calmodulin Inhibits the Binding of Specific [125I]Omega-Conotoxin GVIA to Chick Brain Membranes

The effect of Ca²⁺/calmodulin (CaM) on the specific binding of [¹²⁵I]omega-conotoxin GVIA (¹²⁵I--CTX) to crude membranes from chick brain was investigated. When we examined the effects of the activation of various endogenous protein kinases on specific [¹²⁵I]-CTX binding to crude membranes, we observed that Ca²⁺/CaM had an inhibitory effect regardless of whether or not the standard medium contained ATP (0.5 mM). Ca²⁺/CaM also had an inhibitory effect in a simple binding-assay medium containing HEPES-HCl buffer, BSA, Ca²⁺ and CaM, and this effect was dependent on the concentration of Ca²⁺. The effect of Ca²⁺/CaM was attenuated by the CaM antagonists W-7 and CaM-kinase II fragment (290–309). An experiment with modified ELISA using purified anti -CTX antibody indicated that Ca²⁺/CaM did not affect the direct binding of [¹²⁵I]-CTX and CaM. These results suggest that Ca²⁺/CaM either directly or indirectly affects specific [¹²⁵I]-CTX binding sites, probably N-type Ca²⁺ channels in crude membranes from chick whole brain.     

Biochemical characterization of Ca2+/calmodulin dependent protein kinase from Candida albicans

A multifunctional Ca²⁺/calmodulin dependent protein kinase was purified approximately 650 fold from cytosolic extract of Candida albicans. The purified preparation gave a single band of 69 kDa on sodium dodecyl sulfate polyacrylamide gel electrophoresis with its native molecular mass of 71 kDa suggesting that the enzyme is monomeric. Its activity was dependent on calcium, calmodulin and ATP when measured at saturating histone IIs concentration. The purified Ca²⁺/CaMPK was found to be autophosphorylated at serine residue(s) in the presence of Ca²⁺/calmodulin and enzyme stimulation was strongly inhibited by W-7 (CaM antagonist) and KN-62 (Ca²⁺/CaM dependent PK inhibitor). These results confirm that the purified enzyme is Ca²⁺/CaM dependent protein kinase of Candida albicans. The enzyme phosphorylated a number of exogenous and endogenous substrates in a Ca²⁺/calmodulin dependent manner suggesting that the enzyme is a multifunctional Ca²⁺/calmodulin-dependent protein kinase of Candida albicans.     

Allosteric antagonism of insect odorant receptor ion channels

Background

At a molecular level, insects utilize members of several highly divergent and unrelated families of cell-surface chemosensory receptors for detection of volatile odorants. Most odors are detected via a family of odorant receptors (ORs), which form heteromeric complexes consisting of a well-conserved OR co-receptor (Orco) ion channel and a non-conserved tuning OR that provides coding specificity to each complex. Orco functions as a non-selective cation channel and is expressed in the majority of olfactory receptor neurons (ORNs). As the destructive behaviors of many insects are principally driven by olfaction, Orco represents a novel target for behavior-based control strategies. While many natural and synthetic odorants have been shown to agonize Orco/Or complexes, only a single direct Orco modulator, VUAA1, has been described. In an effort to identify additional Orco modulators, we have investigated the structure/activity relationships around VUAA1.

Results

A search of our compound library identified several VUAA1 analogs that were selected for evaluation against HEK cells expressing Orco from the malaria vector Anopheles gambiae (AgOrco). While the majority of compounds displayed no activity, many of these analogs possess no intrinsic efficacy, but instead, act as competitive VUAA1 antagonists. Using calcium mobilization assays, patch clamp electrophysiology, and single sensillum in vivo recording, we demonstrate that one such candidate, VU0183254, is a specific allosteric modulator of OR signaling, capable of broadly inhibiting odor-mediated OR complex activation.

Conclusions

We have described and characterized the first Orco antagonist, that is capable of non-competitively inhibiting odorant-evoked activation of OR complexes, thereby providing additional insight into the structure/function of this unique family of ligand-gated ion channels. While Orco antagonists are likely to have limited utility in insect control programs, they represent important pharmacological tools that will facilitate the investigation of the molecular mechanisms underlying insect olfactory signal transduction.     

Allosteric effects of the antipsychotic drug trifluoperazine on the energetics of calcium binding by calmodulin

Trifluoperazine (TFP; Stelazine?) is an antagonist of calmodulin (CaM), an essential regulator of calcium‐dependent signal transduction. Reports differ regarding whether, or where, TFP binds to apo CaM. Three crystallographic structures (1CTR, 1A29, and 1LIN) show TFP bound to (Ca²⁺)₄‐CaM in ratios of 1, 2, or 4 TFP per CaM. In all of these, CaM domains adopt the “open” conformation seen in CaM‐kinase complexes having increased calcium affinity. Most reports suggest TFP also increases calcium affinity of CaM. To compare TFP binding to apo CaM and (Ca²⁺)₄‐CaM and explore differential effects on the N‐ and C‐domains of CaM, stoichiometric TFP titrations of CaM were monitored by ¹⁵N‐HSQC NMR. Two TFP bound to apo CaM, whereas four bound to (Ca²⁺)₄‐CaM. In both cases, the preferred site was in the C‐domain. During the titrations, biphasic responses for some resonances suggested intersite interactions. TFP‐binding sites in apo CaM appeared distinct from those in (Ca²⁺)₄‐CaM. In equilibrium calcium titrations at defined ratios of TFP:CaM, TFP reduced calcium affinity at most levels tested; this is similar to the effect of many IQ‐motifs on CaM. However, at the highest level tested, TFP raised the calcium affinity of the N‐domain of CaM. A model of conformational switching is proposed to explain how TFP can exert opposing allosteric effects on calcium affinity by binding to different sites in the “closed,” “semi‐open,” and “open” domains of CaM. In physiological processes, apo CaM, as well as (Ca²⁺)₄‐CaM, needs to be considered a potential target of drug action. Proteins 2010. © 2010 Wiley‐Liss, Inc.     

Calcium/calmodulin-dependent inhibition of microtubule assembly by brain synaptic junction

The effect of synaptic junction (SJ) on microtubule assembly was examined. After preincubation with ATP at 37°C, rat SJ decreased the initial velocity and the extent of the porcine brain microtubule assembly (initiated by the addition of GTP) in a Ca²⁺/calmodulin (CaM)-dependent manner. The degree of the inhibition reached 35% of the control assembly (0-min preincubation) after 20-min preincubation with ATP. There was no inhibition either with heat-treated SJ, at 0°C, or in the presence of EGTA or W-7 (CaM antagonist). The inhibition was due neither to protease(s) nor CaM contaminating the preparations. Free Ca²⁺ concentration level required for the inhibition of microtubule assembly was 10^–6 M. Phosphorylation of microtubule proteins was inhibited by SJ in a Ca²⁺/CaM-dependent manner, and the inhibition occurred in a physiological increase range of intracellular Ca²⁺ concentration (10^–6M) The heat-treated SJ caused no inhibition. The result suggested that the microtubule assembly in the postsynaptic region was regulated by a Ca²⁺/CaM-dependent protein kinase associated with SJ; i. e., major postsynaptic density protein.Abbreviations used CaM calmodulin - DTT dithiothreitol - MAPs microtubule-associated proteins - MES 2-(N-morphorino)ethanesulfonic acid - mPSDp major postsynaptic density protein - PSD postsynaptic density - SDS PAGE sodium dodecyl sulfate polyacrylamide gel electrophoresis - W-7 N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide hydrochloride     

Mechanisms of Calmodulin Regulation of Different Isoforms of Kv7.4 K+ Channels

Calmodulin (CaM), a Ca²⁺-sensing protein, is constitutively bound to IQ domains of the C termini of human Kv7 (hKv7, KCNQ) channels to mediate Ca²⁺-dependent reduction of Kv7 currents. However, the mechanism remains unclear. We report that CaM binds to two isoforms of the hKv7.4 channel in a Ca²⁺-independent manner but that only the long isoform (hKv7.4a) is regulated by Ca²⁺/CaM. Ca²⁺/CaM mediate reduction of the hKv7.4a channel by decreasing the channel open probability and altering activation kinetics. We took advantage of a known missense mutation (G321S) that has been linked to progressive hearing loss to further examine the inhibitory effects of Ca²⁺/CaM on the Kv7.4 channel. Using multidisciplinary techniques, we demonstrate that the G321S mutation may destabilize CaM binding, leading to a decrease in the inhibitory effects of Ca²⁺ on the channels. Our study utilizes an expression system to dissect the biophysical properties of the WT and mutant Kv7.4 channels. This report provides mechanistic insights into the critical roles of Ca²⁺/CaM regulation of the Kv7.4 channel under physiological and pathological conditions.     

Regulation of ryanodine receptors by sphingosylphosphorylcholine: Involvement of both calmodulin-dependent and -independent mechanisms

Sphingosylphosphorylcholine (SPC), a lipid mediator with putative second messenger functions, has been reported to regulate ryanodine receptors (RyRs), Ca²⁺ channels of the sarco/endoplasmic reticulum. RyRs are also regulated by the ubiquitous Ca²⁺ sensor calmodulin (CaM), and we have previously shown that SPC disrupts the complex of CaM and the peptide corresponding to the CaM-binding domain of the skeletal muscle Ca²⁺ release channel (RyR1). Here we report that SPC also displaces Ca²⁺-bound CaM from the intact RyR1, which we hypothesized might lead to channel activation by relieving the negative feedback Ca²⁺CaM exerts on the channel. We could not demonstrate such channel activation as we have found that SPC has a direct, CaM-independent inhibitory effect on channel activity, confirmed by both single channel measurements and [³H]ryanodine binding assays. In the presence of Ca²⁺CaM, however, the addition of SPC did not reduce [³H]ryanodine binding, which we could explain by assuming that the direct inhibitory action of the sphingolipid was negated by the simultaneous displacement of inhibitory Ca²⁺CaM. Additional experiments revealed that RyRs are unlikely to be responsible for SPC-elicited Ca²⁺ release from brain microsomes, and that SPC does not exert detergent-like effects on sarcoplasmic reticulum vesicles. We conclude that regulation of RyRs by SPC involves both CaM-dependent and -independent mechanisms, thus, the sphingolipid might play a physiological role in RyR regulation, but channel activation previously attributed to SPC is unlikely.     

Activity of the yeast vacuolar TRP channel TRPY1 is inhibited by Ca2+–calmodulin binding

Transient receptor potential (TRP) cation channels, which are conserved across mammals, flies, fish, sea squirts, worms, and fungi, essentially contribute to cellular Ca²⁺ signaling. The activity of the unique TRP channel in yeast, TRP yeast channel 1 (TRPY1), relies on the vacuolar and cytoplasmic Ca²⁺ concentration. However, the mechanism(s) of Ca²⁺-dependent regulation of TRPY1 and possible contribution(s) of Ca²⁺-binding proteins are yet not well understood. Our results demonstrate a Ca²⁺-dependent binding of yeast calmodulin (CaM) to TRPY1. TRPY1 activity was increased in the cmd1–6 yeast strain, carrying a non–Ca²⁺-binding CaM mutant, compared with the parent strain expressing wt CaM (Cmd1). Expression of Cmd1 in cmd1–6 yeast rescued the wt phenotype. In addition, in human embryonic kidney 293 cells, hypertonic shock-induced TRPY1-dependent Ca²⁺ influx and Ca²⁺ release were increased by the CaM antagonist ophiobolin A. We found that coexpression of mammalian CaM impeded the activity of TRPY1 by reinforcing effects of endogenous CaM. Finally, inhibition of TRPY1 by Ca²⁺–CaM required the cytoplasmic amino acid stretch E₃₃–Y₉₂. In summary, our results show that TRPY1 is under inhibitory control of Ca²⁺–CaM and that mammalian CaM can replace yeast CaM for this inhibition. These findings add TRPY1 to the innumerable cellular proteins, which include a variety of ion channels, that use CaM as a constitutive or dissociable Ca²⁺-sensing subunit, and contribute to a better understanding of the modulatory mechanisms of Ca²⁺–CaM.     

Caffeine-induced Release of Intracellular Ca2+ from Chinese Hamster Ovary Cells Expressing Skeletal Muscle Ryanodine Receptor : Effects on Full-Length and Carboxyl-Terminal Portion of Ca2+Release Channels

The ryanodine receptor (RyR)/Ca²⁺ release channel is an essential component of excitation–contraction coupling in striated muscle cells. To study the function and regulation of the Ca²⁺ release channel, we tested the effect of caffeine on the full-length and carboxyl-terminal portion of skeletal muscle RyR expressed in a Chinese hamster ovary (CHO) cell line. Caffeine induced openings of the full length RyR channels in a concentration-dependent manner, but it had no effect on the carboxyl-terminal RyR channels. CHO cells expressing the carboxyl-terminal RyR proteins displayed spontaneous changes of intracellular [Ca²⁺]. Unlike the native RyR channels in muscle cells, which display localized Ca²⁺ release events (i.e., “Ca²⁺ sparks” in cardiac muscle and “local release events” in skeletal muscle), CHO cells expressing the full length RyR proteins did not exhibit detectable spontaneous or caffeine-induced local Ca²⁺ release events. Our data suggest that the binding site for caffeine is likely to reside within the amino-terminal portion of RyR, and the localized Ca²⁺ release events observed in muscle cells may involve gating of a group of Ca²⁺ release channels and/or interaction of RyR with muscle-specific proteins.     

An inhibitor of Na/Ca exchange blocks activation of insect olfactory receptors

Earlier we showed that the Na⁺/Ca²⁺ exchanger inhibitor, KB-R7943, potently blocks the odor-evoked activity of lobster olfactory receptor neurons. Here we extend that finding to recombinant mosquito olfactory receptors stably expressed in HEK cells. Using whole-cell and outside-out patch clamping and calcium imaging, we demonstrate that KB-R7943 blocks both the odorant-gated current and the odorant-evoked calcium signal from two different OR complexes from the malaria vector mosquito, Anopheles gambiae, AgOr48 + AgOrco and AgOr65 + AgOrco. Both heteromeric and homomeric (Orco alone) OR complexes were susceptible to KB-R7943 blockade when activated by VUAA1, an agonist that targets the Orco channel subunit, suggesting the Orco subunit may be the target of the drug’s action. KB-R7943 represents a valuable tool to further investigate the functional properties of arthropod olfactory receptors and raises the interesting specter that activation of these ionotropic receptors is directly or indirectly linked to a Na⁺/Ca²⁺ exchanger, thereby providing a template for drug design potentially allowing improved control of insect pests and disease vectors.     

No Evidence for Ionotropic Pheromone Transduction in the Hawkmoth Manduca sexta

Insect odorant receptors (ORs) are 7-transmembrane receptors with inverse membrane topology. They associate with the conserved ion channel Orco. As chaperon, Orco maintains ORs in cilia and, as pacemaker channel, Orco controls spontaneous activity in olfactory receptor neurons. Odorant binding to ORs opens OR-Orco receptor ion channel complexes in heterologous expression systems. It is unknown, whether this also occurs in vivo. As an alternative to this ionotropic transduction, experimental evidence is accumulating for metabotropic odor transduction, implicating that insect ORs couple to G-proteins. Resulting second messengers gate various ion channels. They generate the sensillum potential that elicits phasic-tonic action potentials (APs) followed by late, long-lasting pheromone responses. Because it is still unclear how and when Orco opens after odor-OR-binding, we used tip recordings to examine in vivo the effects of the Orco antagonist OLC15 and the amilorides MIA and HMA on bombykal transduction in the hawkmoth Manduca sexta. In contrast to OLC15 both amilorides decreased the pheromone-dependent sensillum potential amplitude and the frequency of the phasic AP response. Instead, OLC15 decreased spontaneous activity, increased latencies of phasic-, and decreased frequencies of late, long-lasting pheromone responses Zeitgebertime-dependently. Our results suggest no involvement for Orco in the primary transduction events, in contrast to amiloride-sensitive channels. Instead of an odor-gated ionotropic receptor, Orco rather acts as a voltage- and apparently second messenger-gated pacemaker channel controlling the membrane potential and hence threshold and kinetics of the pheromone response.     

Calmodulin Mediates the Ca-Dependent Regulation of Cx44 Gap Junctions

We have shown previously that the Ca²⁺-dependent inhibition of lens epithelial cell-to-cell communication is mediated in part by the direct association of calmodulin (CaM) with connexin43 (Cx43), the major connexin in these cells. We now show that elevation of [Ca²⁺]_i in HeLa cells transfected with the lens fiber cell gap junction protein sheep Cx44 also results in the inhibition of cell-to-cell dye transfer. A peptide comprising the putative CaM binding domain (aa 129-150) of the intracellular loop region of this connexin exhibited a high affinity, stoichiometric interaction with Ca²⁺-CaM. NMR studies indicate that the binding of Cx44 peptide to CaM reflects a classical embracing mode of interaction. The interaction is an exothermic event that is both enthalpically and entropically driven in which electrostatic interactions play an important role. The binding of the Cx44 peptide to CaM increases the CaM intradomain cooperativity and enhances the Ca²⁺-binding affinities of the C-domain of CaM more than twofold by slowing the rate of Ca²⁺ release from the complex. Our data suggest a common mechanism by which the Ca²⁺-dependent inhibition of the α-class of gap junction proteins is mediated by the direct association of an intracellular loop region of these proteins with Ca²⁺-CaM.