Sequence and analysis of the genome of a baculovirus pathogenic for Lymantria dispar

Cyclic nucleotide-gated channels are composed of a core transmembrane domain, structurally homologous to the voltage-gated K+ channels, and a cytoplasmic ligand-binding domain. These two modules are joined by approximately 90 conserved amino acids, the C-linker, whose precise role in the mechanism of channel activation by cyclic nucleotides is poorly understood. We examined cyclic nucleotide-gated channels from bovine photoreceptors and Caenorhabditis elegans sensory neurons that show marked differences in cyclic nucleotide efficacy and sensitivity. By constructing chimeras from these two channels, we identified a region of 30 amino acids in the C-linker (the L2 region) as an important determinant of activation properties. An increase in both the efficacy of gating and apparent affinity for cGMP and cAMP can be conferred onto the photoreceptor channel by the replacement of its L2 region with that of the C. elegans channel. Three residues within this region largely account for this effect. Despite the profound effect of the C-linker region on ligand gating, the identity of the C-linker does not affect the spontaneous, ligand-independent open probability. Based on a cyclic allosteric model of activation, we propose that the C-linker couples the opening reaction in the transmembrane core region to the enhancement of the affinity of the open channel for agonist, which underlies ligand gating.     

Three residues predicted by molecular modeling to interact with the purine moiety alter ligand binding and channel gating in cyclic nucleotide-gated channels

Cytoplasmic cAMP and cGMP are soluble cellular messengers that directly activate cyclic nucleotide-gated (CNG) channels. These channels mediate sensory transduction in photoreceptors and olfactory neurons. The closely related CNG channels in these cell types have different nucleotide activation profiles, and we have investigated the molecular basis of their nucleotide selectivity properties. Previously, we predicted that the purine moiety of the nucleotide interacts with residues F533, K596, and D604 (bovine rod alpha CNG channel subunit sequences) of the nucleotide binding domain. In this study, we replaced these three residues with the corresponding residues of the bovine olfactory CNG channel. Mutations at each position altered the nucleotide activation of the rod CNG channels. In a mutant where K596 was replaced with arginine, cAMP-activated currents were enhanced 8-12-fold, suggesting that residue 596 influences channel gating. Thermodynamic cycle analysis of the data showed that (1) the residues are energetically coupled and (2) energetic coupling exists between the potentiating effects of Ni2+ and the replacement of F533 with tyrosine. These data suggest that changes in one of the residues alter the purine contacts with the other residues and that F533 communicates with the C-linker region of the channel involved in Ni2+ potentiation.     

An isoform of the rod photoreceptor cyclic nucleotide-gated channel beta subunit expressed in olfactory neurons

Sensory transduction in olfactory neurons involves the activation of a cyclic nucleotide-gated (CNG) channel by cAMP. Previous studies identified a CNG channel alpha subunit (CNG2) and a beta subunit (CNG5), which when heterologously expressed form a channel with properties similar but not identical to those of native olfactory neurons. We have cloned a new type of CNG channel beta subunit (CNG4. 3) from rat olfactory epithelium. CNG4.3 derives from the same gene as the rod photoreceptor beta subunit (CNG4.1) but lacks the long, glutamic acid-rich domain found in the N terminus of CNG4.1. Northern blot and in situ hybridization revealed that CNG4.3 is expressed specifically in olfactory neurons. Expression of CNG4.3 in human embryonic kidney 293 cells did not lead to detectable currents. Coexpression of CNG4.3 with CNG2 induced a current with significantly increased sensitivity for cAMP whereas cGMP affinity was not altered. Additionally, CNG4.3 weakened the outward rectification of the current in the presence of extracellular Ca2+, decreased the relative permeability for Ca2+, and enhanced the sensitivity for L-cis diltiazem. Upon coexpression of CNG2, CNG4.3, and CNG5, a conductance with a cAMP sensitivity greater than that of either the CNG2/CNG4.3 or the CNG2/CNG5 channel and near that of native olfactory channel was observed. Our data suggest that CNG4.3 forms a subunit of the native olfactory CNG channel. The expression of various CNG4 isoforms in retina and olfactory epithelium indicates that the CNG4 subunit may be necessary for normal function of both photoreceptor and olfactory CNG channels.     

Subunit interactions in the activation of cyclic nucleotide-gated ion channels

Cyclic nucleotide-gated (CNG) ion channels of retinal photoreceptors and olfactory neurons are multimeric proteins of unknown stoichiometry. To investigate the subunit interactions that occur during CNG channel activation, we have used tandem cDNA constructs of the rod CNG channel to generate heteromultimeric channels composed of wild-type and mutant subunits. We introduced point mutations that affect channel activation: 1) D604M, which alters the relative ability of agonists to promote the allosteric conformational change(s) associated with channel opening, and 2) T560A, which primarily affects the initial binding affinity for cGMP, and to a lesser extent, the allosteric transition. At saturating concentrations of agonist, heteromultimeric channels were intermediate between wild-type and mutant homomultimers in agonist efficacy and apparent affinity for cGMP, cIMP, and cAMP, consistent with a model for the allosteric transition involving a concerted conformational change in all of the channel subunits. Results were also consistent with a model involving independent transitions in two or three, but not one or four, of the channel subunits. The behavior of the heterodimers implies that the channel stoichiometry is some multiple of 2 and is consistent with a tetrameric quaternary structure for the functional channel complex. Steady-state dose-response relations for homomultimeric and heteromultimeric channels were well fit by a Monod, Wyman, and Changeux model with a concerted allosteric opening transition stabilized by binding of agonist.     

Molecular and pharmacological analysis of cyclic nucleotide-gated channel function in the central nervous system

Most functional studies of cyclic nucleotide-gated (CNG) channels have been confined to photoreceptors and olfactory epithelium, in which CNG channels are abundant and easy to study. The widespread distribution of CNG channels in tissues throughout the body has only recently been recognized and the functions of this channel family in many of these tissues remain largely unknown. The molecular biological and pharmacological properties of the CNG channel family are summarized in order to put in context studies aimed at probing CNG channel functions in these tissues using pharmacological and genetic methods. Compounds have now been identified that are useful in distinguishing CNG channel activated pathways from cAMP/cGMP dependent-protein kinases or other pathways. The ways in which these interact with CNG channels are understood and this knowledge is leading to the identification of more potent and more specific CNG channel subtype-specific agonists or antagonists. Recent molecular and genetic analyses have identified novel roles of CNG channels in neuronal development and plasticity in both invertebrates and vertebrates. Targeting CNG channels via specific drugs and genetic manipulation (such as knockout mice) will permit better understanding of the role of CNG channels in both basic and higher orders of brain function.     

Movement of gating machinery during the activation of rod cyclic nucleotide-gated channels

In the visual and olfactory systems, cyclic nucleotide-gated (CNG) ion channels convert stimulus-induced changes in the internal concentrations of cGMP and cAMP into changes in membrane potential. Although it is known that significant activation of these channels requires the binding of three or more molecules of ligand, the detailed molecular mechanism remains obscure. We have probed the structural changes that occur during channel activation by using sulfhydryl-reactive methanethiosulfonate (MTS) reagents and N-ethylmaleimide (NEM). When expressed in Xenopus oocytes, the alpha-subunit of the bovine retinal channel forms homomultimeric channels that are activated by cGMP with a K1/2 of approximately 100 microM. Cyclic AMP, on the other hand, is a very poor activator; a saturating concentration elicits only 1% of the maximum current produced by cGMP. Treatment of excised patches with MTS-ethyltrimethylamine (MTSET) or NEM dramatically potentiated the channel's response to both cyclic nucleotides. After MTSET treatment, the dose-response relation for cGMP was shifted by over two orders of magnitude to lower concentrations. The effect on channel activation by cAMP was even more striking. After modification, the channels were fully activated by cAMP with a K1/2 of approximately 60 microM. This potentiation was abolished by conversion of Cys481 to a nonreactive alanine residue. Potentiation occurred more rapidly in the presence of saturating cGMP, indicating that this region of the channel is more accessible when the channel is open. Cys481 is located in a linker region between the transmembrane and cGMP-binding domains of the channel. These results suggest that this region of the channel undergoes significant movement during the activation process and is critical for coupling ligand binding to pore opening. Potentiation, however, is not mediated by the recently reported interaction between the amino- and carboxy-terminal regions of the alpha-subunit. Deletion of the entire amino-terminal domain had little effect on potentiation by MTSET.     

Cloning and characterization of an olfactory cyclic nucleotide-gated channel expressed in mouse heart

Regulation of ionic currents in the heart is partly achieved by signaling cascades which alter intracellular levels of cyclic nucleotides. Changes in cyclic nucleotide levels can regulate channels either directly, like the direct binding of cAMP to the i(f) channel in pacemaker tissues, or indirectly through phosphorylation of channels by cAMP-dependent, or cGMP-dependent protein kinases. These types of regulation generally alter the voltage sensitivities of channels. A class of voltage-insensitive channels, first discovered in retinal rods and olfactory neurons, were recently identified in the heart. These channels are opened by the direct binding of cyclic nucleotides, providing a means of regulating ionic currents outside the influence of membrane voltage. Since different isoforms have different affinities for cAMP and cGMP, it is important to determine which isoforms are expressed in heart in order to predict their roles in heart function. We have cloned the olfactory channel from mouse heart, and find that although the message is very rare, Western blot analysis indicates the olfactory channel protein is stable in heart sarcolemma. Our data also suggest the olfactory channel protein forms homomeric channels in the heart since other isoforms or splice variants were not detected either by PCR amplification or by RNase protection. In addition, we have isolated and sequenced the mouse olfactory cyclic nucleotide-gated channel gene, and show the genomic organization is remarkably similar to that found in the human retinal channel gene. Part of this work was presented in abstract form.     

Normal standards for cardiopulmonary responses to exercise using a cycle ergometer test

f-channel nucleotide modulation was investigated in sino-atrial (SA) node cells isolated from rabbit hearts, using an inside-out macropatch configuration. Saturating doses (30 microM) of phosphorothioate derivatives of cAMP, Sp-cAMPS and Rp-cAMPS, were tested on the cAMP-induced shift of I(f) activation. Responses were not altered when Sp-cAMPS was combined with cAMP. When Rp-cAMPS was superfused with subsaturating cAMP concentrations (1-10 microM), it inhibited cAMP-induced I(f) activation shift. cGMP and cIMP reversibly shifted the I(f) conductance-voltage curve to more positive values; however they had a lesser specificity than that of cAMP. The efficacy ranking for I(f) activation by cyclic nucleotides was: cAMP > cGMP > cIMP. Non cyclic nucleotides (ATP, ADP and AMP) failed to change I(f) activation, indicating that the cyclic nature of nucleotides seems to be essential to f-channel modulation. Similarities between f-channels and cyclic nucleotide-gated (CNG) channels are discussed.     

Tetracaine reports a conformational change in the pore of cyclic nucleotide-gated channels

Local anesthetics are a diverse group of clinically useful compounds that act as pore blockers of both voltage- and cyclic nucleotide-gated (CNG) ion channels. We used the local anesthetic tetracaine to probe the nature of the conformational change that occurs in the pore of CNG channels during the opening allosteric transition. When applied to the intracellular side of wild-type rod CNG channels expressed in Xenopus oocytes from the alpha subunit, the local anesthetic tetracaine exhibits state-dependent block, binding with much higher affinity to closed states than to open states. Here we show that neutralization of a glutamic acid in the conserved P region (E363G) eliminated this state dependence of tetracaine block. Tetracaine blocked E363G channels with the same effectiveness at high concentrations of cGMP, when the channel spent more time open, and at low concentrations of cGMP, when the channel spent more time closed. In addition, Ni2+, which promotes the opening allosteric transition, decreased the effectiveness of tetracaine block of wild-type but not E363G channels. Similar results were obtained in a chimeric CNG channel that exhibits a more favorable opening allosteric transition. These results suggest that E363 is accessible to internal tetracaine in the closed but not the open configuration of the pore and that the conformational change that accompanies channel opening includes a change in the conformation or accessibility of E363.     

Ca2+ permeation in cyclic nucleotide-gated channels

Cyclic nucleotide-gated (CNG) channels conduct Na+, K+ and Ca2+ currents under the control of cGMP and cAMP. Activation of CNG channels leads to depolarization of the membrane voltage and to a concomitant increase of the cytosolic Ca2+ concentration. Several polypeptides were identified that constitute principal and modulatory subunits of CNG channels in both neurons and non-excitable cells, co-assembling to form a variety of heteromeric proteins with distinct biophysical properties. Since the contribution of each channel type to Ca2+ signaling depends on its specific Ca2+ conductance, it is necessary to analyze Ca2+ permeation for each individual channel type. We have analyzed Ca2+ permeation in all principal subunits of vertebrates and for a principal subunit from Drosophila melanogaster. We measured the fractional Ca2+ current over the physiological range of Ca2+ concentrations and found that Ca2+ permeation is determined by subunit composition and modulated by membrane voltage and extracellular pH. Ca2+ permeation is controlled by the Ca2+-binding affinity of the intrapore cation-binding site, which varies profoundly between members of the CNG channel family, and gives rise to a surprising diversity in the ability to generate Ca2+ signals.     

Molecular determinants of a Ca2+-binding site in the pore of cyclic nucleotide-gated channels: S5/S6 segments control affinity of intrapore glutamates

Cyclic nucleotide-gated (CNG) channels play an important role in Ca2+ signaling in many cells. CNG channels from various tissues differ profoundly in their Ca2+ permeation properties. Using the voltage-dependent Ca2+ blockage of monovalent current in wild-type channels, chimeric constructs and point mutants, we have identified structural elements that determine the distinctively different interaction of Ca2+ with CNG channels from rod and cone photoreceptors and olfactory neurons. Segments S5 and S6 and the extracellular linkers flanking the pore region are the only structural elements that account for the differences between channels. Ca2+ blockage is strongly modulated by external pH. The different pH dependence of blockage suggests that the pKa of intrapore glutamates and their protonation pattern differ among channels. The results support the hypothesis that the S5-pore-S6 module, by providing a characteristic electrostatic environment, determines the protonation state of pore glutamates and thereby controls Ca2+ affinity and permeation in each channel type.     

Predicted ligand interactions of 3'5'-cyclic nucleotide-gated channel binding sites: comparison of retina and olfactory binding site models

Cyclic nucleotide-gated channels (CNGC) open in response to the binding of 3'5'-cyclic nucleotides. Members of the CNGC family vary as much as 100-fold in their ability to respond to cAMP and cGMP. Molecular models of the nucleotide binding domains of the bovine retina and catfish and rat olfactory CNGCs were built from the crystal structure of cAMP bound to catabolite gene activator protein (CAP) with AMMP, a program for molecular mechanics and dynamics. The nucleotide conformation can be predicted from the number of strong and weak interactions between the purine ring and the binding site. The amino acids predicted to be important for determining the nucleotide affinity and specificity are residues 61, 83 (mediated through a water molecule), 119 and 127 (CAP sequence numbers) which interact with the purine ring. These residues also dictate the conformation of the ligand in the binding pocket. cGMP is preferentially bound in the syn conformation in bovine retina, bovine olfactory and rat olfactory CNGCs due to Thr83, while either conformation can bind in catfish olfactory CNGC. cAMP is predicted to bind either in syn or anti conformation, depending on the interaction with residue 119: the anti conformation is preferentially bound in olfactory CNGCs.     

Evidence for distinct signaling mechanisms in two mammalian olfactory sense organs

In mammals, olfactory stimuli are detected by sensory neurons at two distinct sites: the olfactory epithelium (OE) of the nasal cavity and the neuroepithelium of the vomeronasal organ (VNO). While the OE can detect volatile chemicals released from numerous sources, the VNO appears to be specialized to detect pheromones that are emitted by other animals and that convey information of behavioral or physiological importance. The mechanisms underlying sensory transduction in the OE have been well studied and a number of components of the transduction cascade have been cloned. Here, we investigated sensory transduction in the VNO by asking whether VNO neurons express molecules that have been implicated in sensory transduction in the OE. Using in situ hybridization and Northern blot analyses, we found that most of the olfactory transduction components examined, including the guanine nucleotide binding protein alpha subunit (G-alpha-olf), adenylyl cyclase type III, and an olfactory cyclic nucleotide-gated (CNG) channel subunit (oCNC1), are not expressed by VNO sensory neurons. In contrast, VNO neurons do express a second olfactory CNG channel subunit (oCNC2). These results indicate that VNO sensory transduction is distinct from that in the OE but raise the possibility that, like OE sensory transduction, sensory transduction in the VNO might involve cyclic nucleotide-gated ion channels.     

Intestinal bleeding associated with systemic amyloidosis

Total colourblindness (OMIM 216900), also referred to as rod monochromacy (RM) or complete achromatopsia, is a rare, autosomal recessive inherited and congenital disorder characterized by photophobia, reduced visual acuity, nystagmus and the complete inability to discriminate between colours. Electroretinographic recordings show that in RM, rod photoreceptor function is normal, whereas cone photoreceptor responses are absent. The locus for RM has been mapped to chromosome 2q11 (ref. 2), however the gene underlying RM has not yet been identified. Recently, a suitable candidate gene, CNGA3, encoding the alpha-subunit of the cone photoreceptor cGMP-gated cation channel, a key component of the phototransduction pathway, has been cloned and assigned to human chromosome 2q11 (refs 3,4). We report the identification of missense mutations in CNGA3 in five families with RM. Homozygous mutations are present in two families, whereas the remaining families show compound heterozygous mutations. In all cases, the segregation pattern of the mutations is consistent with the autosomal recessive inheritance of the disease and all mutations affect amino acids that are highly conserved among cyclic nucleotide gated channels (CNG) in various species. This is the first report of a colour vision disorder caused by defects other than mutations in the cone pigment genes, and implies at least in this instance a common genetic basis for phototransduction in the three different cone photoreceptors of the human retina.     

Modulation of rod photoreceptor cyclic nucleotide-gated channels by tyrosine phosphorylation

Cyclic nucleotide-gated (CNG) channels in vertebrate photoreceptors are crucial for transducing light-induced changes in cGMP concentration into electrical signals. In this study, we show that both native and exogenously expressed CNG channels from rods are modulated by tyrosine phosphorylation. The cGMP sensitivity of CNG channels, composed of rod alpha-subunits expressed in Xenopus oocytes, gradually increases after excision of inside-out patches from the oocyte membrane. This increase in sensitivity is inhibited by a protein tyrosine phosphatase (PTP) inhibitor and is unaffected by three different Ser/Thr phosphatase inhibitors. Moreover, it is suppressed or reversed by application of ATP but not by a nonhydrolyzable ATP analog. Application of protein tyrosine kinase (PTK) inhibitors causes an increase in cGMP sensitivity, but only in the presence of ATP. Taken together, these results suggest that CNG channels expressed in oocytes are associated with active PTK(s) and PTP(s) that regulate their cGMP sensitivity by changing phosphorylation state. The cGMP sensitivity of native CNG channels from salamander rod outer segments also increases and decreases after incubation with inhibitors of PTP(s) and PTK(s), respectively. These results suggest that rod CNG channels are modulated by tyrosine phosphorylation, which may function as a novel mechanism for regulating the sensitivity of rods to light.     

Mutations in a cyclic nucleotide-gated channel lead to abnormal thermosensation and chemosensation in C. elegans

The C. elegans tax-4 mutants are abnormal in multiple sensory behaviors: they fail to respond to temperature or to water-soluble or volatile chemical attractants. We show that the predicted tax-4 gene product is highly homologous to vertebrate cyclic nucleotide-gated channels. Tax-4 protein expressed in cultured cells functions as a cyclic nucleotide-gated channel. The green fluorescent protein (GFP)-tagged functional Tax-4 protein is expressed in thermosensory, gustatory, and olfactory neurons mediating all the sensory behaviors affected by the tax-4 mutations. The Tax-4::GFP fusion is partly localized at the sensory endings of these neurons. The results suggest that a cyclic nucleotide-gated channel is required for thermosensation and chemosensation and that cGMP is an important intracellular messenger in C. elegans sensory transduction.     

Identification of a high-affinity binding protein for N-acetylchitooligosaccharide elicitor in the plasma membrane of suspension-cultured rice cells by affinity labeling

The carboxyl-terminal 19 amino acids of the type I alpha regulatory subunit (RI alpha) of cAMP-dependent protein kinase (PKA) were investigated to determine their contributions to cAMP selectivity. The parent RI alpha subunit contained an Ala to Thr mutation at position 334 so that it would bind both cAMP and cGMP with high affinity. Stop codons were introduced into the parent cDNA construct at positions corresponding to Val-375, Asn-372, Gln-370, and Cys-360. The purified, bacterially expressed proteins were characterized for their cAMP and cGMP dissociation properties. Site-selective cAMP analogs were used to compete against [3H]cAMP binding to the mutant RI alpha subunits to correctly assign fast and slow dissociation t1/2 values to the A and B domains. A greater than 60-fold drop in B domain t1/2 in the Asn-372-stop to Gln-370-stop transition implicated Tyr-371 as an important cAMP-binding determinant. A similar drop in [3H]cGMP t1/2 for the same transition suggested that the cGMP/cAMP selectivity was not altered. To test this further, Tyr-371 was mutated to Ala, Phe, and Arg in the parent construct. The cAMP and cGMP t1/2 values were determined, as were protein kinase activation constants (Ka) for holoenzymes formed from mutant RI alpha subunits and purified catalytic subunit. The Ka data suggested that mutation of Tyr-371 enhanced B domain cAMP selectivity. Isolated B domains containing Tyr-371-Arg or Tyr-371-Phe mutations were constructed, expressed, and purified to determine their relative inhibition constants (K'I) for cGMP vs cAMP. These data showed that B domain cAMP selectivity was minimally affected by alteration of Tyr-371. Based on these results, it is concluded that aromatic stacking is not important for determining B-domain cyclic nucleotide selectivity. It is proposed that the main function of Tyr-371 is stabilization of the B-domain cAMP-binding pocket through hydrogen bonding with Glu-324.     

Drugs affecting phospholipase C-mediated signal transduction block the olfactory cyclic nucleotide-gated current of adult zebrafish

Amino acid and bile salt odorants are detected by zebrafish with relatively independent odorant receptors, but the transduction cascade(s) subsequently activated by these odorants remains unknown. Electro-olfactogram recording methods were used to determine the effects of two drugs, reported to affect phospholipase C (PLC)/inositol tripohsphate (IP3)-mediated olfactory transduction in other vertebrate species, on amino acid and bile salt-evoked responses. At the appropriate concentrations, either an IP3-gated channel blocker, ruthenium red (0.01-0.1 microM), or a PLC inhibitor, neomycin (50 microM), reduced amino-acid-evoked responses to a significantly greater extent than bile salt-evoked responses. Excised patch recording techniques were used to measure the affects of these drugs on second-messenger-activated currents. Ruthenium red and neomycin are both effective blockers of the olfactory cyclic nucleotide-gated (CNG) current. Both drugs blocked the CNG channel in a voltage-dependent and reversible manner. No IP3-activated currents could be recorded. The differential effects of ruthenium red and neomycin on odor-evoked responses suggest the activation of multiple transduction cascades. The nonspecific actions of these drugs on odor-activated transduction pathways and our inability to record an IP3-activated current do not permit the conclusion that zebrafish, like other fish species, use a PLC/IP3-mediated transduction cascade in the detection of odorants.