Cyclic Nucleotide-gated Channel Family Research Articles

Genome-wide analysis of apple CNGC family allows the identification of MdCNGC15A negatively regulating apple salt tolerance

Calcium (Ca2+) is essential for signal conduction and plant growth. Cyclic nucleotide-gated channels (CNGCs) are Ca2+ transporters that regulate Ca2+ signalling and homeostasis by modulating its transmembrane transport, thereby influencing plant development as well as the biotic and abiotic stress responses. Although identified in numerous plant species, the CNGC family has not been characterized in apple until now. Here, 20 MdCNGCs were identified from the apple genome and were randomly distributed on 13 chromosomes. Phylogenetic analysis classified these MdCNGCs into five groups (I, Ⅱ, Ⅲ, Ⅳ-a, and Ⅳ-b), with five pairs of segmental duplicated genes being detected via collinearity analysis. Sequence alignment and analyses of gene structures, conserved motifs, and 3D structures indicated high structural conservation, particularly within groups. Yeast two-hybrid (Y2H) assays demonstrated interactions between most MdCNGCs and the Ca2+ receptor MdCaM7.1, except for MdCNGC1B and MdCNGC15A. Promoter analysis and expression profiling revealed significant responses to abiotic stress, particularly salt stress, in some MdCNGCs. Silencing MdCNGC15A significantly enhanced apple plants salt tolerance, while its overexpression in apple calli significantly decreased tolerance, as shown by transgenic analysis. Collectively, our results demonstrate the crucial role of MdCNGCs in abiotic stress responses and provide valuable insights for future functional and regulatory studies in apples.

Identification of CNGCs in Glycine max and Screening of Related Resistance Genes after Fusarium solani Infection.

Cyclic nucleotide-gated channels (CNGCs), non-selective cation channels localised on the plasmalemma, are involved in growth, development, and regulatory mechanisms in plants during adverse stress. To date, CNGC gene families in multiple crops have been identified and analysed. However, there have been no systematic studies on the evolution and development of CNGC gene families in legumes. Therefore, in the present study, via transcriptome analysis, we identified 143 CNGC genes in legumes, and thereafter, classified and named them according to the grouping method used for Arabidopsis thaliana. Functional verification for disease stress showed that four GmCNGCs were specifically expressed in the plasmalemma during the stress process. Further, functional enrichment analysis showed that their mode of participation and coordination included inorganic ion concentration regulation inside and outside the membrane via the transmembrane ion channel and participation in stress regulation via signal transduction. The CNGC family genes in G. max involved in disease stress were also identified and physiological stress response and omics analyses were also performed. Our preliminary results revealed the basic laws governing the involvement of CNGCs in disease resistance in G. max, providing important gene resources and a theoretical reference for the breeding of resistant soybean.

Comparative transcriptomic analysis of Glycine soja and G. max and functional identification of GsCNGC20-d interacted with GsCDPK29 under salt stress

Cyclic nucleotide-gated channels (CNGCs) in plants are nonselective Ca2+-permeable cation channels, and are involved in the regulation of plant growth, development and response to stressful environments. However, the functions and molecular mechanisms of CNGCs in soybean under salt stress remain largely unknown. Here, the CNGC family member GsCNGC20-d that is specifically upregulated in the roots of the salt-tolerant G. soja BB52 accession was identified by comparative transcriptomic analysis with the salt-sensitive G. max N23674 cultivar. The GsCNGC20-d protein was localized in the plasma membrane (PM). GsCNGC20-d-overexpressed hairy-root composite soybean plants and transgenic Arabidopsis exhibited enhanced salt tolerance, which was reflected by maintaining higher [Ca2+]cyt and antioxidant activities of POD and CAT as well as lower shoot Na+/K+ ratios, and activating the expression of salt stress-related genes (SOS1–3, SALT3, NHX1, RbohB, CAT1/3 and POD). Moreover, the identified protein kinase GsCDPK29 exhibited an interaction with GsCNGC20-d in Y2H, LUC and BiFC assays. GsCNGC20-d/GsCDPK29 co-overexpressed hairy-root composite soybean plants displayed enhanced salt tolerance through their synergistic effects on the increases of [Ca2+]cyt in root tips and antioxidant activities of POD and CAT, the maintenance of lower MDA content in roots and leaves, and Na+/K+ ratio in shoots. The synergistic regulation of GsCNGC20-d and GsCDPK29 involved in the response and adaptation of salt-tolerant G. soja BB52 to salt stress, will provide new perspectives for further understanding the molecular mechanism of salt tolerance in G. soja and the molecular breeding of salt-tolerant G. max or other crops.

Multiple cyclic nucleotide-gated channels function as ABA-activated Ca2+ channels required for ABA-induced stomatal closure in Arabidopsis.

Abscisic acid (ABA)-activated inward Ca2+-permeable channels in the plasma membrane (PM) of guard cells are required for the initiation and regulation of ABA-specific cytosolic Ca2+ signaling and stomatal closure in plants. But the identities of the PM Ca2+ channels are still unknown. We hypothesized that the ABA-activated Ca2+ channels consist of multiple CYCLIC NUCLEOTIDE-GATED CHANNEL (CNGC) proteins from the CNGC family, which is known as a Ca2+-permeable channel family in Arabidopsis (Arabidopsis thaliana). In this research, we observed high expression of multiple CNGC genes in Arabidopsis guard cells, namely CNGC5, CNGC6, CNGC9, and CNGC12. The T-DNA insertional loss-of-function quadruple mutant cngc5-1 cngc6-2 cngc9-1 cngc12-1 (hereafter c5/6/9/12) showed a strong ABA-insensitive phenotype of stomatal closure. Further analysis revealed that ABA-activated Ca2+ channel currents were impaired, and ABA-specific cytosolic Ca2+ oscillation patterns were disrupted in c5/6/9/12 guard cells compared with in wild-type guard cells. All ABA-related phenotypes of the c5/6/9/12 mutant were successfully rescued by the expression of a single gene out of the four CNGCs under the respective native promoter. Thus, our findings reveal a type of ABA-activated PM Ca2+ channel comprising multiple CNGCs, which is essential for ABA-specific Ca2+ signaling of guard cells and ABA-induced stomatal closure in Arabidopsis.

Dynamic Expression, Differential Regulation and Functional Diversity of the CNGC Family Genes in Cotton.

Cyclic nucleotide-gated channels (CNGCs) constitute a family of non-selective cation channels that are primarily permeable to Ca2+ and activated by the direct binding of cyclic nucleotides (i.e., cAMP and cGMP) to mediate cellular signaling, both in animals and plants. Until now, our understanding of CNGCs in cotton (Gossypium spp.) remains poorly addressed. In the present study, we have identified 40, 41, 20, 20, and 20 CNGC genes in G. hirsutum, G. barbadense, G. herbaceum, G. arboreum, and G. raimondii, respectively, and demonstrated characteristics of the phylogenetic relationships, gene structures, chromosomal localization, gene duplication, and synteny. Further investigation of CNGC genes in G. hirsutum, named GhCNGC1-40, indicated that they are not only extensively expressed in various tissues and at different developmental stages, but also display diverse expression patterns in response to hormones (abscisic acid, salicylic acid, methyl jasmonate, ethylene), abiotic (salt stress) and biotic (Verticillium dahlia infection) stimuli, which conform with a variety of cis-acting regulatory elements residing in the promoter regions; moreover, a set of GhCNGCs are responsive to cAMP signaling during cotton fiber development. Protein–protein interactions supported the functional aspects of GhCNGCs in plant growth, development, and stress responses. Accordingly, the silencing of the homoeologous gene pair GhCNGC1&18 and GhCNGC12&31 impaired plant growth and development; however, GhCNGC1&18-silenced plants enhanced Verticillium wilt resistance and salt tolerance, whereas GhCNGC12&31-silenced plants had opposite effects. Together, these results unveiled the dynamic expression, differential regulation, and functional diversity of the CNGC family genes in cotton. The present work has laid the foundation for further studies and the utilization of CNGCs in cotton genetic improvement.

Three CNGC Family Members, CNGC5, CNGC6, and CNGC9, Are Required for Constitutive Growth of Arabidopsis Root Hairs as Ca2+-Permeable Channels

The genetic identities of Ca2+ channels in root hair (RH) tips essential for constitutive RH growth have remained elusive for decades. Here, we report the identification and characterization of three cyclic nucleotide-gated channel (CNGC) family members, CNGC5, CNGC6, and CNGC9, as Ca2+ channels essential for constitutive RH growth in Arabidopsis. We found that the cngc5-1cngc6-2cngc9-1 triple mutant (designated shrh1) showed significantly shorter and branching RH phenotypes as compared with the wild type. The defective RH growth phenotype of shrh1 could be rescued by either the expression of CNGC5, CNGC6, or CNGC9 single gene or by the supply of high external Ca2+, but could not be rescued by external K+ supply. Cytosolic Ca2+ imaging and patch-clamp data in HEK293T cells showed that these three CNGCs all function as Ca2+-permeable channels. Cytosolic Ca2+ imaging in growing RHs further showed that the Ca2+ gradients and their oscillation in RH tips were dramatically attenuated in shrh1 compared with those in the wild type. Phenotypic analysis revealed that these three CNGCs are Ca2+ channels essential for constitutive RH growth, with different roles in RHs from the conditional player CNGC14. Moreover, we found that these three CNGCs are involved in auxin signaling in RHs. Taken together, our study identified CNGC5, CNGC6, and CNGC9 as three key Ca2+ channels essential for constitutive RH growth and auxin signaling in Arabidopsis. Video Abstract [Display omitted]

Multiple cyclic nucleotide-gated channels coordinate calcium oscillations and polar growth of root hairs.

Calcium gradients underlie polarization in eukaryotic cells. In plants, a tip-focused Ca2+ -gradient is fundamental for rapid and unidirectional cell expansion during epidermal root hair development. Here we report that three members of the cyclic nucleotide-gated channel family are required to maintain cytosolic Ca2+ oscillations and the normal growth of root hairs. CNGC6, CNGC9 and CNGC14 were expressed in root hairs, with CNGC9 displaying the highest root hair specificity. In individual channel mutants, morphological defects including root hair swelling and branching, as well as bursting, were observed. The developmental phenotypes were amplified in the three cngc double mutant combinations. Finally, cngc6/9/14 triple mutants only developed bulging trichoblasts and could not form normal root hair protrusions because they burst after the transition to the rapid growth phase. Prior to developmental defects, single and double mutants showed increasingly disturbed patterns of Ca2+ oscillations. We conclude that CNGC6, CNGC9 and CNGC14 fulfill partially but not fully redundant functions in generating and maintaining tip-focused Ca2+ oscillations, which are fundamental for proper root hair growth and polarity. Furthermore, the results suggest that these calmodulin-binding and Ca2+ -permeable channels organize a robust tip-focused oscillatory calcium gradient, which is not essential for root hair initiation but is required to control the integrity of the root hair after the transition to the rapid growth phase. Our findings also show that root hairs possess a large ability to compensate calcium-signaling defects, and add new players to the regulatory network, which coordinates cell wall properties and cell expansion during polar root hair growth.

Arabidopsis CNGC Family Members Contribute to Heavy Metal Ion Uptake in Plants.

Heavy metal ions, including toxic concentrations of essential ions, negatively affect diverse metabolic and cellular processes. Heavy metal ions are known to enter cells in a non-selective manner; however, few studies have examined the regulation of heavy metal ion transport. Plant cyclic nucleotide-gated channels (CNGCs), a type of Ca2+-permeable-channel, have been suggested to be involved in the uptake of both essential and toxic cations. To determine the candidates responsible for heavy metal ion transport, a series of Arabidopsis CNGC mutants were examined for their response to Pb2+ and Cd2+ ions. The primary focus was on root growth and the analysis of the concentration of heavy metals in plants. Results, based on the analysis of primary root length, indicated that AtCNGC1, AtCNGC10, AtCNGC13 and AtCNGC19 play roles in Pb2+ toxicity, while AtCNGC11, AtCNGC13, AtCNGC16 and AtCNGC20 function in Cd2+ toxicity in Arabidopsis. Ion content analysis verified that the mutations of AtCNGC1 and AtCNGC13 resulted in reduced Pb2+ accumulation, while the mutations of AtCNGC11, AtCNGC15 and AtCNGC19 resulted in less Pb2+ and Cd2+ accumulation in plants. These findings provide functional evidence which support the roles of these AtCNGCs in the uptake and transport of Pb2+ or Cd2+ ion in plants.

Genome-Wide Identification of Cyclic Nucleotide-Gated Ion Channel Gene Family in Wheat and Functional Analyses of TaCNGC14 and TaCNGC16.

Cyclic nucleotide gated channels (CNGCs) play multifaceted roles in plants, particularly with respect to signaling processes associated with abiotic stress signaling and during host-pathogen interactions. Despite key roles during plant survival and response to environment, little is known about the activity and function of CNGC family in common wheat (Triticum aestivum L.), a key stable food around the globe. In this study, we performed a genome-wide identification of CNGC family in wheat and identified a total 47 TaCNGCs in wheat, classifying these genes into four major groups (I–IV) with two sub-groups (IVa and IVb). Sequence analysis revealed the presence of several conserved motifs, including a phosphate binding cassette (PBC) and a “hinge” region, both of which have been hypothesized to be critical for the function of wheat CNGCs. During wheat infection with Pst, the transcript levels of TaCNGC14 and TaCNGC16, both members of group IVb, showed significant induction during a compatible interaction, while a reduction in gene expression was observed in incompatible interactions. In addition, TaCNGC14 and TaCNGC16 mRNA accumulation was significantly influenced by exogenously applied hormones, including abscisic acid (ABA), methyl jasmonate (MeJA), and salicylic acid (SA), suggesting a role in hormone signaling and/or perception. Silencing of TaCNGC14 and TaCNGC16 limited Pst growth and increased wheat resistance against Pst. The results presented herein contribute to our understanding of the wheat CNGC gene family and the mechanism of TaCNGCs signaling during wheat-Pst interaction.

OsCNGC13 promotes seed-setting rate by facilitating pollen tube growth in stylar tissues.

Seed-setting rate is a critical determinant of grain yield in rice (Oryza sativa L.). Rapid and healthy pollen tube growth in the style is required for high seed-setting rate. The molecular mechanisms governing this process remain largely unknown. In this study, we isolate a dominant low seed-setting rate rice mutant, sss1-D. Cellular examination results show that pollen tube growth is blocked in about half of the mutant styles. Molecular cloning and functional assays reveals that SSS1-D encodes OsCNGC13, a member of the cyclic nucleotide-gated channel family. OsCNGC13 is preferentially expressed in the pistils and its expression is dramatically reduced in the heterozygous plant, suggesting a haploinsufficiency nature for the dominant mutant phenotype. We show that OsCNGC13 is permeable to Ca2+. Consistent with this, accumulation of cytoplasmic calcium concentration ([Ca2+]cyt) is defective in the sss1-D mutant style after pollination. Further, the sss1-D mutant has altered extracellular matrix (ECM) components and delayed cell death in the style transmission tract (STT). Based on these results, we propose that OsCNGC13 acts as a novel maternal sporophytic factor required for stylar [Ca2+]cyt accumulation, ECM components modification and STT cell death, thus facilitating the penetration of pollen tube in the style for successful double fertilization and seed-setting in rice.

A Ca2+-Sensitive System Mediates Low-Affinity K+ Uptake in the Absence of AKT1 in Arabidopsis Plants

K(+) acquisition by Arabidopsis roots is mainly mediated by the high-affinity K(+) transporter AtHAK5 and the inward-rectifier K(+) channel AtAKT1. This model is probably universal to plants. Mutant plants lacking these two systems (athak5,atakt1) take up K(+) and grow when the external K(+) concentration is above a certain level, indicating that an additional transport system may compensate for the absence of AtHAK5 and AtAKT1. Here we describe that this alternative system is essential for providing sufficient K(+) to sustain growth of athak5,atakt1 plants. This system is especially sensitive to Ca(2+), Mg(2+), Ba(2+) and La(3+), it transports Cs(+) and its activity is reduced by cyclic nucleotides. These results suggest that a Ca(2+)-permeable voltage-independent non-selective cation channel, probably belonging to the cyclic nucleotide gated channel (CNGC) family, may provide the pathway for K(+) uptake in athak5,atakt1 plants. The genes encoding the two members of the CNGC family that have been described as mediating root K(+) uptake, AtCNGC3 and AtCNGC10, are not up-regulated in athak5,atakt1 plants, excluding overexpression of these genes as a compensatory mechanism. On the other hand, an increased driving force for K(+) in athak5,atakt1 plants due to a hyperpolarization of the membrane potential of its root cells is also discarded. The identification of this unknown system may provide tools to improve plant K(+) nutrition in conditions where AtAKT1 functionality is reduced, such as under salinity. In addition, this system may constitute an important pathway for accumulation of toxic cations such as Cs(+) or radiocesium ((137)Cs(+)), and could play a role in phytoremediation.

The hyperpolarization-activated cyclic nucleotide-gated HCN2 channel transports ammonium in the distal nephron

Recent studies have identified Rhesus proteins as important molecules for ammonia transport in acid-secreting intercalated cells in the distal nephron. Here, we provide evidence for an additional molecule that can mediate NH3/NH4 excretion, the subtype 2 of the hyperpolarization-activated cyclic nucleotide-gated channel family (HCN2), in collecting ducts in rat renal cortex and medulla. Chronic metabolic acidosis in rats did not alter HCN2 protein expression but downregulated the relative abundance of HCN2 mRNA. Its cDNA was identical to the homolog from the brain and the protein was post-translationally modified by N-type glycosylation. Electrophysiological recordings in Xenopus oocytes injected with HCN2 cRNA found that potassium was transported better than ammonium, each of which was transported significantly better than sodium, criteria that are compatible with a role for HCN2 in ammonium transport. In microperfused rat outer medullary collecting duct segments, the initial rate of acidification, upon exposure to a basolateral ammonium chloride pulse, was higher in intercalated than in principal cells. A specific inhibitor of HCN2 (ZD7288) decreased acidification only in intercalated cells from control rats. In rats with chronic metabolic acidosis, the rate of acidification doubled in both intercalated and principal cells; however, ZD7288 had no significant inhibitory effect. Thus, HCN2 is a basolateral ammonium transport pathway of intercalated cells and may contribute to the renal regulation of body pH under basal conditions.

A D262n Mutation in Canine CNGB3 in a Highly Conserved Acidic S2 Region Causes Complete Colorblindness in a Canine Model

Cyclic nucleotide-gated (CNG) channels in cone photoreceptors are composed of CNGA3 (A3) and CNGB3 (B3) subunits. In a canine achromatopsia model, loss of cone ERG function and color vision are associated with the mutation of Asp (D) 262 to Asn (N) in the B3 subunit. This Asp residue is conserved in all CNG channels. We examined the D/N mutation in the human (h) B3 channel co-expressed with a wild-type canine (c) A3 and in a mutant cA3 homomeric channel in which the conserved Asp residue was substituted with Asn. In patch clamp experiments, both heteromeric wild type cA3 co-expressed with D/N hB3 and mutant homomeric D/N cA3 CNG channels had no cGMP-activated currents providing molecular insights into the cause of canine achromatopsia. D262 of the cB3 is located in the S2 transmembrane domain and, along with D252 and D256, these residues comprise an acidic motif conserved in the CNG channel family and extending to some members of the Shaker K superfamily. Hints about the role of this Asp motif have been gained from studies of related channels reporting interactions between the negatively-charged residues in S2 and the conserved positively-charged residues in other transmembrane domains. We generated mutant A3 subunits by substituting each of the three conserved S2 Asp residues with Asn, Cys, or Glu. Patch clamp analysis showed all of the mutant homomeric A3 channels lost function. These YFP-tagged A3 mutant channels exhibited changes in cellular localization compared to wild type channels suggesting that any change in the Asp motif alters subunit trafficking. Future studies will focus on understanding the role of this Asp motif in intra-subunit helical contacts and proper trafficking and assembly of cone CNG channels.

The cyclic nucleotide-gated calmodulin-binding channel AtCNGC10 localizes to the plasma membrane and influences numerous growth responses and starch accumulation in Arabidopsis thaliana

Cyclic nucleotide gated channels (CNGCs) that are regulated by calmodulin (CaM) have been shown to play essential roles in signal transduction, metabolism, and growth in animals. By contrast, very little is known about the subcellular location and the function of these channels in plants. Here we report on the effects of antisense suppression of the expression of AtCNGC10, a putative K+ channel, and the immunolocalization of the protein using an AtCNGC10-specific antiserum. In Arabidopsis thaliana leaves, AtCNGC10 was localized to the plasma membrane of mesophyll and parenchyma cells. Antisense AtCNGC10 plants had 40% of the AtCNGC10 mRNA levels and virtually undetectable protein levels relative to wild type plants. Antisense expression of AtCNGC10 did not affect the mRNA levels of AtCNGC13, the most closely related CNGC family member in the genome. Relative to wild type Columbia, antisense AtCNGC10 plants flowered 10 days earlier, and had a 25% reduction in leaf surface area, thickness and palisade parenchyma cell length. Their roots responded more slowly to gravitropic changes and the chloroplasts accumulated more starch. We propose that AtCNGC10, through interactions with CaM and cGMP, modulates cellular K+ balance across the plasma membrane, and that perturbations of this K+ gradient affect numerous growth and developmental processes.

TRP channels as new pharmacological targets

Changes in intracellular ion concentrations are universal triggers of cell activation. Ca, for instance, is one of the most versatile intracellular second messengers in eukaryotic cells involved in a wide range of cellular processes including exocytosis, contraction, cell proliferation, development and apoptosis. Stimulation of target cells with neurotransmitters, hormones or growth factors leads to an elevation of the intracellular Ca concentration brought about by release from intracellular stores and influx via Ca-permeable cation channels in the cell membrane (Clapham et al. 2001). While excitable cells like neurons are well equipped with voltage-gated ion channels mediating the bulk of cation influx, the molecular identity of cation entry channels in non-excitable cells has been completely mysterious for quite some time. A major leap forward in our understanding of agonist-triggered cation influx was achieved by the discovery of Drosophila melanogaster visual transduction channels associated with the transient receptor potential (trp) and the trp-like (trpl) mutant phenotypes. Since then a new mammalian gene family of TRP homologs has been identified by homology screening (Montell et al. 2002; Clapham et al. 2003). Based on structural homology and on systematic glycosylation scanning, TRP proteins are allocated to the structural superfamily of six-transmembrane spanning ion channels like voltage-gated K channels, the cyclic nucleotide-gated channel family, and single cassettes of voltage-activated Ca and Na channels (Fig. 1). Both Nand C-termini of TRP proteins are assumed to be localised intracellularly, and a channel pore loop appears to be bordered by transmembrane domains 5 and 6. In specialised cells, some members of the TRP family participate in sensory processes like vision and hearing as well as temperature, pain, gustatory and pheromone perception. In addition, many TRP proteins are involved in ubiquitous, very basic cellular processes like sensing of osmotic or oxidative stress and mediating agonist-induced cation entry (Clapham 2003). Based on primary amino acid homology, the conventional TRP proteins can be classified into three subfamilies: TRPC, TRPV, and TRPM (see Fig. 1). Three additional, more distantly related subfamilies have recently been defined: ANKTM1, a Ca-permeant, non-selective cation channel, is the only mammalian member of the TRPA branch of TRP proteins. It is activated by noxious cold temperature, pungent mustard oil compounds and may additionally serve as a mechanosensitive transduction channel in auditory hair cells as well as an ionotropic cannabinoid receptor. The three mucolipins, TRPML1, 2, and 3, appear to be ion channels in intracellular vesicles. Mutations in TRPML1 cause mucolipidosis type IV, a neurodegenerative lysosomal storage disorder, while genetic defects in TRPP2, a member of the TRPP subfamily, are frequently encountered in patients suffering from autosomal dominant polycystic kidney disease. The current compilation of review articles covers salient features of TRPC, TRPV, TRPM, and TRPP proteins at the cutting edge of science. In particular, the authors continue to explore the pharmacotherapeutic potential of this new class of ion channels, a fruitful endeavor that was started at the 25th Symposium at Rauischholzhausen Castle on “TRP channels as Pharmacological Targets” which took place on June 10–12, 2004. TRPC proteins all share a common gating mechanism that involves the activation of phospholipase C isoforms and thus constitute prime molecular candidates responsible for phospholipase C-dependent cation influx. The molecThis revised version was published online in June 2005 with corrections to the Reference list

Cloning and localization of the hyperpolarization-activated cyclic nucleotide-gated channel family in rat brain

Rhythmic firing in brain and heart is mediated by pacemaker channels that are activated by hyperpolarization and regulated directly by cyclic nucleotides. Recent work has identified a new gene family that encodes such channels, which are termed hyperpolarization-activated, cyclic nucleotide-gated (HCN) channels. In this study, we report the molecular cloning and localization by in situ hybridization of HCN1–4 in adult rat brain. The rat HCN1–4 clones show high homology to the deduced amino acid sequence of the mouse channels (>97% identity). The mRNA expression of the four channels in adult brain was evaluated in a systematic manner from the olfactory bulb to lower brain stem nuclei. Each mRNA demonstrated a unique pattern of distribution. HCN1 expression is highly enriched in cerebral cortex, hippocampus, cerebellum, and facial motor nucleus; HCN2 is highly abundant in mamillary bodies, pontine nucleus, ventral cochlear nucleus, and nucleus of the trapezoid body; HCN3 expression is most pronounced in supraoptic nucleus of hypothalamus; and HCN4 expression is most abundant in medial habenula and anterior and principal relay nuclei of the thalamus. These variations in regional specificity of HCN channels could generate important differences in neuronal pacemaker activity across brain systems.

Calcium Imaging in the Olfactory System: New Tools for Visualizing Odor Recognition

Transient elevations of intracellular Ca2+ play an important role in regulating the sensitivity of the sense of smell, both at the level of signal transduction within the cilia of olfactory receptor neurons and at presynaptic sites in the olfactory bulb, but such elevations have not been demonstrated previously because of the small size of these neuronal com partments. Here, we summarize recent progress employing high resolution Ca2+-imaging techniques that permit the visualization of odor-induced neural activity in these critical subcellular compartments of the vertebrate olfactory system. In olfactory neurons, Ca2+ permeable cyclic nucleotide-gated (CNG) cation channels mediate the initial Ca2+ entry during odor transduction. The surprisingly widespread distribution of members of the CNG channel family in the mammalian brain suggests that CNG channel-mediated Ca2+ entry contributes to signal transduction in many CNS neurons. NEUROSCIENTIST 5:4- 7, 1999