Your browser doesn't support javascript.
loading
Mostrar: 20 | 50 | 100
Resultados 1 - 20 de 32
Filtrar
1.
Proc Natl Acad Sci U S A ; 121(30): e2407461121, 2024 Jul 23.
Artigo em Inglês | MEDLINE | ID: mdl-39018191

RESUMO

The Shaker family of voltage-gated K+ channels has been thought of as an animal-specific ion channel family that diversified in concert with nervous systems. It comprises four functionally independent gene subfamilies (Kv1-4) that encode diverse neuronal K+ currents. Comparison of animal genomes predicts that only the Kv1 subfamily was present in the animal common ancestor. Here, we show that some choanoflagellates, the closest protozoan sister lineage to animals, also have Shaker family K+ channels. Choanoflagellate Shaker family channels are surprisingly most closely related to the animal Kv2-4 subfamilies which were believed to have evolved only after the divergence of ctenophores and sponges from cnidarians and bilaterians. Structural modeling predicts that the choanoflagellate channels share a T1 Zn2+ binding site with Kv2-4 channels that is absent in Kv1 channels. We functionally expressed three Shakers from Salpingoeca helianthica (SheliKvT1.1-3) in Xenopus oocytes. SheliKvT1.1-3 function only in two heteromultimeric combinations (SheliKvT1.1/1.2 and SheliKvT1.1/1.3) and encode fast N-type inactivating K+ channels with distinct voltage dependence that are most similar to the widespread animal Kv1-encoded A-type Shakers. Structural modeling of the T1 assembly domain supports a preference for heteromeric assembly in a 2:2 stoichiometry. These results push the origin of the Shaker family back into a common ancestor of metazoans and choanoflagellates. They also suggest that the animal common ancestor had at least two distinct molecular lineages of Shaker channels, a Kv1 subfamily lineage predicted from comparison of animal genomes and a Kv2-4 lineage predicted from comparison of animals and choanoflagellates.


Assuntos
Coanoflagelados , Evolução Molecular , Superfamília Shaker de Canais de Potássio , Animais , Coanoflagelados/genética , Coanoflagelados/metabolismo , Superfamília Shaker de Canais de Potássio/genética , Superfamília Shaker de Canais de Potássio/metabolismo , Filogenia , Sequência de Aminoácidos
2.
Biophys J ; 123(14): 2038-2049, 2024 Jul 16.
Artigo em Inglês | MEDLINE | ID: mdl-38291751

RESUMO

Here we explore the evolutionary origins of fast N-type ball-and-chain inactivation in Shaker (Kv1) K+ channels by functionally characterizing Shaker channels from the ctenophore (comb jelly) Mnemiopsis leidyi. Ctenophores are the sister lineage to other animals and Mnemiopsis has >40 Shaker-like K+ channels, but they have not been functionally characterized. We identified three Mnemiopsis channels (MlShak3-5) with N-type inactivation ball-like sequences at their N termini and functionally expressed them in Xenopus oocytes. Two of the channels, MlShak4 and MlShak5, showed rapid inactivation similar to cnidarian and bilaterian Shakers with rapid N-type inactivation, whereas MlShak3 inactivated ∼100-fold more slowly. Fast inactivation in MlShak4 and MlShak5 required the putative N-terminal inactivation ball sequences. Furthermore, the rate of fast inactivation in these channels depended on the number of inactivation balls/channel, but the rate of recovery from inactivation did not. These findings closely match the mechanism of N-type inactivation first described for Drosophila Shaker in which 1) inactivation balls on the N termini of each subunit can independently block the pore, and 2) only one inactivation ball occupies the pore binding site at a time. These findings suggest classical N-type activation evolved in Shaker channels at the very base of the animal phylogeny in a common ancestor of ctenophores, cnidarians, and bilaterians and that fast-inactivating Shakers are therefore a fundamental type of animal K+ channel. Interestingly, we find evidence from functional co-expression experiments and molecular dynamics that MlShak4 and MlShak5 do not co-assemble, suggesting that Mnemiopsis has at least two functionally independent N-type Shaker channels.


Assuntos
Ctenóforos , Ativação do Canal Iônico , Superfamília Shaker de Canais de Potássio , Animais , Ctenóforos/metabolismo , Ctenóforos/genética , Superfamília Shaker de Canais de Potássio/metabolismo , Superfamília Shaker de Canais de Potássio/genética , Superfamília Shaker de Canais de Potássio/química , Sequência de Aminoácidos , Filogenia , Oócitos/metabolismo
3.
Plant Cell ; 30(12): 2898-2909, 2018 12.
Artigo em Inglês | MEDLINE | ID: mdl-30389753

RESUMO

Plant voltage-gated K+ channels have been referred to as "plant Shakers" in reference to animal Shaker channels, the first K+ channels identified. Recent advances in our knowledge of K+ channel evolution and structure have significantly deepened the divide between these plant and animal K+ channels, suggesting that it is time to completely retire the "plant Shaker" designation. Evolutionary genomics reveals that plant voltage-gated K+ channels and metazoan Shakers derive from distinct prokaryotic ancestors. The plant channels belong to a lineage that includes cyclic nucleotide-gated channels and metazoan ether-à-go-go and hyperpolarization-activated, cyclic nucleotide-gated channels. We refer to this lineage as the CNBD channel superfamily, because all these channels share a cytoplasmic gating domain homologous to cyclic nucleotide binding domains. The first structures of CNBD superfamily channels reveal marked differences in coupling between the voltage sensor and ion-conducting pore relative to metazoan Shaker channels. Viewing plant voltage-gated K+ channel function through the lens of CNBD superfamily structures should lead to insights into how these channels are regulated.


Assuntos
Evolução Molecular , Proteínas de Plantas/genética , Canais de Potássio de Abertura Dependente da Tensão da Membrana/genética , Genômica , Proteínas de Plantas/classificação , Canais de Potássio de Abertura Dependente da Tensão da Membrana/classificação
4.
J Exp Biol ; 223(Pt 21)2020 11 10.
Artigo em Inglês | MEDLINE | ID: mdl-32968001

RESUMO

The centralized nervous systems of bilaterian animals rely on directional signaling facilitated by polarized neurons with specialized axons and dendrites. It is not known whether axo-dendritic polarity is exclusive to bilaterians or was already present in early metazoans. We therefore examined neurite polarity in the starlet sea anemone Nematostella vectensis (Cnidaria). Cnidarians form a sister clade to bilaterians and share many neuronal building blocks characteristic of bilaterians, including channels, receptors and synaptic proteins, but their nervous systems comprise a comparatively simple net distributed throughout the body. We developed a tool kit of fluorescent polarity markers for live imaging analysis of polarity in an identified neuron type, large ganglion cells of the body column nerve net that express the LWamide-like neuropeptide. Microtubule polarity differs in bilaterian axons and dendrites, and this in part underlies polarized distribution of cargo to the two types of processes. However, in LWamide-like+ neurons, all neurites had axon-like microtubule polarity suggesting that they may have similar contents. Indeed, presynaptic and postsynaptic markers trafficked to all neurites and accumulated at varicosities where neurites from different neurons often crossed, suggesting the presence of bidirectional synaptic contacts. Furthermore, we could not identify a diffusion barrier in the plasma membrane of any of the neurites like the axon initial segment barrier that separates the axonal and somatodendritic compartments in bilaterian neurons. We conclude that at least one type of neuron in Nematostella vectensis lacks the axo-dendritic polarity characteristic of bilaterian neurons.


Assuntos
Anêmonas-do-Mar , Animais , Axônios , Citoesqueleto , Microtúbulos , Neurônios
5.
PLoS Genet ; 12(12): e1006457, 2016 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-27911898

RESUMO

In vertebrate neurons, the axon initial segment (AIS) is specialized for action potential initiation. It is organized by a giant 480 Kd variant of ankyrin G (AnkG) that serves as an anchor for ion channels and is required for a plasma membrane diffusion barrier that excludes somatodendritic proteins from the axon. An unusually long exon required to encode this 480Kd variant is thought to have been inserted only recently during vertebrate evolution, so the giant ankyrin-based AIS scaffold has been viewed as a vertebrate adaptation for fast, precise signaling. We re-examined AIS evolution through phylogenomic analysis of ankyrins and by testing the role of ankyrins in proximal axon organization in a model multipolar Drosophila neuron (ddaE). We find giant isoforms of ankyrin in all major bilaterian phyla, and present evidence in favor of a single common origin for giant ankyrins and the corresponding long exon in a bilaterian ancestor. This finding raises the question of whether giant ankyrin isoforms play a conserved role in AIS organization throughout the Bilateria. We examined this possibility by looking for conserved ankyrin-dependent AIS features in Drosophila ddaE neurons via live imaging. We found that ddaE neurons have an axonal diffusion barrier proximal to the cell body that requires a giant isoform of the neuronal ankyrin Ank2. Furthermore, the potassium channel shal concentrates in the proximal axon in an Ank2-dependent manner. Our results indicate that the giant ankyrin-based cytoskeleton of the AIS may have evolved prior to the radiation of extant bilaterian lineages, much earlier than previously thought.


Assuntos
Anquirinas/genética , Segmento Inicial do Axônio/metabolismo , Proteínas de Drosophila/genética , Filogenia , Canais de Potássio Shal/genética , Potenciais de Ação/genética , Animais , Anquirinas/biossíntese , Membrana Celular/genética , Proteínas de Drosophila/biossíntese , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/genética , Drosophila melanogaster/crescimento & desenvolvimento , Neurônios/metabolismo , Canais de Potássio Shal/metabolismo
7.
Proc Natl Acad Sci U S A ; 112(9): E1010-9, 2015 Mar 03.
Artigo em Inglês | MEDLINE | ID: mdl-25691740

RESUMO

We examined the origins and functional evolution of the Shaker and KCNQ families of voltage-gated K(+) channels to better understand how neuronal excitability evolved. In bilaterians, the Shaker family consists of four functionally distinct gene families (Shaker, Shab, Shal, and Shaw) that share a subunit structure consisting of a voltage-gated K(+) channel motif coupled to a cytoplasmic domain that mediates subfamily-exclusive assembly (T1). We traced the origin of this unique Shaker subunit structure to a common ancestor of ctenophores and parahoxozoans (cnidarians, bilaterians, and placozoans). Thus, the Shaker family is metazoan specific but is likely to have evolved in a basal metazoan. Phylogenetic analysis suggested that the Shaker subfamily could predate the divergence of ctenophores and parahoxozoans, but that the Shab, Shal, and Shaw subfamilies are parahoxozoan specific. In support of this, putative ctenophore Shaker subfamily channel subunits coassembled with cnidarian and mouse Shaker subunits, but not with cnidarian Shab, Shal, or Shaw subunits. The KCNQ family, which has a distinct subunit structure, also appears solely within the parahoxozoan lineage. Functional analysis indicated that the characteristic properties of Shaker, Shab, Shal, Shaw, and KCNQ currents evolved before the divergence of cnidarians and bilaterians. These results show that a major diversification of voltage-gated K(+) channels occurred in ancestral parahoxozoans and imply that many fundamental mechanisms for the regulation of action potential propagation evolved at this time. Our results further suggest that there are likely to be substantial differences in the regulation of neuronal excitability between ctenophores and parahoxozoans.


Assuntos
Ctenóforos/genética , Evolução Molecular , Hydra/genética , Canais de Potássio KCNQ/genética , Filogenia , Superfamília Shaker de Canais de Potássio/genética , Sequência de Aminoácidos , Animais , Ctenóforos/metabolismo , Bases de Dados de Proteínas , Humanos , Hydra/metabolismo , Canais de Potássio KCNQ/metabolismo , Camundongos , Dados de Sequência Molecular , Superfamília Shaker de Canais de Potássio/metabolismo , Xenopus laevis
8.
Proc Natl Acad Sci U S A ; 111(15): 5712-7, 2014 Apr 15.
Artigo em Inglês | MEDLINE | ID: mdl-24706772

RESUMO

Mammalian Ether-a-go-go related gene (Erg) family voltage-gated K(+) channels possess an unusual gating phenotype that specializes them for a role in delayed repolarization. Mammalian Erg currents rectify during depolarization due to rapid, voltage-dependent inactivation, but rebound during repolarization due to a combination of rapid recovery from inactivation and slow deactivation. This is exemplified by the mammalian Erg1 channel, which is responsible for IKr, a current that repolarizes cardiac action potential plateaus. The Drosophila Erg channel does not inactivate and closes rapidly upon repolarization. The dramatically different properties observed in mammalian and Drosophila Erg homologs bring into question the evolutionary origins of distinct Erg K(+) channel functions. Erg channels are highly conserved in eumetazoans and first evolved in a common ancestor of the placozoans, cnidarians, and bilaterians. To address the ancestral function of Erg channels, we identified and characterized Erg channel paralogs in the sea anemone Nematostella vectensis. N. vectensis Erg1 (NvErg1) is highly conserved with respect to bilaterian homologs and shares the IKr-like gating phenotype with mammalian Erg channels. Thus, the IKr phenotype predates the divergence of cnidarians and bilaterians. NvErg4 and Caenorhabditis elegans Erg (unc-103) share the divergent Drosophila Erg gating phenotype. Phylogenetic and sequence analysis surprisingly indicates that this alternate gating phenotype arose independently in protosomes and cnidarians. Conversion from an ancestral IKr-like gating phenotype to a Drosophila Erg-like phenotype correlates with loss of the cytoplasmic Ether-a-go-go domain. This domain is required for slow deactivation in mammalian Erg1 channels, and thus its loss may partially explain the change in gating phenotype.


Assuntos
Potenciais de Ação/genética , Canais de Potássio Éter-A-Go-Go/metabolismo , Evolução Molecular , Ativação do Canal Iônico/genética , Animais , Sequência de Bases , Teorema de Bayes , Caenorhabditis , Clonagem Molecular , Biologia Computacional , Daphnia , Ativação do Canal Iônico/fisiologia , Modelos Biológicos , Modelos Genéticos , Dados de Sequência Molecular , Técnicas de Patch-Clamp , Placozoa , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Anêmonas-do-Mar , Análise de Sequência de DNA , Especificidade da Espécie , Xenopus
9.
J Exp Biol ; 218(Pt 4): 572-80, 2015 Feb 15.
Artigo em Inglês | MEDLINE | ID: mdl-25696820

RESUMO

Polarized distribution of signaling molecules to axons and dendrites facilitates directional information flow in complex vertebrate nervous systems. The topic we address here is when the key aspects of neuronal polarity evolved. All neurons have a central cell body with thin processes that extend from it to cover long distances, and they also all rely on voltage-gated ion channels to propagate signals along their length. The most familiar neurons, those in vertebrates, have additional cellular features that allow them to send directional signals efficiently. In these neurons, dendrites typically receive signals and axons send signals. It has been suggested that many of the distinct features of axons and dendrites, including the axon initial segment, are found only in vertebrates. However, it is now becoming clear that two key cytoskeletal features that underlie polarized sorting, a specialized region at the base of the axon and polarized microtubules, are found in invertebrate neurons as well. It thus seems likely that all bilaterians generate axons and dendrites in the same way. As a next step, it will be extremely interesting to determine whether the nerve nets of cnidarians and ctenophores also contain polarized neurons with true axons and dendrites, or whether polarity evolved in concert with the more centralized nervous systems found in bilaterians.


Assuntos
Evolução Biológica , Invertebrados/fisiologia , Neurônios/fisiologia , Animais , Axônios/fisiologia , Citoesqueleto , Dendritos/fisiologia , Microtúbulos
10.
J Exp Biol ; 218(Pt 4): 526-36, 2015 Feb 15.
Artigo em Inglês | MEDLINE | ID: mdl-25696816

RESUMO

We examined the evolutionary origins of the ether-à-go-go (EAG) family of voltage-gated K(+) channels, which have a strong influence on the excitability of neurons. The bilaterian EAG family comprises three gene subfamilies (Eag, Erg and Elk) distinguished by sequence conservation and functional properties. Searches of genome sequence indicate that EAG channels are metazoan specific, appearing first in ctenophores. However, phylogenetic analysis including two EAG family channels from the ctenophore Mnemiopsis leidyi indicates that the diversification of the Eag, Erg and Elk gene subfamilies occurred in a cnidarian/bilaterian ancestor after divergence from ctenophores. Erg channel function is highly conserved between cnidarians and mammals. Here we show that Eag and Elk channels from the sea anemone Nematostella vectensis (NvEag and NvElk) also share high functional conservation with mammalian channels. NvEag, like bilaterian Eag channels, has rapid kinetics, whereas NvElk activates at extremely hyperpolarized voltages, which is characteristic of Elk channels. Potent inhibition of voltage activation by extracellular protons is conserved between mammalian and Nematostella EAG channels. However, characteristic inhibition of voltage activation by Mg(2+) in Eag channels and Ca(2+) in Erg channels is reduced in Nematostella because of mutation of a highly conserved aspartate residue in the voltage sensor. This mutation may preserve sub-threshold activation of Nematostella Eag and Erg channels in a high divalent cation environment. mRNA in situ hybridization of EAG channels in Nematostella suggests that they are differentially expressed in distinct cell types. Most notable is the expression of NvEag in cnidocytes, a cnidarian-specific stinging cell thought to be a neuronal subtype.


Assuntos
Cnidários/genética , Evolução Molecular , Canais de Potássio de Abertura Dependente da Tensão da Membrana/genética , Anêmonas-do-Mar/genética , Animais , Sequência de Bases , Cnidários/fisiologia , Hibridização In Situ , Filogenia , Anêmonas-do-Mar/fisiologia , Xenopus
11.
Nature ; 454(7201): 217-20, 2008 Jul 10.
Artigo em Inglês | MEDLINE | ID: mdl-18548007

RESUMO

Animals from flies to humans are able to distinguish subtle gradations in temperature and show strong temperature preferences. Animals move to environments of optimal temperature and some manipulate the temperature of their surroundings, as humans do using clothing and shelter. Despite the ubiquitous influence of environmental temperature on animal behaviour, the neural circuits and strategies through which animals select a preferred temperature remain largely unknown. Here we identify a small set of warmth-activated anterior cell (AC) neurons located in the Drosophila brain, the function of which is critical for preferred temperature selection. AC neuron activation occurs just above the fly's preferred temperature and depends on dTrpA1, an ion channel that functions as a molecular sensor of warmth. Flies that selectively express dTrpA1 in the AC neurons select normal temperatures, whereas flies in which dTrpA1 function is reduced or eliminated choose warmer temperatures. This internal warmth-sensing pathway promotes avoidance of slightly elevated temperatures and acts together with a distinct pathway for cold avoidance to set the fly's preferred temperature. Thus, flies select a preferred temperature by using a thermal sensing pathway tuned to trigger avoidance of temperatures that deviate even slightly from the preferred temperature. This provides a potentially general strategy for robustly selecting a narrow temperature range optimal for survival.


Assuntos
Comportamento de Escolha/fisiologia , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/fisiologia , Canais de Cátion TRPC/metabolismo , Temperatura , Animais , Aprendizagem da Esquiva , Temperatura Corporal , Proteínas de Drosophila/genética , Drosophila melanogaster/crescimento & desenvolvimento , Feminino , Canais Iônicos , Larva , Dados de Sequência Molecular , Neurônios/metabolismo , Oócitos/metabolismo , Canal de Cátion TRPA1 , Canais de Cátion TRPC/genética , Xenopus laevis
12.
Genome Biol Evol ; 15(3)2023 03 03.
Artigo em Inglês | MEDLINE | ID: mdl-36669828

RESUMO

Ion channels are highly diverse in the cnidarian model organism Nematostella vectensis (Anthozoa), but little is known about the evolutionary origins of this channel diversity and its conservation across Cnidaria. Here, we examined the evolution of voltage-gated K+ channels in Cnidaria by comparing genomes and transcriptomes of diverse cnidarian species from Anthozoa and Medusozoa. We found an average of over 40 voltage-gated K+ channel genes per species, and a phylogenetic reconstruction of the Kv, KCNQ, and Ether-a-go-go (EAG) gene families identified 28 voltage-gated K+ channels present in the last common ancestor of Anthozoa and Medusozoa (23 Kv, 1 KCNQ, and 4 EAG). Thus, much of the diversification of these channels took place in the stem cnidarian lineage prior to the emergence of modern cnidarian classes. In contrast, the stem bilaterian lineage, from which humans evolved, contained no more than nine voltage-gated K+ channels. These results hint at a complexity to electrical signaling in all cnidarians that contrasts with the perceived anatomical simplicity of their neuromuscular systems. These data provide a foundation from which the function of these cnidarian channels can be investigated, which will undoubtedly provide important insights into cnidarian physiology.


Assuntos
Cnidários , Canais de Potássio de Abertura Dependente da Tensão da Membrana , Anêmonas-do-Mar , Animais , Humanos , Cnidários/genética , Canais de Potássio de Abertura Dependente da Tensão da Membrana/genética , Filogenia , Anêmonas-do-Mar/genética , Genoma , Transdução de Sinais
13.
bioRxiv ; 2023 Feb 02.
Artigo em Inglês | MEDLINE | ID: mdl-36778242

RESUMO

Considerable evidence suggests that day-night rhythms in the functional expression of subthreshold potassium (K + ) channels regulate daily oscillations in the rates of spontaneous action potential firing of neurons in the suprachiasmatic nucleus (SCN), the master circadian pacemaker in mammals. The K + conductance(s) driving these daily rhythms in repetitive firing rates, however, have not been identified. To test the hypothesis that subthreshold Kv12.1/Kv12.2-encoded K + channels play a role, we obtained current-clamp recordings from SCN neurons in slices prepared from adult mice harboring targeted disruptions in the Kcnh8 (Kv12.1 -/- ) or Kcnh3 (Kv12.2 -/- ) locus. We found that mean nighttime repetitive firing rates were higher in Kv12.1 -/- and Kv12.2 -/- , than in wild type (WT), SCN neurons. In marked contrast, mean daytime repetitive firing rates were similar in Kv12.1 -/- , Kv12.2 -/- and WT SCN neurons, and the day-night difference in mean repetitive firing rates, a hallmark feature of WT SCN neurons, was eliminated in Kv12.1 -/- and Kv12.2 -/- SCN neurons. Similar results were obtained with in vivo shRNA-mediated acute knockdown of Kv12.1 or Kv12.2 in adult SCN neurons. Voltage-clamp experiments revealed that Kv12-encoded current densities in WT SCN neurons are higher at night than during the day. In addition, pharmacological block of Kv12-encoded currents increased the mean repetitive firing rate of nighttime, but not daytime, in WT SCN neurons. Dynamic clamp-mediated subtraction of modeled Kv12-encoded currents also selectively increased the mean repetitive firing rates of nighttime WT SCN neurons. Despite the elimination of nighttime decrease in the mean repetitive firing rates of SCN neurons, however, locomotor (wheel-running) activity remained rhythmic in Kv12.1 -/- , Kv12.2 -/- , Kv12.1-targeted shRNA-expressing, and Kv12.2-targeted shRNA-expressing animals.

14.
J Gen Physiol ; 155(9)2023 09 04.
Artigo em Inglês | MEDLINE | ID: mdl-37516908

RESUMO

Considerable evidence suggests that day-night rhythms in the functional expression of subthreshold potassium (K+) channels regulate daily oscillations in the spontaneous firing rates of neurons in the suprachiasmatic nucleus (SCN), the master circadian pacemaker in mammals. The K+ conductance(s) driving these daily rhythms in the repetitive firing rates of SCN neurons, however, have not been identified. To test the hypothesis that subthreshold Kv12.1/Kv12.2-encoded K+ channels play a role, we obtained current-clamp recordings from SCN neurons in slices prepared from adult mice harboring targeted disruptions in the Kcnh8 (Kv12.1-/-) or Kcnh3 (Kv12.2-/-) locus. We found that mean nighttime repetitive firing rates were higher in Kv12.1-/- and Kv12.2-/- than in wild type (WT), SCN neurons. In marked contrast, mean daytime repetitive firing rates were similar in Kv12.1-/-, Kv12.2-/-, and WT SCN neurons, and the day-night difference in mean repetitive firing rates, a hallmark feature of WT SCN neurons, was eliminated in Kv12.1-/- and Kv12.2-/- SCN neurons. Similar results were obtained with in vivo shRNA-mediated acute knockdown of Kv12.1 or Kv12.2 in adult SCN neurons. Voltage-clamp experiments revealed that Kv12-encoded current densities in WT SCN neurons are higher at night than during the day. In addition, the pharmacological block of Kv12-encoded currents increased the mean repetitive firing rate of nighttime, but not daytime, in WT SCN neurons. Dynamic clamp-mediated subtraction of modeled Kv12-encoded currents also selectively increased the mean repetitive firing rates of nighttime WT SCN neurons. Despite the elimination of the nighttime decrease in the mean repetitive firing rates of SCN neurons, however, locomotor (wheel-running) activity remained rhythmic in Kv12.1-/-, Kv12.2-/-, and Kv12.1-targeted shRNA-expressing, and Kv12.2-targeted shRNA-expressing animals.


Assuntos
Neurônios do Núcleo Supraquiasmático , Animais , Camundongos , Mamíferos , Neurônios , Potássio , RNA Interferente Pequeno , Núcleo Supraquiasmático
15.
J Gen Physiol ; 153(1)2021 01 04.
Artigo em Inglês | MEDLINE | ID: mdl-33275659

RESUMO

The functionally diverse cyclic nucleotide binding domain (CNBD) superfamily of cation channels contains both depolarization-gated (e.g., metazoan EAG family K+ channels) and hyperpolarization-gated channels (e.g., metazoan HCN pacemaker cation channels and the plant K+ channel KAT1). In both types of CNBD channels, the S4 transmembrane helix of the voltage sensor domain (VSD) moves outward in response to depolarization. This movement opens depolarization-gated channels and closes hyperpolarization-gated channels. External divalent cations and protons prevent or slow movement of S4 by binding to a cluster of acidic charges on the S2 and S3 transmembrane domains of the VSD and therefore inhibit activation of EAG family channels. However, a similar divalent ion/proton binding pocket has not been described for hyperpolarization-gated CNBD family channels. We examined the effects of external Cd2+ and protons on Arabidopsisthaliana KAT1 expressed in Xenopus oocytes and found that these ions strongly potentiate voltage activation. Cd2+ at 300 µM depolarizes the V50 of KAT1 by 150 mV, while acidification from pH 7.0 to 4.0 depolarizes the V50 by 49 mV. Regulation of KAT1 by Cd2+ is state dependent and consistent with Cd2+ binding to an S4-down state of the VSD. Neutralization of a conserved acidic charge in the S2 helix in KAT1 (D95N) eliminates Cd2+ and pH sensitivity. Conversely, introduction of acidic residues into KAT1 at additional S2 and S3 cluster positions that are charged in EAG family channels (N99D and Q149E in KAT1) decreases Cd2+ sensitivity and increases proton potentiation. These results suggest that KAT1, and presumably other hyperpolarization-gated plant CNBD channels, can open from an S4-down VSD conformation homologous to the divalent/proton-inhibited conformation of EAG family K+ channels.


Assuntos
Oócitos , Prótons , Animais , Íons
16.
Biophys J ; 97(1): 110-20, 2009 Jul 08.
Artigo em Inglês | MEDLINE | ID: mdl-19580749

RESUMO

Voltage-gated K+ channels share a common voltage sensor domain (VSD) consisting of four transmembrane helices, including a highly mobile S4 helix that contains the major gating charges. Activation of ether-a-go-go (EAG) family K+ channels is sensitive to external divalent cations. We show here that divalent cations slow the activation rate of two EAG family channels (Kv12.1 and Kv10.2) by forming a bridge between a residue in the S4 helix and acidic residues in S2. Histidine 328 in the S4 of Kv12.1 favors binding of Zn2+ and Cd2+, whereas the homologous residue Serine 321 in Kv10.2 contributes to effects of Mg2+ and Ni2+. This novel finding provides structural constraints for the position of transmembrane VSD helices in closed, ion-bound EAG family channels. Homology models of Kv12.1 and Kv10.2 VSD structures based on a closed-state model of the Shaker family K+ channel Kv1.2 match these constraints. Our results suggest close conformational conservation between closed EAG and Shaker family channels, despite large differences in voltage sensitivity, activation rates, and activation thresholds.


Assuntos
Cátions Bivalentes/metabolismo , Canais de Potássio Éter-A-Go-Go/química , Canais de Potássio Éter-A-Go-Go/metabolismo , Sequência de Aminoácidos , Animais , Cádmio/metabolismo , Canais de Potássio Éter-A-Go-Go/genética , Canal de Potássio Kv1.2/metabolismo , Magnésio/metabolismo , Potenciais da Membrana/fisiologia , Camundongos , Modelos Moleculares , Dados de Sequência Molecular , Mutação , Níquel/metabolismo , Técnicas de Patch-Clamp , Conformação Proteica , Homologia de Sequência de Aminoácidos , Xenopus , Zinco/metabolismo
17.
J Neurosci ; 28(39): 9640-51, 2008 Sep 24.
Artigo em Inglês | MEDLINE | ID: mdl-18815250

RESUMO

TRPA1 is a member of the transient receptor potential (TRP) family of ion channels and is expressed in a subset of nociceptive neurons. An increasing body of evidence suggests that TRPA1 functions as a chemical nocisensor for a variety of reactive chemicals, such as pungent natural compounds and environmental irritants. Activation of TRPA1 by reactive compounds has been demonstrated to be mediated through covalent modification of cytoplasmic cysteines located in the N terminus of the channel, rather than classical lock-and-key binding. TRPA1 activity is also modulated by numerous nonreactive chemicals, but the underlying mechanism is unknown. Menthol, a natural nonreactive cooling compound, is best known as an activator of TRPM8, a related TRP ion channel required for cool thermosensation in vivo. More recently, menthol has been shown to be an activator of mouse TRPA1 at low concentrations, and a blocker, at high concentrations. Here, we show that human TRPA1 is only activated by menthol, whereas TRPA1 from nonmammalian species are insensitive to menthol. Mouse-human TRPA1 chimeras reveal the pore region [including transmembrane domain 5 (TM5) and TM6] as the critical domain determining whether menthol can act as an inhibitor. Furthermore, chimeras between Drosophila melanogaster and mammalian TRPA1 highlight specific residues within TM5 critical for menthol responsiveness. Interestingly, this TM5 region also determines the sensitivity of TRPA1 to other chemical modulators. These data suggest separable structural requirements for modulation of TRPA1 by covalent and nonreactive molecules. Whether this region is involved in binding or gating of TRPA1 channels is discussed.


Assuntos
Antipruriginosos/farmacologia , Proteínas de Choque Térmico HSP90/metabolismo , Potenciais da Membrana/efeitos dos fármacos , Mentol/farmacologia , Animais , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Benzamidas/farmacologia , Linhagem Celular Transformada , Clonagem Molecular/métodos , Relação Dose-Resposta a Droga , Relação Dose-Resposta à Radiação , Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Drosophila melanogaster , Estimulação Elétrica/métodos , Proteínas de Choque Térmico HSP90/agonistas , Proteínas de Choque Térmico HSP90/antagonistas & inibidores , Proteínas de Choque Térmico HSP90/genética , Humanos , Proteínas Luminescentes/genética , Proteínas Luminescentes/metabolismo , Potenciais da Membrana/fisiologia , Mutagênese/fisiologia , Técnicas de Patch-Clamp/métodos , Estrutura Terciária de Proteína/fisiologia , Timol/farmacologia , Transfecção/métodos
18.
Elife ; 72018 12 28.
Artigo em Inglês | MEDLINE | ID: mdl-30592460

RESUMO

Genome sequence data from a range of animal species are raising questions about the origins of glutamate receptors.


Assuntos
Filogenia , Receptores de Glutamato , Animais
19.
J Gen Physiol ; 150(12): 1702-1721, 2018 12 03.
Artigo em Inglês | MEDLINE | ID: mdl-30322883

RESUMO

The Shaker-like family of voltage-gated K+ channels comprises four functionally independent gene subfamilies, Shaker (Kv1), Shab (Kv2), Shaw (Kv3), and Shal (Kv4), each of which regulates distinct aspects of neuronal excitability. Subfamily-specific assembly of tetrameric channels is mediated by the N-terminal T1 domain and segregates Kv1-4, allowing multiple channel types to function independently in the same cell. Typical Shaker-like Kv subunits can form functional channels as homotetramers, but a group of mammalian Kv2-related genes (Kv5.1, Kv6s, Kv8s, and Kv9s) encodes subunits that have a "silent" or "regulatory" phenotype characterized by T1 self-incompatibility. These channels are unable to form homotetramers, but instead heteromerize with Kv2.1 or Kv2.2 to diversify the functional properties of these delayed rectifiers. While T1 self-incompatibility predicts that these heterotetramers could contain up to two regulatory (R) subunits, experiments show a predominance of 3:1R stoichiometry in which heteromeric channels contain a single regulatory subunit. Substitution of the self-compatible Kv2.1 T1 domain into the regulatory subunit Kv6.4 does not alter the stoichiometry of Kv2.1:Kv6.4 heteromers. Here, to identify other channel structures that might be responsible for favoring the 3:1R stoichiometry, we compare the sequences of mammalian regulatory subunits to independently evolved regulatory subunits from cnidarians. The most widespread feature of regulatory subunits is the presence of atypical substitutions in the highly conserved consensus sequence of the intracellular S6 activation gate of the pore. We show that two amino acid substitutions in the S6 gate of the regulatory subunit Kv6.4 restrict the functional stoichiometry of Kv2.1:Kv6.4 to 3:1R by limiting the formation and function of 2:2R heteromers. We propose a two-step model for the evolution of the asymmetric 3:1R stoichiometry, which begins with evolution of self-incompatibility to establish the regulatory phenotype, followed by drift of the activation gate consensus sequence under relaxed selection to limit stoichiometry to 3:1R.


Assuntos
Modelos Moleculares , Canais de Potássio de Abertura Dependente da Tensão da Membrana/fisiologia , Sequência de Aminoácidos , Animais , Cádmio , Camundongos , Microscopia de Fluorescência , Oócitos , Técnicas de Patch-Clamp , Canais de Potássio de Abertura Dependente da Tensão da Membrana/química , Anêmonas-do-Mar , Xenopus
20.
Curr Opin Plant Biol ; 33: 157-167, 2016 10.
Artigo em Inglês | MEDLINE | ID: mdl-27518594

RESUMO

By controlling the opening and closure of the stomatal pores through which gas exchange occurs, guard cells regulate two of the most important plant physiological processes: photosynthesis and transpiration. Accordingly, guard cells have evolved exquisite sensory systems. Here we summarize recent literature on guard cell sensing of light, drought (via the phytohormone abscisic acid (ABA)), and CO2. New advances in our understanding of how guard cells satisfy the energetic and osmotic requirements of stomatal opening and utilize phosphorylation to regulate the anion channels and aquaporins involved in ABA-stimulated stomatal closure are highlighted. Omics and modeling approaches are providing new information that will ultimately allow an integrated understanding of guard cell physiology.


Assuntos
Ácido Abscísico/metabolismo , Dióxido de Carbono/metabolismo , Luz , Reguladores de Crescimento de Plantas/metabolismo , Estômatos de Plantas/fisiologia , Canais Iônicos/metabolismo , Fosforilação
SELEÇÃO DE REFERÊNCIAS
DETALHE DA PESQUISA