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1.
Curr Top Dev Biol ; 102: 385-421, 2013.
Artigo em Inglês | MEDLINE | ID: mdl-23287041

RESUMO

To succeed in fertilization, spermatozoa must decode environmental cues which require a set of ion channels. Recent findings have revealed that K(+) and Cl(-) channels participate in some of the main sperm functions. This work reviews the evidence indicating the involvement of K(+) and Cl(-) channels in motility, maturation, and the acrosome reaction, and the advancement in identifying their molecular identity and modes of regulation. Improving our insight on how these channels operate will strengthen our ability to surmount some infertility problems, improve animal breeding, preserve biodiversity, and develop selective and secure male contraceptives.


Assuntos
Canais de Cloreto/metabolismo , Proteínas de Membrana Transportadoras/metabolismo , Canais de Potássio/metabolismo , Espermatozoides/fisiologia , Reação Acrossômica/fisiologia , Animais , Humanos , Masculino , Capacitação Espermática/fisiologia
2.
WormBook ; : 1-15, 2005 Dec 30.
Artigo em Inglês | MEDLINE | ID: mdl-18050399

RESUMO

Ion channels are the "transistors" (electronic switches) of the brain that generate and propagate electrical signals in the aqueous environment of the brain and nervous system. Potassium channels are particularly important because, not only do they shape dynamic electrical signaling, they also set the resting potentials of almost all animal cells. Without them, animal life as we know it would not exist, much less higher brain function. Until the completion of the C. elegans genome sequencing project the size and diversity of the potassium channel extended gene family was not fully appreciated. Sequence data eventually revealed a total of approximately 70 genes encoding potassium channels out of the more than 19,000 genes in the genome. This seemed to be an unexpectedly high number of genes encoding potassium channels for an animal with a small nervous system of only 302 neurons. However, it became clear that potassium channels are expressed in all cell types, not only neurons, and that many cells express a complex palette of multiple potassium channels. All types of potassium channels found in C. elegans are conserved in mammals. Clearly, C. elegans is "simple" only in having a limited number of cells dedicated to each organ system; it is certainly not simple with respect to its biochemistry and cell physiology.


Assuntos
Caenorhabditis elegans/fisiologia , Canais de Potássio/fisiologia , Animais , Caenorhabditis elegans/genética , Genes de Helmintos , Humanos , Família Multigênica , Canais de Potássio/genética
3.
Proc Natl Acad Sci U S A ; 100(24): 14391-6, 2003 Nov 25.
Artigo em Inglês | MEDLINE | ID: mdl-14612577

RESUMO

GFP-promoter experiments have previously shown that at least nine genes encoding potassium channel subunits are expressed in Caenorhabditis elegans muscle. By using genetic, RNA interference, and physiological techniques we revealed the molecular identity of the major components of the outward K+ currents in body wall muscle cells in culture. We found that under physiological conditions, outward current is dominated by the products of only two genes, Shaker (Kv1) and Shal (Kv4), both expressing voltage-dependent potassium channels. Other channels may be held in reserve to respond to particular circumstances. Because GFP-promoter experiments indicated that slo-2 expression is prominent, we created a deletion mutant to identify the SLO-2 current in vivo. In both whole-cell and single-channel modes, in vivo SLO-2 channels were active only when intracellular Ca2+ and Cl- were raised above normal physiological conditions, as occurs during hypoxia. Under such conditions, SLO-2 is the largest outward current, contributing up to 87% of the total current. Other channels are present in muscle, but our results suggest that they are unlikely to contribute a large outward component under physiological conditions. However, they, too, may contribute currents conditional on other factors. Hence, the picture that emerges is of a complex membrane with a small number of household conductances functioning under normal circumstances, but with additional conductances that are activated during unusual circumstances.


Assuntos
Proteínas de Caenorhabditis elegans/genética , Proteínas de Caenorhabditis elegans/metabolismo , Caenorhabditis elegans/genética , Caenorhabditis elegans/metabolismo , Canais de Potássio de Abertura Dependente da Tensão da Membrana , Canais de Potássio/genética , Canais de Potássio/metabolismo , Animais , Animais Geneticamente Modificados , Genes de Helmintos , Proteínas de Membrana Transportadoras/genética , Proteínas de Membrana Transportadoras/metabolismo , Músculos/metabolismo , Mutação , Interferência de RNA , Superfamília Shaker de Canais de Potássio , Canais de Potássio Shal
4.
Neuron ; 32(5): 867-81, 2001 Dec 06.
Artigo em Inglês | MEDLINE | ID: mdl-11738032

RESUMO

Six mutants of SLO-1, a large-conductance, Ca(2+)-activated K(+) channel of C. elegans, were obtained in a genetic screen for regulators of neurotransmitter release. Mutants were isolated by their ability to suppress lethargy of an unc-64 syntaxin mutant that restricts neurotransmitter release. We measured evoked postsynaptic currents at the neuromuscular junction in both wild-type and mutants and observed that the removal of SLO-1 greatly increased quantal content primarily by increasing duration of release. The selective isolation of slo-1 as the only ion channel mutant derived from a whole genomic screen to detect regulators of neurotransmitter release suggests that SLO-1 plays an important, if not unique, role in regulating neurotransmitter release.


Assuntos
Junção Neuromuscular/metabolismo , Neurotransmissores/metabolismo , Canais de Potássio Cálcio-Ativados/fisiologia , Canais de Potássio/fisiologia , Sequência de Aminoácidos , Animais , Caenorhabditis elegans/genética , Caenorhabditis elegans/metabolismo , Proteínas de Caenorhabditis elegans , Potenciais Pós-Sinápticos Excitadores/fisiologia , Canais de Potássio Ativados por Cálcio de Condutância Alta , Dados de Sequência Molecular , Mutação/genética , Junção Neuromuscular/genética , Neurotransmissores/genética , Canais de Potássio Cálcio-Ativados/genética , Canais de Potássio Cálcio-Ativados/metabolismo , Terminações Pré-Sinápticas/metabolismo , Xenopus
5.
Neuroscience ; 103(4): 853-9, 2001.
Artigo em Inglês | MEDLINE | ID: mdl-11301195

RESUMO

The relationship between the genome and the evolution of the nervous system may differ between an animal like C. elegans with 302 neurons, and mammals with tens of billions of neurons. Here we report that a class of nonconserved potassium channels highly expanded in C. elegans may play a special role in the evolution of its nervous system. The C. elegans genome contains an extended gene family of potassium channels whose members fall into two evolutionary divergent classes. One class constitutes an ancient conserved "set" of K+ channels with orthologues in both humans and Drosophila and a second larger class made up of rapidly evolving genes unique to C. elegans. Chief among this second class are novel potassium channels having four transmembrane domains per subunit that function as regulated leak conductances to modulate cell electrical excitability. This inventory of novel potassium channels is far larger in C. elegans than in humans or Drosophila. We found that, unlike conserved channel genes, the majority of these genes are expressed in very few cells. We also identified DNA enhancer elements associated with these genes that direct gene expression to individual neurons. We conclude that C. elegans may maintain an exceptionally large inventory of these channels (as well as ligand-gated channels) as an adaptive mechanism to "fine tune" individual neurons, making the most of its limited circuitry.


Assuntos
Evolução Biológica , Neurônios/fisiologia , Adaptação Fisiológica , Animais , Sequência de Bases/genética , Caenorhabditis/genética , Caenorhabditis/fisiologia , Caenorhabditis elegans/fisiologia , DNA/genética , Elementos Facilitadores Genéticos/genética , Elementos Facilitadores Genéticos/fisiologia , Expressão Gênica , Dados de Sequência Molecular , Família Multigênica/genética , Canais de Potássio/classificação , Canais de Potássio/genética , Canais de Potássio/fisiologia , Isoformas de Proteínas/genética , Isoformas de Proteínas/fisiologia
6.
J Neurosci ; 20(20): 7517-24, 2000 Oct 15.
Artigo em Inglês | MEDLINE | ID: mdl-11027209

RESUMO

Within the Caenorhabditis elegans genome there exist at least 42 genes encoding TWK (two-P domain K(+)) channels, potassium channel subunits that contain two pore regions and four transmembrane domains. We now report the first functional characterization of a TWK channel from C. elegans. Although potassium channels have been reported to be activated by a variety of factors, TWK-18 currents increase dramatically with increases in temperature. Two mutant alleles of the twk-18 gene confer uncoordinated movement and paralysis in C. elegans. Expression of wild-type and mutant TWK-18 channels in Xenopus oocytes showed that mutant channels express much larger potassium currents than wild-type channels. Promoter-green fluorescent protein fusion experiments indicate that TWK-18 is expressed in body wall muscle. Our genetic and physiological data suggest that the movement defects observed in mutant twk-18 animals may be explained by an increased activity of the mutant TWK-18 channels.


Assuntos
Proteínas de Caenorhabditis elegans , Mutação , Canais de Potássio/genética , Temperatura , Animais , Caenorhabditis elegans , Células Cultivadas , Mapeamento Cromossômico , Expressão Gênica , Proteínas de Fluorescência Verde , Locomoção/genética , Proteínas Luminescentes/genética , Dados de Sequência Molecular , Músculos/metabolismo , Mutagênese , Oócitos/citologia , Oócitos/metabolismo , Especificidade de Órgãos , Técnicas de Patch-Clamp , Potássio/metabolismo , Canais de Potássio/metabolismo , Regiões Promotoras Genéticas/genética , Estrutura Terciária de Proteína/genética , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Cromossomo X/genética , Xenopus
7.
Neuron ; 26(3): 647-57, 2000 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-10896160

RESUMO

TRP and TRPL are two light-sensitive cation channel subunits required for the Drosophila photoresponse; however, our understanding of the identities, subunit composition, and function of the light-responsive channels is incomplete. To explain the residual photoresponse that remains in the trp mutant, a third TRP-related subunit has previously been proposed to function with TRPL. Here, we identify such a subunit, TRPgamma. We show that TRPgamma is highly enriched in photoreceptor cells and preferentially heteromultimerizes with TRPL in vitro and in vivo. The N-terminal domain of TRPgamma dominantly suppressed the TRPL-dependent photoresponse, indicating that TRPgamma-TRPL heteromultimers contribute to the photoresponse. While TRPL and TRPgamma homomultimers are constitutively active, we demonstrate that TRPL-TRPgamma heteromultimers form a regulated phospholipase C- (PLC-) stimulated channel.


Assuntos
Canais de Cálcio/fisiologia , Proteínas de Ligação a Calmodulina/metabolismo , Cátions/metabolismo , Proteínas de Drosophila , Proteínas de Insetos/fisiologia , Canais Iônicos/metabolismo , Canais Iônicos/fisiologia , Proteínas de Membrana/metabolismo , Isoformas de Proteínas/fisiologia , Sequência de Aminoácidos/genética , Animais , Canais de Cálcio/genética , Proteínas de Ligação a Calmodulina/genética , Drosophila/fisiologia , Eletrorretinografia , Proteínas de Insetos/genética , Canais Iônicos/genética , Luz , Proteínas de Membrana/genética , Dados de Sequência Molecular , Organelas/metabolismo , Células Fotorreceptoras de Invertebrados/metabolismo , Isoformas de Proteínas/genética , Retina/efeitos da radiação , Canais de Potencial de Receptor Transitório
8.
Nat Neurosci ; 3(8): 771-9, 2000 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-10903569

RESUMO

The gating of different potassium channels depends on many diverse factors. We now report a unique example of a K+ channel with a Cl - dependence. The slo-2 gene was cloned from Caenorhabditis elegans and is widely expressed in both neurons and muscles; it was highly abundant, as suggested by its high representation in the C. elegans EST database. SLO-2, like its paralogue, SLO-1, was also dependent on Ca 2+. We show by site-directed mutagenesis that its requirements for both Cl- and Ca2+ are synergistic and associated with the same functional domain. SLO-2's dependence on Cl - implies that intracellular Cl- homeostasis may be important in regulating cellular excitability through this unusual K+ channel.


Assuntos
Proteínas de Caenorhabditis elegans , Cloretos/fisiologia , Proteínas de Membrana Transportadoras , Neurônios/fisiologia , Canais de Potássio Cálcio-Ativados , Canais de Potássio/fisiologia , Sequência de Aminoácidos , Animais , Animais Geneticamente Modificados , Caenorhabditis elegans , Cálcio/farmacologia , Cloretos/farmacologia , Sequência Conservada , Proteínas de Fluorescência Verde , Proteínas de Helminto/fisiologia , Canais de Potássio Ativados por Cálcio de Condutância Alta , Proteínas Luminescentes/genética , Mamíferos , Dados de Sequência Molecular , Músculos/fisiologia , Canais de Potássio/química , Canais de Potássio/genética , Alinhamento de Sequência , Homologia de Sequência de Aminoácidos
9.
J Neurosci ; 19(22): 9831-40, 1999 Nov 15.
Artigo em Inglês | MEDLINE | ID: mdl-10559392

RESUMO

K(+) channels are key regulators of cellular excitability. Mutations that activate K(+) channels can lower cellular excitability, whereas those that inhibit K(+) channels may increase excitability. We show that the Caenorhabditis elegans egl-2 gene encodes an eag K(+) channel and that a gain-of-function mutation in egl-2 blocks excitation in neurons and muscles by causing the channel to open at inappropriately negative voltages. Tricyclic antidepressants reverse egl-2(gf) mutant phenotypes, suggesting that EGL-2 is a tricyclic target. We verified this by showing that EGL-2 currents are inhibited by imipramine. Similar inhibition is observed with the mouse homolog MEAG, suggesting that inhibition of EAG-like channels may mediate some clinical side effects of this class of antidepressants.


Assuntos
Caenorhabditis elegans/fisiologia , Imipramina/farmacologia , Neurônios/fisiologia , Canais de Potássio/fisiologia , Sequência de Aminoácidos , Animais , Caenorhabditis elegans/genética , Quimiotaxia , Canais de Potássio Éter-A-Go-Go , Metanossulfonato de Etila , Feminino , Genótipo , Proteínas de Helminto/genética , Camundongos , Dados de Sequência Molecular , Atividade Motora , Mutagênese , Oviposição , Fenótipo , Bloqueadores dos Canais de Potássio , Canais de Potássio/química , Alinhamento de Sequência , Homologia de Sequência de Aminoácidos
10.
Ann N Y Acad Sci ; 868: 286-303, 1999 Apr 30.
Artigo em Inglês | MEDLINE | ID: mdl-10414302

RESUMO

As many as 50 genes in the C. elegans genome may encode K+ channels belonging to the novel structural class of two-pore (4TM) channels. Many 4TM channels can be grouped into channel subfamilies. We analyzed 4TM channels in C. elegans using methods made possible by having complete genomic sequence. Two genes were chosen for comprehensive analysis, n2P16 and n2P17. By comparing the pattern of conservation in genomic DNA sequences between C. elegans and a closely related species, C. briggsae, we were able to identify all coding regions and predict the gene structure for these two genes. Given the extent of the 4TM channel family, we were surprised to discover that n2P17 produced at least six alternative transcripts encoding a constant central region and variable amino- and carboxyl-termini. Blocks of highly conserved DNA sequences in noncoding regions were also apparent and most likely confer important regulatory functions. The interspecies comparison of the deduced channel proteins revealed that the extracellular loop between M1 and P1 is an apparent hot spot for evolutionary change in both channels. This contrasts with the membrane-spanning domains that are highly conserved. Analysis of intron positions for 36 channels revealed that introns are frequently present at an identical position within the pore region, but very few are located in membrane-spanning domains.


Assuntos
Caenorhabditis elegans/genética , Canais de Potássio/genética , Processamento Alternativo , Sequência de Aminoácidos , Animais , Sequência de Bases , Sequência Conservada , Bases de Dados Factuais , Evolução Molecular , Genes de Helmintos/genética , Íntrons , Dados de Sequência Molecular , Filogenia , Canais de Potássio/classificação , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Alinhamento de Sequência
11.
Nat Neurosci ; 2(5): 416-21, 1999 May.
Artigo em Inglês | MEDLINE | ID: mdl-10321244

RESUMO

Both intracellular calcium and voltage activate Slo1, a high-conductance potassium channel, linking calcium with electrical excitability. Using molecular techniques, we created a calcium-insensitive variant of this channel gated by voltage alone. Calcium sensitivity was restored by adding back small portions of the carboxyl (C)-terminal 'tail' domain. Two separate regions of the tail independently conferred different degrees of calcium sensitivity; together, they restored essentially wild-type calcium dependence. These results suggest that, in the absence of calcium, the Slo1 tail inhibits voltage-dependent gating, and that calcium removes this inhibition. Slo1 may have evolved from an ancestral voltage-sensitive potassium channel represented by the core; the tail may represent the more recent addition of a calcium-dependent modulatory domain.


Assuntos
Cálcio/fisiologia , Canais de Potássio Cálcio-Ativados , Canais de Potássio/fisiologia , Transplante , Sequência de Aminoácidos , Animais , Ativação do Canal Iônico , Canais de Potássio Ativados por Cálcio de Condutância Alta , Potenciais da Membrana/fisiologia , Dados de Sequência Molecular , Técnicas de Patch-Clamp , Estrutura Terciária de Proteína , Xenopus
12.
J Physiol ; 514 ( Pt 3): 639-53, 1999 Feb 01.
Artigo em Inglês | MEDLINE | ID: mdl-9882736

RESUMO

1. A low stringency polymerase chain reaction (PCR) homology screening procedure was used to probe a mouse liver cDNA library to identify novel inward rectifier K+ channel genes. A single gene (mLV1) was identified that exhibited extensive sequence homology with previously cloned inward rectifier K+ channel genes. The mLV1 gene showed greatest sequence identity with genes belonging to the Kir4 subfamily. The amino acid sequence of mLV1 was 96 % identical to a Kir channel cloned from human kidney (hKir4.2), and approximately 60 % identical to the Kir4.1 channel cloned from human and rat, so that mLV1 was classified as mKir4.2. 2. Xenopus oocytes injected with cRNA encoding mKir4.2 displayed a large inwardly rectifying K+ current, while control oocytes injected with H2O displayed no similar K+ current. The current was blocked by Ba2+ and Cs+ in a voltage-dependent fashion and displayed inward rectification that was intermediate between that of the strong inward rectifier Kir2.1 and the weak inward rectifier Kir1.1. The current was weakly blocked by TEA in a voltage-independent fashion. 3. mKir4.2 current was subject to modulation by several distinct mechanisms. Intracellular acidification decreased mKir4.2 current in a reversible fashion, while activation of protein kinase C decreased mKir4.2 current in a manner that was not rapidly reversible. Incubation of oocytes in elevated [K+] produced a slowly developing enhancement of current. 4. Oocytes co-injected with cRNA for mKir4.2 and Kir5.1, a protein that does not form functional homomeric channels, displayed membrane currents with properties distinct from those expressing mKir4.2 alone. Co-injected oocytes displayed larger currents than mKir4.2, with novel kinetic properties and an increased sensitivity to Ba2+ block at negative potentials, suggesting that mKir4.2 forms functional heteromultimeric channels with Kir5.1, as has been shown for Kir4.1 5. These results demonstrate for the first time that a Kir4.2 channel gene product forms functional channels in Xenopus oocytes, that these Kir channels display novel properties, and that Kir4.2 subunits may be responsible for physiological modulation of functional Kir channels.


Assuntos
Fígado/metabolismo , Canais de Potássio Corretores do Fluxo de Internalização , Canais de Potássio/biossíntese , Sequência de Aminoácidos , Animais , Clonagem Molecular , Estimulação Elétrica , Eletrofisiologia , Biblioteca Gênica , Humanos , Concentração de Íons de Hidrogênio , Potenciais da Membrana/fisiologia , Camundongos , Dados de Sequência Molecular , Oócitos/metabolismo , Técnicas de Patch-Clamp , Reação em Cadeia da Polimerase , Bloqueadores dos Canais de Potássio , Canais de Potássio/genética , RNA Mensageiro/biossíntese , Ratos , Especificidade da Espécie , Xenopus laevis
13.
Am J Physiol ; 275(2): H600-8, 1998 08.
Artigo em Inglês | MEDLINE | ID: mdl-9683449

RESUMO

The Ca2+-sensitive K+ channel (maxi-K+) is an important modulator of corporal smooth muscle tone. The goal of these studies was twofold: 1) to determine the feasibility of transfecting corporal smooth muscle cells in vivo with the hSlo cDNA, which encodes for the human smooth muscle maxi-K+ channel, and 2) to determine whether transfection of the maxi-K+ channel would affect the physiological response to cavernous nerve stimulation in a rat model in vivo. Intracorporal microinjection of pCMVbeta/Lac Z DNA in 10-wk-old rats resulted in significant incorporation and expression of beta-galactosidase activity in 10 of 12 injected animals for up to 75 days postinjection. Moreover, electrical stimulation of the cavernous nerve revealed that, relative to the responses obtained in age-matched control animals (N = 12), intracavernous injection of naked pcDNA/hSlo DNA was associated with a statistically significant elevation in the mean amplitude of the intracavernous pressure response at all levels of current stimulation (range 0.5-10 mA) at both 1 mo (N = 5) and 2 mo (N = 8) postinjection. Furthermore, qualitatively similar observations were made at 3 mo (N = 2) and 4 mo (N = 2) postinjection. These data indicate that naked hSlo DNA is quite easily incorporated into corporal smooth muscle and, furthermore, that expression is sustained for at least 2 mo in corporal smooth muscle cells in vivo. Finally, after expression, hSlo is capable of measurably altering nerve-stimulated penile erection. Taken together, these data provide compelling evidence for the potential utility of gene therapy in the treatment of erectile dysfunction.


Assuntos
Músculo Liso/fisiologia , Ereção Peniana/fisiologia , Pênis/fisiologia , Canais de Potássio Cálcio-Ativados , Canais de Potássio/fisiologia , Transfecção/métodos , Animais , Clonagem Molecular , DNA Complementar , Estimulação Elétrica , Técnicas de Transferência de Genes , Humanos , Canais de Potássio Ativados por Cálcio de Condutância Alta , Masculino , Microinjeções , Músculo Liso/inervação , Pênis/inervação , Canais de Potássio/biossíntese , Canais de Potássio/genética , Pressão , Ratos , Ratos Sprague-Dawley , Proteínas Recombinantes de Fusão/biossíntese , beta-Galactosidase/biossíntese , beta-Galactosidase/genética
14.
J Biol Chem ; 273(6): 3509-16, 1998 Feb 06.
Artigo em Inglês | MEDLINE | ID: mdl-9452476

RESUMO

Potassium channels have evolved to play specialized roles in both excitable and inexcitable tissues. Here we describe the cloning and expression of Slo3, a novel potassium channel abundantly expressed in mammalian spermatocytes. Slo3 represents a new and unique type of potassium channel regulated by both intracellular pH and membrane voltage. Reverse transcription-polymerase chain reaction, Northern analysis, and in situ hybridization show that Slo3 is primarily expressed in testis in both mouse and human. Because of its sensitivity to both pH and voltage, Slo3 could be involved in sperm capacitation and/or the acrosome reaction, essential steps in fertilization where changes in both intracellular pH and membrane potential are known to occur. The protein sequence of mSlo3 (the mouse Slo3 homologue) is similar to Slo1, the large conductance, calcium- and voltage-gated potassium channel. These results suggest that Slo channels comprise a multigene family, defined by a combination of sensitivity to voltage and a variety of intracellular factors. Northern analysis from human testis indicates that a Slo3 homologue is present in humans and conserved with regard to sequence, transcript size, and tissue distribution. Because of its high testis-specific expression, pharmacological agents that target human Slo3 channels may be useful in both the study of fertilization as well as in the control or enhancement of fertility.


Assuntos
Canais de Potássio Cálcio-Ativados , Canais de Potássio/genética , Espermatócitos/metabolismo , Sequência de Aminoácidos , Animais , Clonagem Molecular , Humanos , Concentração de Íons de Hidrogênio , Ativação do Canal Iônico , Canais de Potássio Ativados por Cálcio de Condutância Alta , Masculino , Camundongos , Dados de Sequência Molecular , Canais de Potássio/química , Canais de Potássio/metabolismo , RNA Complementar/genética , Homologia de Sequência de Aminoácidos
15.
Biophys J ; 73(3): 1355-63, 1997 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-9284303

RESUMO

The high-conductance Ca2+-activated K+ channel (mSlo) plays a vital role in regulating calcium entry in many cell types. mSlo channels behave like voltage-dependent channels, but their voltage range of activity is set by intracellular free calcium. The mSlo subunit has two parts: a "core" resembling a subunit from a voltage-dependent K+ channel, and an appended "tail" that plays a role in calcium sensing. Here we present evidence for a site on the tail that interacts with calcium. This site, the "calcium bowl," is a novel calcium-binding motif that includes a string of conserved aspartate residues. Mutations of the calcium bowl fall into two categories: 1) those that shift the position of the G-V relation a similar amount at all [Ca2+], and 2) those that shift the position of the G-V relation only at low [Ca2+]. None of these mutants alters the slope of the G-V curve. These mutant phenotypes are apparent in calcium ion, but not in cadmium ion, where mutant and wild type are indistinguishable. This suggests that the calcium bowl is sensitive to calcium ion, but insensitive to cadmium ion. The presence and independence of a second calcium-binding site is inferred because channels still respond to increasing levels of [Ca2+] or [Cd2+], even when the calcium bowl is mutationally deleted. Thus a low level of activation in the absence of divalent cations is identical in mutant and wild-type channels, possibly because of activation of this second Ca2+-binding site.


Assuntos
Cálcio/farmacologia , Canais de Potássio Cálcio-Ativados , Canais de Potássio/química , Canais de Potássio/fisiologia , Sequência de Aminoácidos , Animais , Cádmio/farmacologia , Dípteros , Feminino , Humanos , Canais de Potássio Ativados por Cálcio de Condutância Alta , Potenciais da Membrana/efeitos dos fármacos , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Nematoides , Oócitos/fisiologia , Técnicas de Patch-Clamp , Mutação Puntual , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Alinhamento de Sequência , Homologia de Sequência de Aminoácidos , Xenopus laevis
16.
Neuron ; 19(1): 151-64, 1997 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-9247271

RESUMO

Mutations in the C. elegans egl-36 gene result in defective excitation of egg-laying and enteric muscles. Dominant gain-of-function alleles inhibit enteric and egg-laying muscle contraction, whereas a putative null mutation has no observed phenotype. egl-36 encodes a Shaw-type (Kv3) voltage-dependent potassium channel subunit. In Xenopus oocytes, wild-type egl-36 expresses noninactivating channels with slow activation kinetics. One gain-of-function mutation causes a single amino acid substitution in S6, and the other causes a substitution in the cytoplasmic amino terminal domain. Both mutant alleles produce channels dramatically shifted in their midpoints of activation toward hyperpolarized voltages. An egl-36::gfp fusion is expressed in egg-laying muscles and in a pair of enteric muscle motor neurons. The mutant egl-36 phenotypes can thus be explained by expression in these cells of potassium channels that are inappropriately opened at hyperpolarized potentials, causing decreased excitability due to increased potassium conductance.


Assuntos
Genes/genética , Potenciais da Membrana/fisiologia , Mutação/genética , Canais de Potássio/genética , Animais , Sequência de Bases , Dados de Sequência Molecular , Oócitos , Técnicas de Patch-Clamp , Fenótipo , Xenopus
17.
J Biol Chem ; 272(18): 11710-7, 1997 May 02.
Artigo em Inglês | MEDLINE | ID: mdl-9115223

RESUMO

cDNA libraries from rat chromaffin cells and PC12 cells were screened for homologs to the mouse mSlo gene that encodes a large conductance, calcium (Ca2+)- and voltage-activated potassium channel (BK channel). One Slo variant contained sequence encoding a cysteine-rich, 59-amino acid insert for a previously described site of alternative splicing. This insert is reminiscent of zinc-finger domains. The exon was found in RNA from pancreas, anterior pituitary, cerebellum, and hippocampus. Expression in Xenopus oocytes of a Slo construct containing this exon conferred a -30 to -20 mV shift of the conductance-voltage curve. A previously uncharacterized alternative splice junction near the C-terminal end of Slo was also identified. In contrast to BK channels in rat chromaffin cells, none of the Slo variants exhibited inactivation when expressed in Xenopus oocytes. PCR screening of chromaffin cell RNA failed to reveal a homolog of an accessory beta subunit known to influence Slo channel function. Furthermore, a beta-subunit-dependent Slo channel activator, dehydrosoyasaponin I, was without effect on chromaffin cell BK current. The results argue that an accessory subunit may not be a required component of the native chromaffin cell BK channel.


Assuntos
Medula Suprarrenal/metabolismo , Processamento Alternativo , Células Cromafins/metabolismo , Cisteína , Éxons , Canais de Potássio Cálcio-Ativados , Canais de Potássio/genética , Canais de Potássio/fisiologia , Sequência de Aminoácidos , Animais , Sequência de Bases , Cálcio/farmacologia , Bovinos , Células Cultivadas , Primers do DNA , Feminino , Variação Genética , Subunidades alfa do Canal de Potássio Ativado por Cálcio de Condutância Alta , Subunidades beta do Canal de Potássio Ativado por Cálcio de Condutância Alta , Canais de Potássio Ativados por Cálcio de Condutância Alta , Potenciais da Membrana/efeitos dos fármacos , Camundongos , Dados de Sequência Molecular , Oócitos/efeitos dos fármacos , Oócitos/fisiologia , Especificidade de Órgãos , Células PC12 , Técnicas de Patch-Clamp , Reação em Cadeia da Polimerase , Canais de Potássio/biossíntese , Ratos , Proteínas Recombinantes/metabolismo , Homologia de Sequência de Aminoácidos , Xenopus laevis
18.
J Neurosci ; 17(1): 32-44, 1997 Jan 01.
Artigo em Inglês | MEDLINE | ID: mdl-8987734

RESUMO

Shal (Kv4) potassium channel genes encode classical subthreshold A-currents, and their regulation may be a key factor in determining neuronal firing frequency. The inactivation rate of Shal channels is increased by a presently unidentified class of proteins in both Drosophila and mammals. We have cloned a novel Shal channel subunit (jShalgamma1) from the jellyfish Polyorchis penicillatus that alters Shal currents from both invertebrates and vertebrates. When co-expressed with the conserved jellyfish Shal homolog jShal1, jShalgamma1 dramatically changes both the rate of inactivation and voltage range of activation and steady-state inactivation. jShalgamma1 provides fast inactivation by a classic N-type mechanism, which is independent of its effects on voltage dependence. jShalgamma1 forms functional channels only as a heteromultimer, and jShalgamma1 + jShal1 heteromultimers are functional only in a 2:2 subunit stoichiometry.


Assuntos
Fragmentos de Peptídeos/fisiologia , Canais de Potássio/genética , Canais de Potássio/fisiologia , Cifozoários/genética , Sequência de Aminoácidos , Animais , Clonagem Molecular , Sequência Conservada , Eletrofisiologia , Matemática , Dados de Sequência Molecular , Canais de Potássio/química
19.
Diabetologia ; 39(8): 891-8, 1996 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-8858210

RESUMO

Insulin secretion from pancreatic beta cells is dependent on membrane potential changes that result from the concerted regulation of multiple ion channels. Among the distinct K+ channels known to be expressed in beta cells, large conductance Ca(2+)-activated K+ channels have been suggested to play an important role in stimulus-secretion coupling. In the course of a strategy to identify transcripts that are enriched in human pancreatic islet cells, we isolated a partial cDNA encoding a human large conductance Ca(2+)-activated K+ channel mRNA (hSlo). Northern analysis of mRNA showed that among a panel of human tissues hSlo is expressed at its highest levels in pancreatic islets. Screening of human insulinoma and islet cDNA libraries with the partial cDNA resulted in the isolation of 19 hSlo cDNAs. These comprised three splice variants: one shared the common underlying structure of previously reported Slo cDNAs, another variant encoded a novel 60-amino acid insertion in the putative Ca(2+)-sensing domain of hSlo, while the third group of clones had an alternate exon encoding eight amino acids in the predicted COOH-terminal end. Analysis of somatic-cell hybrids containing different portions of chromosome 10 indicated that hSlo maps to chromosome 10q22.2-q23.1. Furthermore, high resolution localization was obtained by analysis of genome-wide radiation hybrids and the CEPH "B" mega-YAC library, both of which identified for the first time a highly polymorphic genetic marker (D10S195) linked to hSlo. These studies provide tools with which to explore the physiological role of Ca(2+)-activated K+ channel proteins in pancreatic islets, and also to investigate the contribution of this locus to the inherited susceptibility to non-insulin-dependent diabetes mellitus.


Assuntos
Clonagem Molecular , DNA Recombinante/genética , Ilhotas Pancreáticas/metabolismo , Canais de Potássio Cálcio-Ativados , Canais de Potássio/genética , Processamento Alternativo , Sequência de Aminoácidos , Sequência de Bases , Northern Blotting , Mapeamento Cromossômico , Cromossomos Humanos Par 10 , Primers do DNA/química , Diabetes Mellitus Tipo 2/genética , Expressão Gênica/genética , Marcadores Genéticos , Humanos , Canais de Potássio Ativados por Cálcio de Condutância Alta , Masculino , Dados de Sequência Molecular , Reação em Cadeia da Polimerase , Canais de Potássio/química , RNA Mensageiro/genética , Sequências Repetitivas de Ácido Nucleico
20.
Neuropharmacology ; 35(7): 805-29, 1996.
Artigo em Inglês | MEDLINE | ID: mdl-8938713

RESUMO

The wealth of accumulating data from the Caenorhabditis elegans genome sequencing project has rapidly accelerated the discovery of novel potassium channel genes and now places within reach the possibility of describing the total complement of potassium channels used by an individual species. Using annotated GenBank sequences, BLAST searches of unfinished sequences and degenerate oligonucleotide polymerase chain reaction (PCR) screens, we have identified and compiled genes for 38 C. elegans potassium channel and two cyclic nucleotide-gated cation channel subunits, representing eight conserved multigene families. Novel families of potassium channel genes were revealed, as well as conserved homologues of all known vertebrate families. Two separate families represent C. elegans homologues for human potassium channels recently implicated in hereditary long QT arrhythmias. Of particular note is an exceptionally large class of at least 23 genes with a novel subunit structure having two tandem 'P' domains; these channels may form as dimers in contrast to all other potassium channel types which form as tetramers. The 40 potassium channel genes are evenly distributed on all six C. elegans chromosomes, with the exception of the instances of gene clustering on the fifth and X chromosomes.


Assuntos
Caenorhabditis elegans/genética , Genes de Helmintos , Canais de Potássio/genética , Sequência de Aminoácidos , Animais , Sequência Conservada , Humanos , Ativação do Canal Iônico , Dados de Sequência Molecular
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