RESUMO
The strength of synaptic transmission between a neuron and multiple postsynaptic partners can vary considerably. We have studied synaptic heterogeneity using the glutamatergic Drosophila neuromuscular junction (NMJ), which contains multiple synaptic connections of varying strengths between a motor axon and muscle fiber. In larval NMJs, there is a gradient of synaptic transmission from weak proximal to strong distal boutons. We imaged synaptic transmission with the postsynaptically targeted fluorescent calcium sensor SynapCam, to investigate the molecular pathways that determine synaptic strength and set up this gradient. We discovered that mutations in the Bone Morphogenetic Protein (BMP) signaling pathway disrupt production of strong distal boutons. We find that strong connections contain unbundled microtubules in the boutons, suggesting a role for microtubule organization in transmission strength. The spastin mutation, which disorganizes microtubules, disrupted the transmission gradient, supporting this interpretation. We propose that the BMP pathway, shown previously to function in the homeostatic regulation of synaptic growth, also boosts synaptic transmission in a spatially selective manner that depends on the microtubule system.
Assuntos
Proteínas Morfogenéticas Ósseas/metabolismo , Microtúbulos/metabolismo , Junção Neuromuscular/fisiologia , Terminações Pré-Sinápticas/fisiologia , Transmissão Sináptica/fisiologia , Adenosina Trifosfatases/genética , Adenosina Trifosfatases/metabolismo , Animais , Animais Geneticamente Modificados , Proteínas Morfogenéticas Ósseas/genética , Cálcio/metabolismo , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Drosophila , Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Feminino , Corantes Fluorescentes , Imuno-Histoquímica , Proteínas Associadas aos Microtúbulos/genética , Proteínas Associadas aos Microtúbulos/metabolismo , Técnicas de Patch-Clamp , Receptores de Superfície Celular/genética , Receptores de Superfície Celular/metabolismo , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismoRESUMO
A tetra-ortho-chloro substituted azobenzene unit was incorporated into a photoswitchable tethered ligand for ionotropic glutamate receptors. This compound confers the modified protein with the unusual optical responses of the substituted azo scaffold permitting channel opening with yellow and red light and channel closing with blue light.
Assuntos
Compostos Azo/química , Canais Iônicos/metabolismo , Canais Iônicos/efeitos da radiação , Receptores de Glutamato/metabolismo , Receptores de Glutamato/efeitos da radiação , Células HEK293 , Humanos , Canais Iônicos/química , Ligantes , Estrutura Molecular , Fenômenos Ópticos , Receptores de Glutamato/químicaRESUMO
Glutamate is the major excitatory neurotransmitter in the mammalian central nervous system and gates non-selective cation channels. The origins of glutamate receptors are not well understood as they differ structurally and functionally from simple bacterial ligand-gated ion channels. Here we report the discovery of an ionotropic glutamate receptor that combines the typical eukaryotic domain architecture with the 'TXVGYG' signature sequence of the selectivity filter found in K(+) channels. This receptor exhibits functional properties intermediate between bacterial and eukaryotic glutamate-gated ion channels, suggesting a link in the evolution of ionotropic glutamate receptors.
Assuntos
Ativação do Canal Iônico/fisiologia , Filogenia , Receptores Ionotrópicos de Glutamato/metabolismo , Proteínas Recombinantes/metabolismo , Rotíferos/metabolismo , Motivos de Aminoácidos , Sequência de Aminoácidos , Animais , Bactérias , Bases de Dados Genéticas , Expressão Gênica , Ácido Glutâmico/metabolismo , Dados de Sequência Molecular , Oócitos/citologia , Oócitos/fisiologia , Plantas , Potássio/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Receptores Ionotrópicos de Glutamato/genética , Proteínas Recombinantes/genética , Rotíferos/química , Rotíferos/genética , Alinhamento de Sequência , Transfecção , Xenopus laevisRESUMO
Imaging activity of neurons in intact brain tissue was conceived several decades ago and, after many years of development, voltage-sensitive dyes now offer the highest spatial and temporal resolution for imaging neuronal functions in the living brain. Further progress in this field is expected from the emergent development of genetically encoded fluorescent sensors of membrane potential. These fluorescent protein (FP) voltage sensors overcome the drawbacks of organic voltage sensitive dyes such as non-specificity of cell staining and the low accessibility of the dye to some cell types. In a transgenic animal, a genetically encoded sensor could in principle be expressed specifically in any cell type and would have the advantage of staining only the cell population determined by the specificity of the promoter used to drive expression. Here we critically review the current status of these developments.
Assuntos
Técnicas Biossensoriais/métodos , Proteínas de Fluorescência Verde/metabolismo , Canais Iônicos/fisiologia , Rede Nervosa/fisiologia , Animais , Linhagem Celular , Proteínas de Fluorescência Verde/química , Proteínas de Fluorescência Verde/genética , Humanos , Canais Iônicos/genética , Canais Iônicos/metabolismo , Potenciais da Membrana/fisiologia , Camundongos , Camundongos TransgênicosRESUMO
Three first-generation fluorescent protein voltage sensitive probes (FP-voltage sensors) were characterized in mammalian cells. Flare, a Kv1.4 variant of FlaSh [Siegel MS, Isacoff EY. Neuron 1997;19(October (4)):735-41], SPARC [Ataka K, Pieribone VA. Biophys J 2002;82(January (1 Pt 1)):509-16], and VSFP-1 [Sakai R, Repunte-Canonigo V, Raj CD, Knopfel T. Eur J Neurosci 2001;13(June (12)):2314-18] were expressed, imaged and voltage clamped in HEK 293 cells and in dissociated hippocampal neurons. We were unable to detect a signal in response to changes in membrane potential after averaging16 trials with any of the three constructs. Using the hydrophobic voltage sensitive dye, di8-ANEPPS, as a surface marker, confocal analyses demonstrated poor plasma membrane expression for Flare, SPARC and VSFP-1 in both HEK 293 cells and dissociated hippocampal neurons. Almost all of the expressed FP-voltage sensors reside in internal membranes in both cell types. This internal expression generates a background fluorescence that increases the noise in the optical measurement.
Assuntos
Membrana Celular/metabolismo , Expressão Gênica/fisiologia , Proteínas Luminescentes/metabolismo , Neurônios/ultraestrutura , Animais , Células Cultivadas , Técnicas de Cocultura , Estimulação Elétrica/métodos , Embrião de Mamíferos , Corantes Fluorescentes/metabolismo , Hipocampo/citologia , Humanos , Ativação do Canal Iônico/fisiologia , Canal de Potássio Kv1.4/genética , Proteínas Luminescentes/genética , Potenciais da Membrana/fisiologia , Potenciais da Membrana/efeitos da radiação , Camundongos , Camundongos Transgênicos , Técnicas de Patch-Clamp/métodos , Transfecção/métodosRESUMO
Fluorescent proteins (FPs) have been engineered to produce an optical report in response to cellular signals. FP fluorescence can be made directly sensitive to the chemical environment, via specific mutations of or around the chromophore. Alternatively, FPs can be made indirectly sensitive to cellular signals by their fusion to 'detector' proteins that respond to specific cellular signals with structural rearrangements that act on the FP to alter fluorescence. These optical sensors of membrane voltage, neurotransmitter release, and intracellular messengers, including powerful new sensors of Ca(2+), cyclic nucleotides and nitric oxide, are likely to provide new insights into the workings of cellular signals and of information processing in neural circuits.
Assuntos
Engenharia Genética/métodos , Proteínas Luminescentes/genética , Neurônios/fisiologia , Óptica e Fotônica , Transdução de Sinais/genética , Animais , Proteínas de Fluorescência Verde , Humanos , Indicadores e Reagentes , Proteínas Luminescentes/análise , Neurônios/químicaAssuntos
Bioquímica/métodos , Corantes Fluorescentes/farmacologia , Canais Iônicos/metabolismo , Proteínas Luminescentes/química , Proteínas Luminescentes/metabolismo , Proteínas Recombinantes de Fusão/química , Transdução de Sinais , Animais , Eletrofisiologia , Proteínas de Fluorescência Verde , Modelos Biológicos , Mutagênese , Canais de Potássio/metabolismo , Proteínas Recombinantes de Fusão/metabolismo , Fatores de Tempo , XenopusRESUMO
X-ray crystallography has made considerable recent progress in providing static structures of ion channels. Here we describe a complementary method-systematic fluorescence scanning-that reveals the structural dynamics of a channel. Local protein motion was measured from changes in the fluorescent intensity of a fluorophore attached at one of 37 positions in the pore domain and in the S4 voltage sensor of the Shaker K+ channel. The local rearrangements that accompany activation and slow inactivation were mapped onto the homologous structure of the KcsA channel and onto models of S4. The results place clear constraints on S4 location, voltage-dependent movement, and the mechanism of coupling of S4 motion to the operation of the slow inactivation gate in the pore domain.
Assuntos
Proteínas de Bactérias , Ativação do Canal Iônico/genética , Canais de Potássio/química , Canais de Potássio/genética , Corantes Fluorescentes/química , Modelos Moleculares , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Estrutura Secundária de Proteína , Estrutura Terciária de Proteína , Homologia de Sequência de Aminoácidos , Espectrometria de Fluorescência/métodos , Relação Estrutura-AtividadeRESUMO
The mechanism by which physiological signals regulate the conformation of molecular gates that open and close ion channels is poorly understood. Voltage clamp fluorometry was used to ask how the voltage-sensing S4 transmembrane domain is coupled to the slow inactivation gate in the pore domain of the Shaker K(+) channel. Fluorophores attached at several sites in S4 indicate that the voltage-sensing rearrangements are followed by an additional inactivation motion. Fluorophores attached at the perimeter of the pore domain indicate that the inactivation rearrangement projects from the selectivity filter out to the interface with the voltage-sensing domain. Some of the pore domain sites also sense activation, and this appears to be due to a direct interaction with S4 based on the finding that S4 comes into close enough proximity to the pore domain for a pore mutation to alter the nanoenvironment of an S4-attached fluorophore. We propose that activation produces an S4-pore domain interaction that disrupts a bond between the S4 contact site on the pore domain and the outer end of S6. Our results indicate that this bond holds the slow inactivation gate open and, therefore, we propose that this S4-induced bond disruption triggers inactivation.
Assuntos
Ativação do Canal Iônico/fisiologia , Potenciais da Membrana/fisiologia , Canais de Potássio/fisiologia , Eletrofisiologia , Fluorometria , Humanos , Mutagênese Sítio-Dirigida , Técnicas de Patch-Clamp , Conformação Proteica , Superfamília Shaker de Canais de PotássioRESUMO
Voltage-gated potassium channels are composed of four subunits. Voltage-dependent activation of these channels consists of a depolarization-triggered series of charge-carrying steps that occur in each subunit. These major charge-carrying steps are followed by cooperative step(s) that lead to channel opening. Unlike the late cooperative steps, the major charge-carrying steps have been proposed to occur independently in each of the channel subunits. In this paper, we examine this further. We showed earlier that the two major charge-carrying steps are associated with two sequential outward transmembrane movements of the charged S4 segment. We now use voltage clamp fluorometry to monitor these S4 movements in individual subunits of heterotetrameric channels. In this way, we estimate the influence of one subunit's S4 movement on another's when the energetics of their transmembrane movements differ. Our results show that the first S4 movement occurs independently in each subunit, while the second occurs cooperatively. At least part of the cooperativity appears to be intrinsic to the second S4 charge-carrying rearrangement. Such cooperativity in gating of voltage-dependent channels has great physiological relevance since it can affect both action potential threshold and rate of propagation.
Assuntos
Ativação do Canal Iônico/fisiologia , Canais de Potássio/química , Canais de Potássio/genética , Animais , Estimulação Elétrica , Eletroquímica , Fluorometria , Mutagênese Sítio-Dirigida , Oócitos/fisiologia , Técnicas de Patch-Clamp , Canais de Potássio/metabolismo , Conformação Proteica , Superfamília Shaker de Canais de Potássio , XenopusRESUMO
The vertebrate olfactory system utilizes odorant receptors to receive and discriminate thousands of different chemical stimuli. An understanding of how these receptors encode information about an odorant's molecular structure requires a characterization of their ligand specificities. We employed an expression cloning strategy to identify a goldfish odorant receptor that is activated by amino acids-potent odorants for fish. Structure-activity analysis indicates that the receptor is preferentially tuned to recognize basic amino acids. The receptor is a member of a multigene family of G protein-coupled receptors, sharing sequence similarities with the calcium sensing, metabotropic glutamate, and V2R class of vomeronasal receptors. The ligand tuning properties of the goldfish amino acid odorant receptor provide information for unraveling the molecular mechanisms underlying olfactory coding.
Assuntos
Carpa Dourada/genética , Receptores Odorantes/genética , Animais , Arginina/metabolismo , Arginina/farmacologia , Northern Blotting , Células Cultivadas , Clonagem Molecular , Eletrofisiologia , Proteínas de Ligação ao GTP/fisiologia , Expressão Gênica/fisiologia , Hibridização In Situ , Rim/citologia , Potenciais da Membrana/fisiologia , Dados de Sequência Molecular , Família Multigênica , Oócitos/fisiologia , RNA Mensageiro/análise , Receptores de Detecção de Cálcio , Receptores de Superfície Celular/genética , Receptores Odorantes/metabolismo , Homologia de Sequência de Aminoácidos , Olfato/fisiologia , Trítio , XenopusRESUMO
The glutamatergic neuromuscular junction (NMJ) in Drosophila adds new boutons and branches during larval development. We generated transgenic fruit flies that express a novel green fluorescent membrane protein at the postsynaptic specialization, allowing for repeated noninvasive confocal imaging of synapses in live, developing larvae. As synapses grow, existing synaptic boutons stretch apart and new boutons insert between them; in addition, new boutons are added at the ends of existing strings of boutons. Some boutons are added de novo, while others bud from existing boutons. New branches form as multiple boutons bud from existing boutons. Nascent boutons contain active zones, T bars, and synaptic vesicles; we observe no specialized growth structures. Some new boutons exhibit a lower level of Fasciclin II, suggesting that the levels of this synaptic cell adhesion molecule vary locally during synaptic growth.
Assuntos
Drosophila/crescimento & desenvolvimento , Animais , Animais Geneticamente Modificados , Moléculas de Adesão Celular Neuronais/fisiologia , Proteínas de Fluorescência Verde , Indicadores e Reagentes , Larva , Proteínas Luminescentes/análise , Microscopia Confocal , Proteínas Recombinantes de Fusão/análise , Sinapses/fisiologiaRESUMO
Voltage-gated ion channels underlie the generation of action potentials and trigger neurosecretion and muscle contraction. These channels consist of an inner pore-forming domain, which contains the ion permeation pathway and elements of its gates, together with four voltage-sensing domains, which regulate the gates. To understand the mechanism of voltage sensing it is necessary to define the structure and motion of the S4 segment, the portion of each voltage-sensing domain that moves charged residues across the membrane in response to voltage change. We have addressed this problem by using fluorescence resonance energy transfer as a spectroscopic ruler to determine distances between S4s in the Shaker K+ channel in different gating states. Here we provide evidence consistent with S4 being a tilted helix that twists during activation. We propose that helical twist contributes to the movement of charged side chains across the membrane electric field and that it is involved in coupling voltage sensing to gating.
Assuntos
Ativação do Canal Iônico , Canais de Potássio/química , Animais , Eletroquímica , Mutagênese Sítio-Dirigida , Canais de Potássio/genética , Canais de Potássio/metabolismo , Conformação Proteica , Superfamília Shaker de Canais de Potássio , Espectrometria de Fluorescência/métodos , XenopusRESUMO
Voltage-dependent ion channels transduce changes in the membrane electric field into protein rearrangements that gate their transmembrane ion permeation pathways. While certain molecular elements of the voltage sensor and gates have been identified, little is known about either the nature of their conformational rearrangements or about how the voltage sensor is coupled to the gates. We used voltage clamp fluorometry to examine the voltage sensor (S4) and pore region (P-region) protein motions that underlie the slow inactivation of the Shaker K+ channel. Fluorescent probes in both the P-region and S4 changed emission intensity in parallel with the onset and recovery of slow inactivation, indicative of local protein rearrangements in this gating process. Two sequential rearrangements were observed, with channels first entering the P-type, and then the C-type inactivated state. These forms of inactivation appear to be mediated by a single gate, with P-type inactivation closing the gate and C-type inactivation stabilizing the gate's closed conformation. Such a stabilization was due, at least in part, to a slow rearrangement around S4 that stabilizes S4 in its activated transmembrane position. The fluorescence reports of S4 and P-region fluorophore are consistent with an increased interaction of the voltage sensor and inactivation gate upon gate closure, offering insight into how the voltage-sensing apparatus is coupled to a channel gate.
Assuntos
Ativação do Canal Iônico/fisiologia , Canais de Potássio/química , Canais de Potássio/genética , Animais , Estimulação Elétrica , Eletrofisiologia , Corantes Fluorescentes , Mutagênese Sítio-Dirigida/fisiologia , Oócitos/fisiologia , Técnicas de Patch-Clamp , Canais de Potássio/metabolismo , Conformação Proteica , Superfamília Shaker de Canais de Potássio , Fatores de TempoRESUMO
We have acquired structural evidence that two components evident previously in the depolarization-evoked gating currents from voltage-gated Shaker K+ channels have their origin in sequential, two-step outward movements of the S4 protein segments. A point mutation greatly destabilizes the "fully retracted" state of S4 transmembrane translocation, causing instead an intermediate state to predominate at resting potentials. This state is distinguishable topologically and fluorometrically. That a point mutation effectively excludes half the range of S4 motion from physiological voltages suggests that the diverse sensitivities among voltage-gated channels might reflect not only differences in S4 valence, but also displacement. Existence of an intermediate subunit state helps explain why modeling channel activation has required positing greater than four closed states.
Assuntos
Ativação do Canal Iônico/fisiologia , Canais de Potássio/química , Canais de Potássio/genética , Sequência de Aminoácidos , Animais , Estimulação Elétrica , Metanossulfonato de Etila/análogos & derivados , Metanossulfonato de Etila/farmacologia , Corantes Fluorescentes , Indicadores e Reagentes/farmacologia , Ativação do Canal Iônico/efeitos dos fármacos , Mutagênese , Oócitos/fisiologia , Técnicas de Patch-Clamp , Conformação Proteica , Estrutura Terciária de Proteína , Superfamília Shaker de Canais de PotássioRESUMO
K+ channels can be occupied by multiple permeant ions that appear to bind at discrete locations in the conduction pathway. Neither the molecular nature of the binding sites nor their relation to the activation or inactivation gates that control ion flow are well understood. We used the permeant ion Ba2+ as a K+ analog to probe for K+ ion binding sites and their relationship to the activation and inactivation gates. Our data are consistent with the existence of three single-file permeant-ion binding sites: one deep site, which binds Ba2+ with high affinity, and two more external sites whose occupancy influences Ba2+ movement to and from the deep site. All three sites are accessible to the external solution in channels with a closed activation gate, and the deep site lies between the activation gate and the C-type inactivation gate. We identify mutations in the P-region that disrupt two of the binding sites, as well as an energy barrier between the sites that may be part of the selectivity filter.
Assuntos
Canais de Potássio/metabolismo , Animais , Bário/metabolismo , Sítios de Ligação/genética , Fenômenos Biofísicos , Biofísica , Eletroquímica , Feminino , Técnicas In Vitro , Ativação do Canal Iônico , Cinética , Potenciais da Membrana , Modelos Biológicos , Oócitos/metabolismo , Mutação Puntual , Potássio/metabolismo , Canais de Potássio/química , Canais de Potássio/genética , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Deleção de Sequência , Superfamília Shaker de Canais de Potássio , Xenopus laevisRESUMO
Previous studies have shown that both the Fasciclin II (Fas II) cell adhesion molecule and the Shaker potassium channel are localized at the Drosophila neuromuscular junction, where they function in the growth and plasticity of the synapse. Here, we use the GAL4-UAS system to drive expression of the chimeric proteins CD8-Fas II and CD8-Shaker and show that the C-terminal sequences of both Fas II and Shaker are necessary and sufficient to drive the synaptic localization of a heterologous protein. Moreover, we show that the PDZ-containing protein Discs-Large (Dlg) controls the localization of these proteins, most likely through a direct interaction with their C-terminal amino acids. Finally, transient expression studies show that the pathway these proteins take to the synapse involves either an active clustering or a selective stabilization in the synaptic membrane.
Assuntos
Moléculas de Adesão Celular Neuronais/metabolismo , Proteínas de Drosophila , Drosophila/fisiologia , Marcação de Genes , Proteínas de Insetos/fisiologia , Canais de Potássio/metabolismo , Sinapses/metabolismo , Proteínas Supressoras de Tumor , Sequência de Aminoácidos , Animais , Antígenos CD8/genética , Moléculas de Adesão Celular Neuronais/genética , Quimera , Músculos/metabolismo , Canais de Potássio/genética , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Superfamília Shaker de Canais de PotássioRESUMO
Measuring electrical activity in large numbers of cells with high spatial and temporal resolution is a fundamental problem for the study of neural development and information processing. To address this problem, we have constructed a novel, genetically encoded probe that can be used to measure transmembrane voltage in single cells. We fused a modified green fluorescent protein (GFP) into a voltage-sensitive K+ channel so that voltage-dependent rearrangements in the K+ channel would induce changes in the fluorescence of GFP. The probe has a maximal fractional fluorescence change of 5.1%, making it comparable to some of the best organic voltage-sensitive dyes. Moreover, the fluorescent signal is expanded in time in a way that makes the signal 30-fold easier to detect. A voltage sensor encoded into DNA has the advantage that it may be introduced into an organism noninvasively and targeted to specific developmental stages, brain regions, cell types, and subcellular compartments.
Assuntos
Membrana Celular/fisiologia , Neurônios/fisiologia , Canais de Potássio/fisiologia , Animais , Proteínas de Fluorescência Verde , Cinética , Proteínas Luminescentes/análise , Proteínas Luminescentes/biossíntese , Proteínas Luminescentes/química , Potenciais da Membrana , Modelos Estruturais , Oócitos/fisiologia , Técnicas de Patch-Clamp , Canais de Potássio/biossíntese , Canais de Potássio/química , Estrutura Secundária de Proteína , Proteínas Recombinantes de Fusão/biossíntese , Superfamília Shaker de Canais de Potássio , XenopusRESUMO
The permeation pathways of the voltage-gated K+ channels Kv3.1 and ShakerB delta 6-46 (ShB delta) were studied using Mg2+ block. Internal Mg2+ blocked both channels in a voltage-dependent manner, and block was partially relieved by external K+, consistent with Mg2+ binding within the pore. The kinetics of Mg2+ block was much faster for Kv3.1 than for ShB delta. Fast block of Kv3.1 was transferred to ShB delta with transplantation of the P-region, but not of S6. The difference in the P-region, causing the change in Mg2+ binding kinetics, was attributed to ShB delta (V443) and its analog Kv3.1(L401), because in both channels leucine at this position gave fast block, whereas valine gave slow block. For Kv3.1 the major determinant of the voltage dependence of Mg2+ binding resided primarily in the off rate, whereas for Kv3.1(L401V) the voltage dependence resided primarily in the on rate, consistent with a change in the rate-limiting barrier for Mg2+ binding. Our data suggest that hydrophobic residues at positions 401 of Kv3.1 and 443 of ShB delta act as barriers to the movement of Mg2+ in the pore.
Assuntos
Magnésio/metabolismo , Neuropeptídeos/genética , Neuropeptídeos/metabolismo , Mutação Puntual , Canais de Potássio de Abertura Dependente da Tensão da Membrana , Canais de Potássio/genética , Canais de Potássio/metabolismo , Sequência de Aminoácidos , Animais , Sítios de Ligação/genética , Fenômenos Biofísicos , Biofísica , Feminino , Técnicas In Vitro , Ativação do Canal Iônico , Cinética , Magnésio/farmacologia , Potenciais da Membrana , Modelos Biológicos , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Neuropeptídeos/química , Oócitos/efeitos dos fármacos , Oócitos/metabolismo , Canais de Potássio/química , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Superfamília Shaker de Canais de Potássio , Canais de Potássio Shaw , Xenopus laevisRESUMO
We have probed internal and external accessibility of S4 residues to the membrane-impermeant thiol reagent methanethiosulfonate-ethyltrimethlammonium (MTSET) in both open and closed, cysteine-substituted Shaker K+ channels. Our results indicate that S4 traverses the membrane with no more than 5 amino acids in the closed state, and that the distribution of buried residues changes when channels open. This change argues for a displacement of S4 through the plane of the membrane in which an initially intracellular residue moves to within 3 amino acids of the extracellular solution. These results demonstrate that the putative voltage-sensing charges of S4 actually reside in the membrane and that they move outward when channels open. We consider constraints placed on channel structure by these results.