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1.
Voltage-dependent gating of Shaker A-type potassium channels in Drosophila muscle 总被引:26,自引:6,他引:20
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The voltage-dependent gating mechanism of A1-type potassium channels coded for by the Shaker locus of Drosophila was studied using macroscopic and single-channel recording techniques on embryonic myotubes in primary culture. From a kinetic analysis of data from single A1 channels, we have concluded that all of the molecular transitions after first opening, including the inactivation transition, are voltage independent and therefore not associated with charge movement through the membrane. In contrast, at least some of the activation transitions leading to first opening are considerably voltage dependent and account for all of the voltage dependence seen in the macroscopic currents. This mechanism is similar in many ways to that of vertebrate neuronal voltage-sensitive sodium channels, and together with the sequence similarities in the S4 region suggests a conserved mechanism for voltage-dependent gating among channels with different selectivities. By testing independent and coupled models for activation and inactivation we have determined that the final opening transition and inactivation are not likely to arise from the independent action of multiple subunits, each with simple gating transitions, but rather come about through their aggregate properties. A partially coupled model accurately reproduces all of the single-channel and macroscopic data. This model will provide a framework on which to organize and understand alterations in gating that occur in Shaker variants and mutants. 相似文献
2.
3.
C Y Lee 《FEBS letters》1992,306(2-3):95-97
The 'ball-and-chain' model suggests the existence of a negative site which may attract the positively charged inactivation ball to occlude the pore when the channel is in the open state. For Shaker K+ channels, we propose that the state-dependent negative site be tryptophan-435, which becomes negatively charged after receiving an electron from tyrosine-445. The kinetic scheme for the channel's activation-inactivation coupling as derived from the YW-gated model resembles a successful 'scheme 8' proposed by Zagotta and Aldrich. Our model suggests that the final rapid voltage-independent transition to the open state is due to the deprotonation of tyrosine-445. 相似文献
4.
The steady-state slope conductance of Limulus ventral photoreceptors increases markedly when the membrane is depolarized from rest. The ionic basis of this rectification has been examined with a voltage-clamp technique. Tail currents that occur when membrane potential is repolarized after having been depolarized have been identified. The tail currents reverse direction at a voltage that becomes more positive when Ko is increased. Rectification is reduced by extracellular 4-aminopyridine and by intracellular injection of tetra-ethyl-ammonium (TEA). These results indicate that the membrane rectification around resting potential is due primarily to voltage-sensitive K+ channels. The increase in gK caused by depolarization is not mediated by a voltage-dependent rise in in Cai++, since intracellular injection of Ca++ causes a decrease rather than an increase in slope conductance. TEA can be used to examine the functional role of the K+ channels because it blocks them without substantially affecting the light-activated Na+ conductance. The effect of TEA on response-intensity curves shows that the K+ channels serve to compress the voltage range of receptor potentials. 相似文献
5.
Various experiments have suggested that the S4 segment in voltage-dependent Na(+) and K(+) channels is in contact with a solvent-accessible cavity. We explore the consequences of the existence of such a cavity through the electrostatic effects on the gating currents of Shaker K(+) channels under conditions of reduced ionic strength S. We observe that approximately 10-fold reductions of intracellular S produce reductions of the measured gating charge of approximately 10%. These effects continue at even lower values of S. The reduction of gating charge when S is reduced by 10-fold at the extracellular surface is much smaller (approximately 2%). Shifts of the Q(V) curve because of a reduced S are small (<10 mV in size), which is consistent with very little fixed surface charge. Continuum electrostatic calculations show that the S effects on gating charge can be explained by the alteration of the local potential in an intracellular conical cavity of 20-24-A depth and 12-A aperture, and a smaller extracellular cavity of 3-A depth and the same aperture. In this case, the attenuation of the membrane potential at low S leads to reduction of the apparent gating charge. We suggest that this cavity is made by a bundle of transmembrane helices, and that the gating charge movement occurs by translocation of charged residues across a thin septum of approximately 3-7 A thickness. 相似文献
6.
Four cDNA clones from the Shaker locus of Drosophila induce kinetically distinct A-type potassium currents in Xenopus oocytes 总被引:13,自引:0,他引:13
The Shaker gene encodes the A-channel of larval and pupal muscle, or one or more of its subunits. Alternative splicing produces messages for several different proteins; two mRNA species have previously been shown to induce the expression of A-currents in Xenopus oocytes. Two additional mRNAs have now been tested and found to produce A-currents in oocytes. The four currents differ in kinetics of inactivation, indicating that the Shaker products may contribute to kinetic diversity in A-channels of the fly and that sequences in both the amino- and carboxy-terminal regions are important for inactivation. 相似文献
7.
TRP channels in Drosophila photoreceptors: the lipid connection 总被引:2,自引:0,他引:2
Hardie RC 《Cell calcium》2003,33(5-6):385-393
The light-sensitive current in Drosophila photoreceptors is mediated by transient receptor potential (TRP) channels, at least two members of which (TRP and TRPL) are activated downstream of phospholipase C (PLC) in response to light. Recent evidence is reviewed suggesting that Drosophila TRP channels are activated by one or more lipid products of PLC activity: namely diacylglycerol (DAG), its metabolites (polyunsaturated fatty acids) or the reduction in phosphatidylinositol 4,5-bisphosphate (PIP(2)). The most compelling evidence for this view comes from analysis of rdgA mutants which are unable to effectively metabolise DAG due to a defect in DAG kinase. The rdgA mutation leads to constitutive activation of both TRP and TRPL channels and dramatically increases sensitivity to light in hypomorphic mutations of PLC and G protein. 相似文献
8.
Maurotoxin (alpha-KTx6.2) is a toxin derived from the Tunisian chactoid scorpion Scorpio maurus palmatus, and it is a member of a new family of toxins that contain four disulfide bridges (, Eur. J. Biochem. 254:468-479). We investigated the mechanism of the maurotoxin action on voltage-gated K(+) channels expressed in Xenopus oocytes. Maurotoxin blocks the channels in a voltage-dependent manner, with its efficacy increasing with greater hyperpolarization. We show that an amino acid residue in the external mouth of the channel pore segment that is known to be involved in the actions of other peptide toxins is also involved in maurotoxin's interaction with the channel. We conclude that, despite the unusual disulfide bridge pattern, the mechanism of the maurotoxin action is similar to those of other K(+) channel toxins with only three disulfide bridges. 相似文献
9.
L K Kaczmarek 《The New biologist》1991,3(4):315-323
In the last 4 years, the molecular identity of several types of voltage-dependent potassium channels has been discovered. These include channels that terminate action potentials and control repetitive neuronal firing, as well as channels whose biological role is not yet understood. The majority of these are encoded by genes related to the Drosophila Shaker gene. The large number of genes comprising the Shaker gene family, coupled with the existence of different channels that result from alternatively spliced messages from the same gene, provide both vertebrates and invertebrates with a wide selection of channels whose voltage-dependence and kinetics can be tailored to the needs of a specific cell. Mutagenesis experiments on such channels are providing new information on those regions of the protein that govern essential aspects of channel activity, such as gating by voltage and ion permeation. Another gene, unrelated to the Shaker family, encodes a voltage-dependent potassium channel that activates much more slowly than the Shaker channels. This has been termed the MinK channel. 相似文献
10.
Modulation of voltage-dependent Shaker family potassium channels by an aldo-keto reductase 总被引:2,自引:0,他引:2
The beta subunit (Kvbeta) of the Shaker family voltage-dependent potassium channels (Kv1) is a cytosolic protein that forms a permanent complex with the channel. Sequence and structural conservation indicates that Kvbeta resembles an aldo-keto reductase (AKR), an enzyme that catalyzes a redox reaction using an NADPH cofactor. A putative AKR in complex with a Kv channel has led to the hypothesis that intracellular redox potential may dynamically influence the excitability of a cell through Kvbeta. Since the AKR function of Kvbeta has never been demonstrated, a direct functional coupling between the two has not been established. We report here the identification of Kvbeta substrates and the demonstration that Kvbeta is a functional AKR. We have also found that channel function is modulated when the Kvbeta-bound NADPH is oxidized. Further studies of the enzymatic properties of Kvbeta seem to favor the role of Kvbeta as a redox sensor. These results suggest that Kvbeta may couple the excitability of the cell to its metabolic state and present a new avenue of research that may lead to understanding of the physiological functions of Kvbeta. 相似文献
11.
《The Journal of general physiology》1994,103(3):389-407
In Drosophila photoreceptors light induces phosphoinositide hydrolysis and activation of Ca(2+)-permeable plasma membrane channels, one class of which is believed to be encoded by the trp gene. We have investigated the properties of the light-sensitive channels under conditions where they are activated independently of the transduction cascade. Whole-cell voltage clamp recordings were made from photoreceptors in a preparation of dissociated Drosophila ommatidia. Within a few minutes of establishing the whole-cell configuration, there is a massive spontaneous activation of cation-permeable channels. When clamped near resting potential, this "rundown current" (RDC) accelerates over several seconds, peaks, and then relaxes to a steady- state which lasts indefinitely (many minutes). The RDC is invariably associated with a reduction in sensitivity to light by at least 100- fold. The RDC has a similar absolute magnitude, reversal potential, and voltage dependence to the light-induced current, suggesting that it is mediated by the same channels. The RDC is almost completely (> or = 98%) blocked by La3+ (10-20 microM) and is absent, or reduced and altered in the trp mutant (which lacks a La(3+)-sensitive light- dependent Ca2+ channel), suggesting that it is largely mediated by the trp-dependent channels. Power spectra of the steady-state noise in the RDC can be fitted by simple Lorentzian functions consistent with random channel openings. The variance/mean ratio of the RDC noise suggests the underlying events (channels) have conductances of approximately 1.5-4.5 pS in wild-type (WT), but 12-30 pS in trp photoreceptors. Nevertheless, the power spectra of RDC noise in WT and trp are indistinguishable, in both cases being fitted by the sum of two Lorentzians with a major time constant (effective "mean channel open time") of 1-2 ms and a minor component at higher frequencies (approximately 0.2 ms). This implies that the noise in the WT RDC may actually be dominated by non-trp- dependent channels and that the trp-dependent channels may be of even lower unit conductance. 相似文献
12.
《The Journal of general physiology》1994,103(3):409-427
Whole-cell voltage clamp recordings were made from photoreceptors of dissociated Drosophila ommatidia under conditions when the light- sensitive channels activate spontaneously, generating a "rundown current" (RDC). The Ca2+ and voltage dependence of the RDC was investigated by applying voltage steps (+80 to -100 mV) at a variety of extracellular Ca2+ concentrations (0-10 mM). In Ca(2+)-free Ringer large currents are maintained tonically throughout 50-ms-long voltage steps. In the presence of external Ca2+, hyperpolarizing steps elicit transient currents which inactivate increasingly rapidly as Ca2+ is raised. On depolarization inactivation is removed with a time constant of approximately 10 ms at +80 mV. The Ca(2+)-dependent inactivation is suppressed by 10 mM internal BAPTA, suggesting it requires Ca2+ influx. The inactivation is absent in the trp mutant, which lacks one class of Ca(2+)-selective, light-sensitive channel, but appears unaffected by the inaC mutant which lacks an eye-specific protein kinase C. Hyperpolarizing voltage steps applied during light responses in wild- type (WT) flies before rundown induce a rapid transient facilitation followed by slower inhibition. Both processes accelerate as Ca2+ is raised, but the time constant of inhibition (12 ms with 1.5 mM external Ca2+ at -60 mV) is approximately 10 times slower than that of the RDC inactivation. The Ca(2+)-mediated inhibition of the light response recovers in approximately 50-100 ms on depolarization, recovery being accelerated with higher external Ca2+. The Ca2+ and voltage dependence of the light-induced current is virtually eliminated in the trp mutant. In inaC, hyperpolarizing voltage steps induced transient currents which appeared similar to those in WT during early phases of the light response. However, 200 ms after the onset of light, the currents induced by voltage steps inactivated more rapidly with time constants similar to those of the RDC. It is suggested that the Ca(2+)-dependent inactivation of the light-sensitive channels first occurs at some concentration of Ca2+ not normally reached during the moderate illumination regimes used, but that the defect in inaC allows this level to be reached. 相似文献
13.
We previously concluded that the Kv2.1 K(+) channel inactivates preferentially from partially activated closed states. We report here that the Kv3.1 channel also exhibits two key features of this inactivation mechanism: a U-shaped voltage dependence measured at 10 s and stronger inactivation with repetitive pulses than with a single long depolarization. More surprisingly, slow inactivation of the Kv1 Shaker K(+) channel (Shaker B Delta 6--46) also has a U-shaped voltage dependence for 10-s depolarizations. The time and voltage dependence of recovery from inactivation reveals two distinct components for Shaker. Strong depolarizations favor inactivation that is reduced by K(o)(+) or by partial block by TEA(o), as previously reported for slow inactivation of Shaker. However, depolarizations near 0 mV favor inactivation that recovers rapidly, with strong voltage dependence (as for Kv2.1 and 3.1). The fraction of channels that recover rapidly is increased in TEA(o) or high K(o)(+). We introduce the term U-type inactivation for the mechanism that is dominant in Kv2.1 and Kv3.1. U-type inactivation also makes a major but previously unrecognized contribution to slow inactivation of Shaker. 相似文献
14.
The similarity in structure of potassium (K(+)) channels from different families has been revealed by only recently available crystallographic 3D structural data. The hydropathic analysis presented in this work illuminates whether homologous residues perform the same functions in channels that use different gating mechanisms. We calculated and compared the hydropathic profiles of two K(+) channels, KcsA and Kv1.2 (the latter a member of the Shaker family), at their pore-forming domain. Quantitative information describing important interactions stabilizing the protein beyond obvious secondary-structure elements was extracted from the analysis and applied as a template for subsequent molecular-dynamics (MD) analyses. For example, two key groups of interactions, defining the turns that connect the transmembrane helices and responsible for the orientation of the pore helix, were identified. Our results also indicate that Asp(80) and Asp(379) play a similar role in stabilizing the P-loop of KcsA and Kv1.2, respectively, but to significantly different extents. 相似文献
15.
External Ba2+ speeds the OFF gating currents (IgOFF) of Shaker K+ channels but only upon repolarization from potentials that are expected to open the channel pore. To study this effect we used a nonconducting and noninactivating mutant of the Shaker K+ channel, ShH4-IR (W434F). External Ba2+ slightly decreases the quantity of ON gating charge (QON) upon depolarization to potentials near -30 mV but has little effect on the quantity of charge upon stepping to more hyperpolarized or depolarized potentials. More strikingly, Ba2+ significantly increases the decay rate of IgOFF upon repolarization to -90 mV from potentials positive to approximately -55 mV. For Ba2+ to have this effect, the depolarizing command must be maintained for a duration that is dependent on the depolarizing potential (> 4 ms at -30 mV and > 1 ms at 0 mV). The actions of Ba2+ on the gating current are dose-dependent (EC50 approximately 0.2 mM) and are not produced by either Ca2+ or Mg2+ (2 mM). The results suggest that Ba2+ binds to a specific site on the Shaker K+ channel that destabilizes the open conformation and thus facilitates the return of gating charge upon repolarization. 相似文献
16.
Unilateral exposure of Shaker B potassium channels to hyperosmolar solutions. 总被引:3,自引:0,他引:3
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This study tests the hypothesis that ion channels will be affected differently by external (extracellular) versus internal (cytoplasmic) exposure to hyperosmolar media. We looked first for effects on inactivation kinetics in wild-type Shaker B potassium channels. Although external hyperosmolar exposure did not alter the inactivation rate, internal exposure slowed both onset and recovery from fast inactivation. Differential effects on activation kinetics were then characterized by using a noninactivating Shaker B mutant. External hyperosmolar exposure slowed the late rising phase of macroscopic current without affecting the initial delay or early rising phase kinetics. By contrast, internal exposure slowed the initial steps in channel activation with only minimal changes in the later part of the rising phase. Neither external nor internal hyperosmolar exposure affected tail current rates in these noninactivating channels. Additionally, suppression of peak macroscopic current was approximately twofold smaller during external, as compared with internal, hyperosmolar exposure. Single-channel currents, observed under identical experimental conditions, showed a differential suppression equivalent to that seen in macroscopic currents. Apparently, during unilateral hyperosmolar exposure, changes in macroscopic peak current arise primarily from changes in single-channel conductance rather than from changes in equilibrium channel gating. We conclude that unilateral hyperosmolar exposure can provide information concerning the potential structural localization of functional components within ion-channel molecules. 相似文献
17.
Niven JE Vähäsöyrinki M Juusola M 《Proceedings. Biological sciences / The Royal Society》2003,270(Z1):S58-S61
Shaker K(+)-channels are one of several voltage-activated K(+)-channels expressed in Drosophila photoreceptors. We have shown recently that Shaker channels act as selective amplifiers, attenuating some signals while boosting others. Loss of these channels reduces the photoreceptor information capacity (bits s(-1)) and induces compensatory changes in photoreceptors enabling them to minimize the impact of this loss upon coding natural-like stimuli. Energy as well as coding is also an important consideration in understanding the role of ion channels in neural processing. Here, we use a simple circuit model that incorporates the major ion channels, pumps and exchangers of the photoreceptors to derive experimentally based estimates of the metabolic cost of neural information in wild-type (WT) and Shaker mutant photoreceptors. We show that in WT photoreceptors, which contain Shaker K(+)-channels, each bit of information costs approximately half the number of ATP molecules than each bit in Shaker photoreceptors, in which lack of the Shaker K(+)-channels is compensated by increased leak conductance. Additionally, using a Hodgkin-Huxley-type model coupled to the circuit model we show that the amount of leak present in both WT and Shaker photoreceptors is optimized to both maximize the available voltage range and minimize the metabolic cost. 相似文献
18.
The Drosophila phototransduction cascade transforms light into depolarizations that are further shaped by activation of voltage-dependent K+ (Kv) channels. In whole-cell recordings of isolated photoreceptors, we show that light selectively modulated the delayed rectifier (Shab) current. Shab currents were increased by light with similar kinetics to the light-induced current itself (latency approximately 20 ms), recovering to control values with a t(1/2) of approximately 60 s in darkness. Genetic disruption of PLCbeta4, responsible for light-induced PIP(2) hydrolysis, abolished this light-dependent modulation. In mutants of CDP-diaclyglycerol synthase (cds(1)), required for PIP(2) resynthesis, the modulation became irreversible, but exogenously applied PIP(2) restored reversibility. The modulation was accurately and reversibly mimicked by application of PIP(2) to heterologously expressed Shab channels in excised inside-out patches. The results indicate a functionally implemented mechanism of Kv channel modulation by PIP(2) in photoreceptors, which enables light-dependent regulation of signal processing by direct coupling to the phototransduction cascade. 相似文献
19.
Multiple products of the Drosophila Shaker gene may contribute to potassium channel diversity 总被引:25,自引:0,他引:25
K+ channels are known through electrophysiology and pharmacology to be an exceptionally diverse group of channels. Molecular studies of the Shaker (Sh) locus in Drosophila have provided the first glimpse of K+ channel structure. The sequences of several Sh cDNA clones have been reported; none are identical. We have isolated and examined 18 additional Sh cDNAs in an attempt to understand the origin, extent, and significance of the variability. The diversity is extensive: we have already identified cDNAs representing at least nine distinct types, and Sh could potentially encode 24 or more products. This diversity, however, fits a simple pattern in which variable 3' and 5' ends are spliced onto a central constant region to yield different cDNA types. These different Sh cDNAs encode proteins with distinct structural features. 相似文献
20.
The members of the voltage-dependent potassium channel family subserve a variety of functions and are expected to have voltage sensors with different sensitivities. The Shaker channel of Drosophila, which underlies a transient potassium current, has a high voltage sensitivity that is conferred by a large gating charge movement, approximately 13 elementary charges. A Shaker subunit's primary voltage-sensing (S4) region has seven positively charged residues. The Shab channel and its homologue Kv2.1 both carry a delayed-rectifier current, and their subunits have only five positively charged residues in S4; they would be expected to have smaller gating-charge movements and voltage sensitivities. We have characterized the gating currents and single-channel behavior of Shab channels and have estimated the charge movement in Shaker, Shab, and their rat homologues Kv1.1 and Kv2.1 by measuring the voltage dependence of open probability at very negative voltages and comparing this with the charge-voltage relationships. We find that Shab has a relatively small gating charge, approximately 7.5 e(o). Surprisingly, the corresponding mammalian delayed rectifier Kv2.1, which has the same complement of charged residues in the S2, S3, and S4 segments, has a gating charge of 12.5 e(o), essentially equal to that of Shaker and Kv1.1. Evidence for very strong coupling between charge movement and channel opening is seen in two channel types, with the probability of voltage-independent channel openings measured to be below 10(-9) in Shaker and below 4 x 10(-8) in Kv2.1. 相似文献