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1.
D. T. Edmonds 《European biophysics journal : EBJ》1987,15(1):27-33
In this paper it is shown that the very different kinetics measured for the rise of the sodium current which follows a depolarization of the membrane in the squid giant axon, the frog node and the frog node treated with Batrachotoxin may be accurately predicted using only the measured equilibrium and static characteristics for the three preparations and the kinetics measured for the gating charge transfer.The kinetic predictions follow the use of the silent gate model for ion channel gating. The model is electrostatic and its chief assumptions are that the channel gate, called here the N-system, has fast kinetics and responds to the gating charge that transfers but not directly to the trans-membrane voltage applied. Because channel gating, corresponding here to the motion of the N-system, does not change its energy in the trans-membrane applied electric field the gating is electrically silent as far as gating charge transfer measurement is concerned. However the probability of gating rises with the quantity of gating charge that transfers due to the electrostatic interaction between the N-system and the gating charge, redistributed under the influence of the applied trans-membrane electric field. With these assumptions the kinetics of sodium channel gating are predictable using only the static and equilibrium characteristics of gating charge and channel activation measured as a function of membrane voltage, and the kinetics of the gating charge transfer. Because of the fast kinetics assumed for the N-system the predicted kinetics are the same for channels with any number of equivalent and independent N-systems or gates acting in parallel.The model predictions for sodium permeability kinetics are compared in detail with those recently measured for the frog node treated with Batrachotoxin and excellent agreement is obtained. 相似文献
2.
Sodium channel inactivation in the crayfish giant axon. Must channels open before inactivating? 总被引:9,自引:1,他引:8
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B P Bean 《Biophysical journal》1981,35(3):595-614
Experiments on sodium channel inactivation kinetics were performed on voltage-clamped crayfish giant axons. The primary goal was to investigate whether channels must open before inactivating. Voltage-clamp artifacts were minimized by the use of low-sodium solutions and full series resistance compensation, and the spatial uniformity of the currents was checked with a closely spaced pair of electrodes used to measure local current densities. For membrane potentials between -40 and +40 mV, sodium currents decay to zero with a single exponential time-course. The time constant for decay is a steep function of membrane potential. The time-course of inactivation measured with the double-pulse method is very similar to the decay of current at the same potential. Steady-state inactivation curves measured with different test pulses are identical. The time-course of double pulse inactivation shows a lag that roughly correlates with the opening of sodium channels, but detailed comparisons with the time course of the prepulse current suggest that it is not strictly necessary for channels to open before inactivating. Measurements of the potential dependence of the integral of sodium conductance area also inconsistent with the simplest cases of models in which channels must open before inactivating. 相似文献
3.
The effects of ryanodine, 9,21-didehydroryanodine and 9,21-didehydroryanodol on two types of K+ channel (a maxi, Ca2+-activated, 170 pS channel (BK channel) and an inward rectifier, stretch-sensitive channel of 35 pS conductance (IK channel) found in the plasma membrane of locust skeletal muscle have been investigated. 10–9M-10–5M ryanodine irreversibly induced a dose-dependent reduction of the reversal potential (Vrev) of the currents of both channels, i.e. from 60 mV in the absence of the alkaloid to 15 mV for 10–5M ryanodine, measured under physiologically normal K+ and Na+ gradients. In both cases the change in the ionic selectivity was Ca2+-independent. 9,21-didehydroryanodine and 9,21-didehyroryanodol also reduced Vrev, but only to 35 mV during application of 10–5M of these compounds. Additionally, 9,21-didehydroryanodine reversibly diminished the conductances of the two K+ channels. To test the hypothesis that ryanoids increase Na+ permeability by enlarging the K+ channels, the channels were probed with quaternary ammonium ions during ryanoid application. When applied to the cytoplasmic face of inside-out patches exised from locust muscle membrane, TEA blocked the K+ channels in a voltage-dependent fashion. The dissociation constant (Kd(0)) for TEA block of the IK channel was reduced from 44 mM to 1 mM by 10–7 M ryanodine, but the voltage-dependence of the block was unaffected. Qualitatively similar data were obtained for the BK channel. Ryanodine had no effect on the Kd for cytoplasmically-applied TMA. However, the voltage-dependence for TMA block was increased for both K+ channels, from 0.47 to 0.8 with 10–6M ryanodine. The effects of ryanodine on TEA and TMA block support the hypothesis that ryanodine enlarges the K+ channels so as to facilitate permeation of partially hydrated Na+ ions. 相似文献
4.
The β-subunit of the voltage-sensitive K+ channels shares 15–30% amino acid identity with the sequences of aldo–keto reductases (AKR) genes. However, the AKR properties of the protein remain unknown. To begin to understand its oxidoreductase properties, we examine the pyridine coenzyme binding activity of the protein in vitro. The cDNA of Kvβ2.1 from rat brain was subcloned into a prokaryotic expression vector and overexpressed in Escherichia coli. The purified protein was tetrameric in solution as determined by size exclusion chromatography. The protein displayed high affinity binding to NADPH as determined by fluorometric titration. The KD values for NADPH of the full-length wild-type protein and the N-terminus deleted protein were 0.1±0.007 and 0.05±0.006 M, respectively — indicating that the cofactor binding domain is restricted to the C-terminus, and is not drastically affected by the absence of the N-terminus amino acids, which form the ball and chain regulating voltage-dependent inactivation of the α-subunit. The protein displayed poor affinity for other coenzymes and the corresponding values of the KD for NADH and NAD were between 1–3 μM whereas the KD for FAD was >10 μM. However, relatively high affinity binding was observed with 3-acetyl pyridine NADP, indicating selective recognition of the 2′ phosphate at the binding site. The selectivity of Kvβ2.1 for NADPH over NADP may be significant in regulating the K+ channels as a function of the cellular redox state. 相似文献
5.
Teruyama R Sakuraba M Wilson LL Wandrey NE Armstrong WE 《American journal of physiology. Endocrinology and metabolism》2012,302(3):E273-E285
The epithelial Na? channels (ENaCs) are present in kidney and contribute to Na? and water homeostasis. All three ENaC subunits (α, β, and γ) were demonstrated in the cardiovascular regulatory centers of the rat brain, including the magnocellular neurons (MNCs) in the supraoptic nucleus (SON) and the paraventricular nucleus (PVN). However, the functional significance of ENaCs in vasopressin (VP) and oxytocin (OT) synthesizing MNCs is completely unknown. In this study, we show with immunocytochemical double-labeling that the α-ENaC is colocalized with either VP or OT in MNCs in the SON and PVN. In addition, parvocellular neurons in the dorsal, ventrolateral, and posterior subregions of the PVN (not immunoreactive to VP or OT) are also immunoreactive for α-ENaC. In contrast, immunoreactivity to β- and γ-ENaC is colocalized with VP alone within the MNCs. Furthermore, immunoreactivity for a known target for ENaC expression, the mineralcorticoid receptor (MR), is colocalized with both VP and OT in MNCs. Using single-cell RT-PCR, we detected mRNA for all three ENaC subunits and MR in cDNA libraries derived from single MNCs. In whole cell voltage clamp recordings, application of the ENaC blocker benzamil reversibly reduced a steady-state inward current and decreased cell membrane conductance approximately twofold. Finally, benzamil caused membrane hyperpolarization in a majority of VP and about one-half of OT neurons in both spontaneously firing and quiet cells. These results strongly suggest the presence of functional ENaCs that may affect the firing patterns of MNCs, which ultimately control the secretion of VP and OT. 相似文献
6.
In chemostat cultures of Bacillus caldolyticus, adaptation in a single step from 70–100°C was followed under aerobic and oxygen-limited conditions and was found to proceed more smoothly under the latter circumstances. Variations of the medium (e.g. yeast extract or silicate concentrations) showed that growth at 100°C is in all respects similar to that of cultures at moderate temperatures.Enzyme preparations derived from cultures at 5°C intervals between 70 and 100°C were used to determine the temperature range. For all nine enzymes tested, the optimum temperature was found to be 67°C; the latter was independent of the growth temperature. Differences were found, however, with respect to the maximum temperature of individual enzymes, and three groups, with maxima between 70 and 80°C, 80 and 90°C and 90 and 100°C can be distinguished. Again, there was no correlation with the growth temperature.Stability experiments also revealed that enzymes from the same organism can have different thermal properties: Some were found to be quite thermolabile (e.g. the pyruvate kinase), while others (e.g. hexokinase and glutamate-pyruvate transaminase) exhibited a high thermostability. These properties were not related to the growth temperature within the 70–100°C range, too.Six of the enzymes tested could be stabilized by their respective substrates, but the degree of protection varied for individual enzymes. Three enzymes (acetate kinase, glutamate dehydrogenase and myokinase) could not be stabilized by their substrates.Comparative experiments with the hexokinase suggested, that the thermal integrity of the enzymes is better protected within the cell as compared to the stability of the enzyme preparations.Abbreviations used AK
acetate kinase
- Ala-DH
alanine dehydrogenase
- Ald
aldolase
- GIDH
glutamate dehydrogenase
- G6P-DH
glucose-6-phosphate dehydrogenase
- GTP
glutamate-pyruvate transaminase
- HK
hexokinase
- ICDH
isocitrate dehydrogenase
- MK
myokinase
- PK
pyruvate kinase 相似文献
7.
G. H. Giebisch 《The Yale journal of biology and medicine》1997,70(4):311-322
Potassium channels in the apical and basolateral membranes of tubule cells serve several important functions. They contribute to the generation of the cell-negative potential, mediate volume reductions following cell swelling and play a key role in secretion of potassium in both the thick ascending limb of Henle''s loop and principal tubule cells of the initial and cortical collecting tubules. Secretion of potassium occurs via a well-defined class of potassium channels distinguished by their low single channel conductance, mild inward rectification, high sensitivity to inhibition by low pH, millimolar concentrations of ATP, arachidonic acid and PKC, and stimulation by vasopressin and pretreatment with a high potassium diet. Genes encoding several isoforms of this channel have been cloned and the proteins located to the apical membranes of cells lining the thick ascending limb of Henle''s loop and the collecting tubules, and progress made concerning their structure-function relationship. 相似文献
8.
Johannessen M Fontanilla D Mavlyutov T Ruoho AE Jackson MB 《American journal of physiology. Cell physiology》2011,300(2):C328-C337
σ-Receptors are integral membrane proteins that have been implicated in a number of biological functions, many of which involve the modulation of ion channels. A wide range of synthetic ligands activate σ-receptors, but endogenous σ-receptor ligands have proven elusive. One endogenous ligand, dimethyltryptamine (DMT), has been shown to act as a σ-receptor agonist. Progesterone and other steroids bind σ-receptors, but the functional consequences of these interactions are unclear. Here we investigated progesterone binding to σ(1)- and σ(2)-receptors and evaluated its effect on σ-receptor-mediated modulation of voltage-gated Na(+) channels. Progesterone binds both σ-receptor subtypes in liver membranes with comparable affinities and blocks photolabeling of both subtypes in human embryonic kidney 293 cells that stably express the human cardiac Na(+) channel Na(v)1.5. Patch-clamp recording in this cell line tested Na(+) current modulation by the σ-receptor ligands ditolylguanidine, PB28, (+)SKF10047, and DMT. Progesterone inhibited the action of these ligands to varying degrees, and some of these actions were reduced by σ(1)-receptor knockdown with small interfering RNA. Progesterone inhibition of channel modulation by drugs was consistent with stronger antagonism of σ(2)-receptors. By contrast, progesterone inhibition of channel modulation by DMT was consistent with stronger antagonism of σ(1)-receptors. Progesterone binding to σ-receptors blocks σ-receptor-mediated modulation of a voltage-gated ion channel, and this novel membrane action of progesterone may be relevant to changes in brain and cardiovascular function during endocrine transitions. 相似文献
9.
10.
Computational models of single pacemaker neuron and neural population in the pre-Bötzinger Complex (pBC) were developed based on the previous models by Butera et al. (1999a,b). Our modeling study focused on the conditions that could define endogenous bursting vs. tonic activity in single pacemaker neurons and population bursting vs. asynchronous firing in populations of pacemaker neurons. We show that both bursting activity in single pacemaker neurons and population bursting activity may be released or suppressed depending on the expression of persistent sodium (INaP) and delayed-rectifier potassium (IK) currents. Specifically, a transition from asynchronous firing to population bursting could be induced by a reduction of IK via a direct suppression of the potassium conductance or through an elevation of extracellular potassium concentration. Similar population bursting activity could be triggered by an augmentation of INaP. These findings are discussed in the context of the possible role of population bursting activity in the pBC in the respiratory rhythm generation in vivo vs. in vitro and during normal breathing in vivo vs. gasping. 相似文献
11.
Marta Goberna Maria Gadermaier Carlos García Bernhard Wett Heribert Insam 《Applied and environmental microbiology》2010,76(19):6564-6571
The acclimatization of methanogens to two-phase olive mill wastes (TPOMW) was investigated in pilot fermenters started up with cattle excreta (37°C) and after changing their feed to excreta plus TPOMW (37°C or 55°C) or TPOMW alone (37°C) until a steady state was reached (28 days). Methanogenic diversity was screened using a phylogenetic microarray (AnaeroChip), and positive targets were quantified by real-time PCR. Results revealed high phylogenetic richness, with representatives of three out of the four taxonomic orders found in digesters. Methanosarcina dominated in the starting excreta (>96% of total 16S rRNA gene copies; over 45 times more abundant than any other methanogen) at high acetate (0.21 g liter−1) and ammonia N concentrations (1.3 g liter−1). Codigestion at 37°C induced a 6-fold increase of Methanosarcina numbers, correlated with CH4 production (rPearson = 0.94; P = 0.02). At 55°C, the rise in temperature and H2 partial pressure induced a burst of Methanobacterium, Methanoculleus, Methanothermobacter, and a group of uncultured archaea. The digestion of excreta alone resulted in low but constant biogas production despite certain oscillations in the methanogenic biomass. Unsuccessful digestion of TPOMW alone was attributed to high Cu levels inducing inhibition of methanogenic activity. In conclusion, the versatile Methanosarcina immediately adapted to the shift from excreta to excreta plus TPOMW and was responsible for the stimulated CH4 production at 37°C. Higher temperatures (55°C) fostered methanogenic diversity by promoting some H2 scavengers while yielding the highest CH4 production. Further testing is needed to find out whether there is a link between increased methanogenic diversity and reactor productivity.Turning residues into energy is a societal and scientific priority due to climate change, fossil fuel exhaustion, and waste accumulation. In 2006, in Europe (EU27), less than 3% of electricity production came from biomass and wastes (11). Biogas plants, which anaerobically treat organic wastes to produce energy, are increasingly promoted in Europe, but their distribution is highly biased (35). While thousands of full- and farm-scale biogas plants are spread over central and northern Europe, anaerobic digestion technology in Mediterranean countries—Portugal, Spain, Italy, Greece, and Turkey—is in its early stages (35). These nations and other circum-Mediterranean countries lead in the production of olive oil and thus in olive mill wastes and wastewaters, which have a huge biogas production potential due to their lipid composition (1). Spain alone generates one-third of the world''s oil production and millions of tons of two-phase olive mill wastes (TPOMW) per year. TPOMW are mostly burned or composted (28), hence releasing methane into the atmosphere. This compels a change in strategy: methane production from TPOMW should be optimized in engineered environments and transformed into energy.TPOMW is a humid residue containing the olive pulp and stone. Its anaerobic digestibility is hampered by its low pH, low ammonia N, and high content in antimicrobial substances (1). However, it has been successfully fermented under laboratory conditions by supplementing it with nutrients and increasing the reactor organic loading rate stepwise (2) or by codigesting it with residues with a high buffering capacity, e.g., cattle excreta (17). These approaches seem to facilitate the adaptation of the methane-producing anaerobic community to the environmental conditions that TPOMW impose.Methanogenic archaea—microbes clustered within five orders of the Euryarchaeota—constitute the last step in the trophic chain of decomposers degrading organic matter in oxygen-free environments (36). Methanogenesis is often the rate-limiting step of anaerobic digestion of organic wastes (3) due to the fast duplication times of bacteria, which generate all substrates for the slow-growing methane-producing archaea. It is also the most sensitive step in processing imbalances (4), likely due to the lack of functional redundancy among methanogens (8). High concentrations of volatile fatty acids, salts, ammonia, and heavy metals can be inhibitory for methanogens (5, 22) and are the most common reasons for reactor failure (3). Our objective was to understand the adaptation of methanogenic communities to TPOMW. We investigated methanogenic diversity and abundance in pilot digesters fed with cattle excreta and after changing their feed to TPOMW or TPOMW plus excreta. We expected that mixing both residues would allow a faster adaptation and more efficient performance of the methanogenic communities in digesting TPOMW. The cofermentation was evaluated at 37°C and 55°C. During an acclimatization period of 28 days, we screened the methanogenic diversity using an in-house-devised phylogenetic microarray, the AnaeroChip (13), and quantified dominant genera by real-time quantitative PCR (qPCR). We have taken primers from the literature, and we present four new sets of genus-specific primers and SYBR green I-optimized assays for quantifying methanogens in anaerobic environments. 相似文献
12.
The osmotic and permeability parameters of a cell membrane are essential physico-chemical properties of a cell and particularly important with respect to cell volume changes and the regulation thereof. Here, we report the hydraulic conductivity, L(p), the non-osmotic volume, V(b), and the Arrhenius activation energy, E(a), of mammalian COS-7 cells. The ratio of V(b) to the isotonic cell volume, V(c iso), was 0.29. E(a), the activation energy required for the permeation of water through the cell membrane, was 10,700, and 12,000 cal/mol under hyper- and hypotonic conditions, respectively. Average values for L(p) were calculated from swell/shrink curves by using an integrated equation for L(p). The curves represented the volume changes of 358 individually measured cells, placed into solutions of nonpermeating solutes of 157 or 602 mOsm/kg (at 0, 10 or 22°C) and imaged over time. L(p) estimates for all six combinations of osmolality and temperature were calculated, resulting in values of 0.11, 0.21, and 0.10 μm/min/atm for exosmotic flow and 0.79, 1.73 and 1.87 μm/min/atm for endosmotic flow (at 0, 10 and 22°C, respectively). The unexpected finding of several fold higher L(p) values for endosmotic flow indicates highly asymmetric membrane permeability for water in COS-7. This phenomenon is known as rectification and has mainly been reported for plant cell, but only rarely for animal cells. Although the mechanism underlying the strong rectification found in COS-7 cells is yet unknown, it is a phenomenon of biological interest and has important practical consequences, for instance, in the development of optimal cryopreservation. 相似文献
13.
Claire Bagnéris Claire E. Naylor Emily C. McCusker B.A. Wallace 《The Journal of general physiology》2015,145(1):5-16
In excitable cells, the initiation of the action potential results from the opening of voltage-gated sodium channels. These channels undergo a series of conformational changes between open, closed, and inactivated states. Many models have been proposed for the structural transitions that result in these different functional states. Here, we compare the crystal structures of prokaryotic sodium channels captured in the different conformational forms and use them as the basis for examining molecular models for the activation, slow inactivation, and recovery processes. We compare structural similarities and differences in the pore domains, specifically in the transmembrane helices, the constrictions within the pore cavity, the activation gate at the cytoplasmic end of the last transmembrane helix, the C-terminal domain, and the selectivity filter. We discuss the observed differences in the context of previous models for opening, closing, and inactivation, and present a new structure-based model for the functional transitions. Our proposed prokaryotic channel activation mechanism is then compared with the activation transition in eukaryotic sodium channels.Eukaryotic sodium channels are complex, multidomain proteins that assemble into pseudotetrameric structures. Sodium channels are also present in some prokaryotes (Ren et al., 2001), where they are homologous but simpler, single-domain proteins that assemble to form functional homotetramers (Nurani et al., 2008). Both eukaryotic and prokaryotic sodium channels consist of voltage-sensor (VS) and pore regions that are physically separated in three dimensions. Pore-only channels can be constructed, which are capable of undergoing opening and closing transitions and support ion flux in a manner similar to that of intact channels (McCusker et al., 2011; Shaya et al., 2011). Although the kinetics of their processes of conversion between states are different, both eukaryotic and prokaryotic channels exhibit activation, recovery, and slow inactivation transitions (Koishi et al., 2004; Charalambous and Wallace, 2011); in addition, eukaryotic channels undergo a fast inactivation process that is not observed in prokaryotic channels (Pavlov et al., 2005). Prokaryotic sodium channels have also been shown to be blocked by eukaryotic sodium channel antagonists (Bagnéris et al., 2014).Many models have been proposed for the structural changes that result in the open, closed, and slow inactivated states of prokaryotic sodium channels (e.g., Kuzmenkin et al., 2004; Zhao et al., 2004; Pavlov et al., 2005; Shafrir et al., 2008). Although there are, as yet, no crystal structures of eukaryotic sodium channels, the structures of several prokaryotic sodium channel orthologues have recently been determined by x-ray crystallography (Payandeh et al., 2011, 2012; McCusker et al., 2012; Zhang et al., 2012; Bagnéris et al., 2013; Shaya et al., 2014). These include the voltage-gated sodium channels from Magnetococcus marinus, formerly known as Magnetococcus spirillium (NavMs); from Arcobacter butzler (NavAb); from Rickettsiales sp. HIMB114 (NavRh); and from Alkalilimnicola ehrlichei (NavAe). These structures now make it possible to observe various states of prokaryotic sodium channels because they have apparently captured the pore regions in partially and fully open (NavMs), closed (NavAb and NavAe), and inactivated (NavAb and NavRh) conformations, thereby enabling structural comparisons that provide new insights into the transition processes.Because the transmembrane pore regions of the NavMs and NavAb pores have very high sequence identities (∼69%; McCusker et al., 2012; Fig. 1), our comparisons will primarily be confined to these structures because with this level of similarity, differences seen are likely to be state dependent rather than a consequence of sequence dissimilarities. They enable comparisons of structures that we designate as the following: partially (pOpenMs; Protein Data Bank [PDB] accession no. 4F4L; 3.5-Å resolution; McCusker et al., 2012) and fully open (OpenMs; PDB accession no. 3ZJZ, 2.9-Å resolution; Bagnéris et al., 2013), closed (ClosedAb; PBD accession no. 3RVY, an I217C mutant; 2.7-Å resolution; Payandeh et al., 2011), and inactivated (InactivatedAb; PDB accession no. 4EKW, 3.2-Å resolution; Payandeh et al., 2012) forms. The other inactivated form for which there is a crystal structure, NavRh (PDB accession no. 4DXW; 3.0-Å resolution; Zhang et al., 2012), and the other closed form, NavAe (PDB accession no. 4LTO; 3.5-Å resolution; Shaya et al., 2014), are more distant homologues (only 42 and 45% identity, respectively, in the pore region; Fig. 1). In addition, although both the NavAb and NavMs homologues have been shown to support sodium flux (Payandeh et al., 2011; D’Avanzo et al., 2013), neither NavRh nor NavAe has yet been shown to exhibit functional activity; therefore, we considered them to be less suitable for detailed comparisons. Although the resolutions of the structures are modest, all are sufficient to define the types and magnitudes of the conformational differences described in the comparisons made in this Review. In addition to these crystal structures, however, two structures of another orthologue, NavCt (PDB accession no. 4BGN, ∼9-Å resolution; Tsai et al., 2013), have been determined by electron crystallography, but those structures are at too low a resolution to make viable comparisons.Open in a separate windowFigure 1.Sequence alignment, using Clustal Omega (Sievers et al., 2011), of the prokaryotic sodium channel orthologues: NavMs from Magnetococcus marinus MC-1 (UniProt accession no. A0L5S6), NavAb from Arcobacter butzleri RM4018 (UniProt accession no. A8EVM5), NavAe from Alkalilimnicola ehrlichii MLHE-1 (UniProt accession no. ), NavCt from Caldalkalibacillus thermarum TA2.A1 (UniProt accession no. Q0ABW0F5L478), NavRh from Rickettsiales sp. HIMB114 (UniProt accession no. D0RMU8), and domain IV (DIV) of the human Nav1.4 sodium channel (UniProt accession no. ). The identities of the helical regions (transmembrane helices S1–S6), the N-terminal intracellular helix S1N, the S4–S5 linker helix, the P1 and P2 pore helices, and the intracellular C-terminal coiled-coil (CC) helix are indicated in the horizontal colored tubes above the sequences and are based on the crystal structures of NavAb for the VS region (cyan), on the crystal structure of NavMs for the pore region (green), and on the site-directed electron paramagnetic resonance spectroscopy of NavMs and circular dichroism truncation studies of NaChBac for the CTD (gray). The vertical magenta bar indicates the extracellular negatively charged (ENC) region formed by D49 (in S2), and the red bars are the intracellular negatively charged (INC) region formed by E59 (in S2) and D81 (in S3) involved in the gating charge transfer across the membrane through sequential interactions with four arginine residues in S4 (indicated by the cyan vertical bars); in the “up” conformation, which corresponds to the activated state, residues E59 and D81 form the salt-bridged pairs. The residues comprising the SF are highlighted in light green vertical bars. The residues in purple are proposed to be the start of the twist in S6 that is implicated in activation gate opening (light purple from the partially open structure and dark purple in the fully open structure). The residue in yellow indicates the location of the final hydrophobic constriction region (HC3) in the closed structure, which effectively corresponds to the location of the activation gate.This Review focuses on comparisons of the ClosedAb–pOpenMs–OpenMs–InactivatedAb crystal structures, developing a structure-based model for the functional transitions. Although these crystal structures provide an important new structural context for understanding the nature of the transitions, it must be remembered that any models developed from them will ultimately require verification by functional studies under voltage-clamp conditions that probe native structures in real time and in the context of cell membranes. P35499
Defining the conformational states represented in the crystal structures
The full-length NavAb channel has its VS in the “up” conformation (with residues E59 and D81 forming salt-bridged pairs to arginines in S4; Fig. 1) (Payandeh et al., 2011). This conformation of the VS is considered to be associated with the activated or fully open state as defined by disulfide cross-linking experiments (DeCaen et al., 2008, 2009). In addition, because in crystals there is no transmembrane potential, we expected that the structure would include an open form of the pore, as would be seen when the channel gate opens in response to membrane depolarization. However, in these crystals, the activation gate, and thus the pore region, appears to be in the closed conformation: the pore has no continuous transmembrane pathway that would enable the passage of sodium ions from the extracellular to the intracellular surface (McCusker et al., 2012; Fig. 2, A and B). Although the selectivity filter (SF) is sufficiently open to enable ions to enter the pore, the bottom of its cavity (at the activation gate) is blocked (Payandeh et al., 2011) so ions cannot exit. This structure has thus been described as being in a “pre-open” state, in other words, with a VS conformation that has primed the gate for opening but in which the gate is actually closed. This demonstrates that the structures of the VS and pore regions can be uncoupled, and may indicate the presence of an additional conformation intermediate between the “open” and “closed” states. In the comparisons in this Review, which focuses on the pore region responsible for the opening and closing of the transmembrane pathway, we have designated this structure as “closed” (ClosedAb) based on the state of the pore region. It is very similar in dimensions and features to the NavAe closed pore (Shaya et al., 2014), a structure that does not contain a VS region, another indication that the pore region state is not solely determined by the VS state.Open in a separate windowFigure 2.Accessible surface differences between the open and closed structures. (A; middle) Accessible surface plots (made using CAVER software; Chovancova et al., 2012) showing radius versus distance through the central axis of the pore for ClosedAb-I217C (slate blue), ClosedAe (red), InactivatedAb (gray), OpenMs (light green), and pOpenMs (turquoise). (Left and right) Slab surface mode/cartoon depictions of the pores, sliced along the transmembrane direction for the ClosedAb (left, slate blue) and OpenMs (right, light green) pores, highlighting the sites of the first minor constriction (blue underlay: present for both forms with the responsible residues, V213 in the closed form and I215 in the open form), the second minor construction (orange underlay: present only in the closed form, residue I217C), and the third major constriction (yellow underlay: present only for the closed form, residue M221) at the intracellular end of the cavity. I217C is the mutation in the closed structure that enabled crystallization at higher resolution. (B) Detailed view of the three “hydrophobic constriction” (HC) regions noted above shown in cartoon and stick mode for the ClosedAb (slated blue) and OpenMs (light green) structures. The distances shown were measured between two diagonally opposite residues. It is clear that the narrow constrictions at HC2 and HC3 (6.62 and 4.81 Å) seen in the closed structure are not present in the open structure (where the equivalent distances are 14.84 and 17.18 Å).One of the two NavMs pore structures (Bagnéris et al., 2013) has been designated to be in the fully open state (OpenMs) by virtue of its transmembrane pathway being of sufficient diameter along the full length of the pore, from the extracellular surface to the intracellular surface (Fig. 2, A and B), to enable the translocation of sodium ions across the cell membrane (Ulmschneider et al., 2013). It has all four monomers with a splayed conformation at their intracellular ends. In contrast, in the partially open (pOpenMs) structure (McCusker et al., 2012) (which lacks the last 37 residues of its C-terminal domain [CTD]), only one of the four monomers of the tetramer adopts an open splayed conformation, with the result that the activation gate is only partially open; it is not sufficient to permit ion translocation but is more widely open than the closed structure (Fig. 2 A). The other three monomers in this structure are effectively equivalent to the closed-state pore conformations. A model “open” structure was constructed (McCusker et al., 2012) based on using the most open of the four monomers to produce a symmetric tetramer, which was very similar to the structure later determined of the fully open state (Bagnéris et al., 2013). In the partially open structure, the distal end of the CTD, which has been proposed to form a stabilizing four-helix coiled-coil (Powl et al., 2010; Irie et al., 2012; Bagnéris et al., 2013; Shaya et al., 2014), is absent, although it is present in the fully open structure; this may have enabled the uncoupled opening of the four S6 helices, and this asymmetry suggests there may be an asynchrony in the functional transition between states.The two inactivated forms of NavAb (InactivatedAb; Payandeh et al., 2012) and NavRh (InactivatedRh; Zhang et al., 2012) have been so designated based on their closed pores and collapsed SFs (although the authors of the NavAb structure were careful to describe it as being in a “potentially” inactivated state, largely because there was no confirming functional data). Both of these structures have closed activation gates, which are very similar to the ClosedAb structure, but their SFs are unlike any of the other structures, and would be too narrow to enable ions to enter the pore (Figs. 2 A, middle, and 4, C and D, and Table S1).Open in a separate windowFigure 4.Comparisons of the SF regions. (Left and middle columns) Views from the extracellular surface showing the size of the SF central holes, defined as the white areas in the middle of each structure. (Right column) Side view superpositions of the SFs in cartoon and stick depictions, in each case comparing the OpenMs structure (light green) with the corresponding structure in that row. The identities of the SF residues (TLES) are indicated. Only two monomers are shown for clarity. (A; left) OpenMs (pale green) and (middle) pOpenMs (turquoise) structures. (B; middle) ClosedAb (slate) and overlay (right) comparison of ClosedAb and OpenMs. (C) As in B for InactivatedAb (AB tetramer; gray). (D) As in B for InactivatedAb (CD tetramer; gray). (E) As in B for ClosedAe (raspberry). The distances between the narrowest parts of the SFs in each case are given in Table S1.Structural transitions associated with opening and closing
The extracellular turret and vestibule surfaces (comprised of the S5–S6 linker, including the P1, P2, and SF regions; Fig. 1) are very similar in the OpenMs and ClosedAb pores, suggesting that these are not substantially involved in the gating transition. In addition, the N-terminal ends of their S6 helices (Fig. 3 B) are virtually superimposable, as are the C-terminal ends of the S5 helices. However, the intracellular surface features differ substantially (Fig. 3 A) as a result of differences in the relative orientations and splaying of the C-terminal ends of their S6 helices away from the pore axis. Also, the N-terminal ends of the S5 helices appear to have moved slightly with respect to each other. Had the open pore structure had its VS attached, this latter movement would have impinged on the S4–S5 linker (Fig. 3 A, circle) of an adjacent monomer. We speculate that the reason the NavAb pore is closed is that the linker region is in the closed conformation, and that if the linker had been more closely coupled to the VS in its open conformation in the crystal, the NavAb pore structure would also have been of an open conformation.Open in a separate windowFigure 3.Differences between the open and closed structures. (A) Comparison of the OpenMs (green) pore and the ClosedAb (slate blue) crystal structures, depicted in cylindrical mode viewed from the intracellular surface. The equivalent residue numbers for the pore domain were G129 to M221 in NavMs and G130 to M222 in NavAb. Three-dimensional alignments (in all cases the least-squares superpositions were done using residues 145–198 [or their sequence equivalents] at the top of helices S5 and S6) and figures were made using PyMOL software (Schrödinger, LLC). The motions associated with the S5 and S6 helices are indicated by the small and large arrows, respectively. One of the ClosedAb monomers is shown in gray, so that it can be seen that the region of the S4–S5 linker that the S5 helix in the open state would impinge on (magenta circle) is in the adjacent, not the same, monomer. (B) The Cα carbons of the S6 helixes (in stick motif) showing that the ClosedAb (slate blue), InactivatedAb (gray), and ClosedAe (red) structures overlay closely, but that the OpenMs (green) deviates from the other structures starting at residue T206. (C) Plot of the delta phi (blue) and delta psi (red) angles in the S6 helix as a function of residue number. Values are those of OpenMs structure (PDB accession no. 3ZJZ-A chain) minus those of the ClosedAb structure (3RVY-A chain), demonstrating that the differences start after residue T206 in NavMs and continue to the end of the S6 helix. The single peak at around residue 155 is not related to the transition but simply arises from different interactions of the two proteins, with the different crystallization detergent molecules present adjacent to this site. (D) Secondary structure alignments compared using the 2Struc server (Klose et al., 2010). The position corresponding to the T206 residue in helix S6 is indicated by the black box in both parts. (Top) OpenMs versus ClosedAb. The locations of the S5 and S6 helices are indicated by the horizontal green bars. Both structures have essentially identical secondary structures, even around T206. (Bottom) OpenMs versus InactivatedAb. The biggest differences are at the top of S5 (purple box) and in the turret loop (cyan box), not in helix S6 nor the region around T206.When the structures of the S6 pore helices of all the conformations are examined in detail (Fig. 3 B), the differences at the quaternary structural level between the open and closed (and inactivated) structures appear to arise from a twist in the middle of the S6 helix structures beginning at residue T206. The extracellular ends of all of the S6 helices in all of the closed and inactivated structures superimpose closely (Fig. 3 B), but the OpenMs structure is an outlier. This results from a change in the backbone Ramachandran angles between the open and closed forms, which perpetrates along the C-terminal end of S6 starting at T206 (Fig. 3 C). The consequence of this deviation is that the C-terminal end of the S6 helix in the OpenMs structure moves away from the axis of the top part of the helix, thereby opening the activation gate. In the lower resolution pOpenMs structure, residue T209 appeared to be the focal point of the bend, but in the higher resolution fully OpenMs structure, it is clear that the beginning of the bend occurs one turn earlier in S6, at T206. Notably, the changes between open and closed structures are subtle enough so that they do not change the secondary structure (Fig. 3 D) of the S6 helix. What starts as a relatively small change in the middle of the helix is translated to a motion of >5 Å at the end of S6 that forms the activation gate. This suggests a simple (energetically inexpensive) mechanism for opening and closing: a twist in the backbone that does not disrupt the hydrogen bonding pattern is sufficient to open the gate. It does not require major rearrangements of the rest of the structure and is thus compatible with a rapid opening, such as that which gives rise to the initial phase of the action potential in excitable cells. The asynchrony of the motions of the four monomers, as suggested by the partially open structure, suggests that there may also be an asynchrony of the process of opening.The open and closed structures have very similar cavity regions (Fig. 2 A), with a minor hydrophobic constriction (designated HC1) near the bottom of the cavity (Fig. 2 A) near residue I215 in NavMs (equivalent to V213 in NavAb) that is not sufficient to prevent ions continuing their passage. However, the ClosedAb form has two additional major hydrophobic constrictions (HC2 and HC3) further down toward the extracellular surface (Fig. 2 A) at approximately the level of residues M221 and I217 (numbering according to NavAb sequence; notably, the latter corresponds to the site of the mutation to a cysteine in the NavAb construct). Neither of these constrictions is seen in the open NavMs structure. HC3 (which obscures the exit) would exclude passage of even totally dehydrated sodium ions in the closed structure.Because the SFs of NavMs and NavAb have identical sequences (TLESWS; Fig. 1), they can be directly compared. The SFs of the OpenMs (Fig. 4 A) and ClosedAb (Fig. 4 B) structures are superimposable (Fig. 4 B, right), thus indicating that the closing of the activation gate, which prevents ion translocation, does not affect the potential entry of ions into the pore. In contrast, the SFs of the inactivated forms (Fig. 4, C and D) are very different from those of both the open and closed states, suggesting that they are altered during the inactivation process.The structure and role of the CTD in opening and closing
All of the initial crystal structures determined (NavAb, NavMs, and NavRh) have elucidated the nature of the transmembrane domains (effectively to the end of the S6 helix) of the channels, but the structures of their CTDs (beyond the end of helix S6) were not interpretable, although these regions were present in the proteins used to produce the crystals. The structure of the CTD of the open conformation was first determined not by crystallography but by a combination of spectroscopic methods (Powl et al., 2010; Bagnéris et al., 2013), and shown to have a disordered region adjacent to the activation gate and a distal coiled-coil region (Figs. 1 and and5);5); molecular dynamics calculations (Bagnéris et al., 2013) suggested that flexibility of the region adjacent to the activation gate in the open state could account for the lack of defined structure in the crystal. The CTD in the closed NavAe pore (Shaya et al., 2014) is the only other such domain that has been defined structurally, this time by crystallographic means. Like the open CTD, its distal end formed a coiled-coil, but unlike the open CTD, the proximal end of the structure, nearest to the activation gate, was helical and relatively well ordered. These observations suggested that although the C-terminal end of the CTD remains unchanged in the two states, the end adjacent to the activation gate undergoes an ordered-to-disordered transition when the gate is opened (Fig. 5), thereby accommodating the separation movement at the end of the S6 helix that forms the gate, without uncoiling the distal coiled-coil that has been proposed to have a structural role in stabilizing the tetrameric structure in the membrane (Mio et al., 2010; Powl et al., 2010). Functional studies on the NavMs channel (Bagnéris et al., 2013), as well as on another orthologue (NavSulP from Sulfitobacer pontiacus; Irie et al., 2012), have indicated that this region (especially the EEE motif near the top of the CTD) plays a role in inactivation, recovery, and closing, perhaps by promotion of the conformational change of the S6 helix. In addition, in the NavAe pore structure, there is some density in the extracellular region beyond the end of the transmembrane segments that could be a calcium or other cation. Domain DIV of the human Nav1.4 sodium channel also possesses a similar negatively charged region just after its activation gate (Fig. 1), which appears to be a common feature in the prokaryotic sodium channels.Open in a separate windowFigure 5.Schematic diagram of (1) closed, (2) open, and (3) inactivated states of prokaryotic sodium channels based on crystallographic studies. Only two monomers are shown in each figure for clarity. The color scheme is as in Fig. 1. The VS S1–S4 helices are depicted as cyan bars, the S4–S5 linker is an orange bar, the S5–S6 helices of the pore region are green bars, and the coiled-coil region of the CTD is gray. The extracellular negatively charged region in S2 is in a magenta circle, and the intracellular negatively charged regions in S2 and S3 are in red circles. The four arginines in S4 involved in the gating charge transfer across the membrane through sequential interactions, with the extracellular negatively charged region and the intracellular negatively charged region represented by a “+.” The residues forming the SF are indicated by red boxes. The residue in purple is the start of the S6 twist that results in the open gate; the residue in yellow is the third hydrophobic constriction site in the closed form, which is not constricted in the open form, and indicates the position of the activation gate. The white bars represent the helical region present in the CTD in the closed and inactivated structures, and the corresponding dotted gray lines are the disordered CTD linker region in the open conformation.What produces the open and closed states in the crystals?
An obvious suggestion as to what causes NavMs pore structure to be open is that it is missing the constraints provided by the VS and S4–S5 linker regions present in the full-length channel structures. However, this cannot be the only reason, as the two structures of pore-only constructs, NavMs and NavAe, are in different states (NavMs is open, whereas NavAe is closed). It is also not because of the presence of CTD structures because, again, the CTDs are present in both of these two structures, albeit in different conformations. Furthermore, the partially open NavMs construct is missing the distal end of its CTD, yet one of its monomers is in the open state. The CTDs are present but not visible in any of the other structures. It is possible that the different CTD structures have a role in driving the transition (for example because of the different conformations at their N-terminal ends), but this cannot be determined from the structures in hand. It could also be because of the differences in crystal packing that enable the NavMs pores to open: in the NavMs crystals, the transmembrane regions of different pores are aligned next to each other, as if in a membrane bilayer, with a large enough gap to fit the CTD between two separate bilayers and thus allow sufficient room for the bottom of the pore to open. In the NavAe crystals, the CTDs are sandwiched between other CTDs and pore regions of symmetrically related molecules, rigidifying the whole structure and potentially preventing the opening.The different structures in the crystals may be caused by (or alternatively, result in) the presence or absences of ions within the pore, as sodium ions are visible in the SF of only the NavMs open-state crystal forms. However, in a version of the NavAb orthologue engineered to be calcium selective (Tang et al., 2014), the presence of calcium ions in that pore did not produce an open state, so the mere presence of ions appears not to be sufficient to produce an open pore. The most likely candidate in vivo is the VS-pore linker between helices S4 and S5, which could act as a lever, either pushing or pulling the pore domain when the VS is activated to its “up” position. But the linker is not present in either the NavMs open or partially open structures, nor in the NavAe closed structures. And although it is present in the NavAb structures, it is in the “wrong” position to influence the opening of the pore, so it may be a contributing, but not the only, driving force. This is an important issue and will need to be resolved in the future to enable a full understanding of the opening and closing processes.Structural transitions associated with inactivation
Comparisons of the structure of the InactivatedAb channel with both the ClosedAb and OpenMs structures indicate areas of the protein primarily involved in the inactivation transition. The InactivatedAb structure has a very similar upper cavity region to both the open and closed structures, and an asymmetric inactivation gate conformation that, like the symmetrical gate of the closed state, is occluded. The activation gate structures of the S6 helices of both the InactivatedAb and InactivatedRh channels closely overlay the ClosedAb (Fig. 3 B) but not the OpenMs structures, suggesting that the hinge necessary to open the gate has not been activated. Although their S6 conformations appear to be the same, the structures of the two types of InactivatedAb tetramers (“AB” and “CD”) and the ClosedAb form do exhibit differences at their C-terminal ends: the inactivated forms have between two and eight fewer ordered residues visible in the crystals, suggesting that they exhibit more mobility or flexibility in this region. Because the CTD has not been resolved in any of the inactivated form crystals, we cannot make any conclusions about whether the regions adjacent to the transmembrane helices are disordered (as in the open state) or helical (as in the closed state). Nor can we discern even if they exhibit the same coiled-coil structures at their distal ends that are present in both the open and closed forms.In contrast, the SF region of the InactivatedAb structure (as well as that of the inactivated NavRh structure) is very different from either the open or closed states (Fig. 4). It has been described as “collapsed,” and indeed it is of insufficient diameter to enable a full dehydrated sodium ion to enter into the pore, much less exit it. Because in this case comparisons can be made for two crystal structures of the same orthologue (NavAb), the differences in the inactivated and closed structures cannot be attributed to SF sequence differences. The changes involve both the P1 and P2 helices as well as the SF itself, and involve modest differences in the secondary structures of the turret and top of S5 (Fig. 3 D). The observation that structural changes associated with the inactivated structure primarily involve the SF region corresponds well with earlier functional studies suggesting that the region close to the SF is responsible for inactivation (Pavlov et al., 2005). Interestingly, too, is the correspondence with studies done many years ago using circular dichroism spectroscopy (Cronin et al., 2003) on eukaryotic (electric eel) sodium channels that were induced to adopt open, closed, and inactivated states through the use of toxins and drugs. Those functional studies indicated that the secondary structures of the open and closed channels were surprisingly very similar but that the secondary structures of the inactivated form was considerably different. This also corresponds with the structural observations for the prokaryotic channels, where there are no apparent changes in secondary structure between open and closed states but significant differences between the open and the inactivated state.The structural comparisons thus suggest that inactivation closes not only the extracellular but also the intracellular ends of the transmembrane passageway, although the extracellular changes appear to be the defining ones for the inactivation state.Structural models for sodium channel opening–closing–inactivation mechanisms
Early models (e.g., Armstrong and Bezanilla, 1973; Hille, 1975; Lehmann-Horn and Jurkat-Rott, 1999; see also Hille, 2001, and Armstrong, 2007) proposed for eukaryotic sodium channel charge movement and gating were based primarily on functional observations and were produced, for the most part, before either any sodium channel sequences were determined or any crystal structures of any members of the voltage-gated cation channel superfamily were available. Structural models included suggestions such as the constriction of the SF or the shutting of two flaps (Lehmann-Horn and Jurkat-Rott, 1999), one at the extracellular surface and one at the intracellular surface, as being responsible for inactivation and closing, respectively. With the sequencing of the first eukaryotic sodium channel (Noda et al., 1984), three-dimensional models were developed that proposed the involvement of specific regions for the activation and inactivation processes (Guy and Seetharamulu, 1986; Yu et al., 2005). Many other approaches, including mutational effects, interactions with toxins, drug and ligand binding, and various spectroscopic studies, have since contributed to structure–function models of sodium channels (Catterall, 2012). More recently, the crystal structures of potassium channels (Doyle et al., 1998; Long et al., 2007) provided structural templates and information for understanding the electromechanical coupling and mechanism of their opening and closing. The sequence homology of sodium channels to these other members of the voltage-gated ion channel superfamily suggested that they would share a common architecture, albeit with very different SF regions, and led to more detailed models for sodium channel structures and functions (Yarov-Yarovoy et al., 2001; Shafrir et al., 2008; Zarrabi et al., 2010).Soon after the prokaryotic sodium channels were identified as simpler target molecules for biophysical studies (Ren et al., 2001), and well before any of the prokaryotic orthologue crystal structures were determined, a hinge model for NaChBac (the first sodium channel orthologue whose sequence was determined) was proposed. This model for opening of the activation gate, like the models for the MthK potassium channel (Jiang et al., 2002), was based on the presence of a glycine residue in the middle of the S6 helix. Because glycines lack side chains, this type of residue could act as a flexible pivot for the top and bottom of the S6 helix, which could then move to open the gate. This model was supported by functional studies that showed that mutations of the glycine altered the rate of inactivation and recovery (Ito et al., 2004; Zhao et al., 2004), and spectroscopic studies that showed that changing the glycine to a conformationally less flexible serine produced a more thermally stable protein (O’Reilly et al., 2008); later molecular dynamics calculations (Barber et al., 2012) also supported a hinge-bending model involving this glycine. This model for the mechanism became less favored, however, after the determination of the sequences of other orthologues, as they did not have a glycine in the equivalent position.The solution of the crystal structure of the prokaryotic sodium channel NavAb, with sequence and structural homology (especially in the VS regions) to single-domain tetrameric prokaryotic and eukaryotic potassium channel structures, prompted the suggestion (Payandeh et al., 2011) that the mechanism behind the coupling and pore opening might be similar to another model proposed for K+ channels (Long et al., 2007). Indeed, the VS of the ClosedAb structure aligns well with the VS of the open Kv1.2 structure, although they diverge in the pore, specifically at the beginning of the base of the S5 helix, and their SFs are completely different both in sequence and in structure (one using side chains and backbone carbonyls, and the other narrower one using only backbone carbonyls to create the ion-binding sites). That model had the VS and pore regions moving as modular units, with the S4–S5 linker movement causing the conformational change. In the sodium channel case, the proposed change was a rotation-like motion of the S5 and S6 helices relative to each other, opening the pore in an iris-like dilation motion. This bears semblance to the twisting mechanism proposed for the opening and closing of the nonspecific ion channel in nicotinic acetylcholine receptors (Unwin, 2003), where the transmembrane helices of adjacent subunits change positions relative to each other, primarily on one surface, producing a rotational squeezing and release mechanism that changes the size of the pore along its length.A new structure-based model for the opening–closing–inactivation mechanisms
The observed structural differences described above between OpenMs and ClosedAb do not support a model based on a modular S5–S6 iris-like motion. In the prokaryotic sodium channel crystal structures, the C-terminal end of the S5 helix exhibits very little change between the two different states, and nothing suggests its movement is coupled with that of the S6 helix in either the same or an adjacent subunit within the tetramer. Instead, the opening and closing appears to involve a twist/bend in the middle of the S6 helix, which moves the activation gate to occlude or open the intracellular surface, enabling ion egress from the cavity (McCusker et al., 2012; Bagnéris et al., 2013; Video 1). This is remarkably similar to the earlier hinge model, except that there is no glycine at the hinge position. Instead, a threonine (T206 in the NavMs numbering scheme) is located at the juncture, in an equivalent position in the sequence to the glycine found in the NaChBac orthologue. The structure of NavMs shows that there would be sufficient space to enable the backbone motion even with the threonine side chain, as the Cβ substituent does not impinge on the region that moves. However, the enhanced flexibility of a glycine residue in NaChBac could be responsible for its faster rate of opening (Ren et al., 2001) than NavMs (Ulmschneider et al., 2013).Because the S1–S4 helices of the VS structure are already in the open conformation in the ClosedAb and InactivatedAb structures, it initially seemed likely that the S4–S5 linker in these structures would be in a conformation similar to that which it would adopt in the open pore state. However, comparisons of the OpenMs and ClosedAb structures indicate that the positions of especially the S5 helices in the open pore would mitigate against the linker in the ClosedAb structure being completely in the activated state because the end of the open state S5 helix would impinge on the linker. It is therefore suggested that the linker is in a “primed” state but that it moves in concert with the S5 and S6 helices as the activation gate is opened.The structure-based model for the slow inactivation process, like several of the early models (Lehmann-Horn and Jurkat-Rott, 1999), appears to primarily involve a closing of the SF to ion entry on the extracellular surface (Fig. 5). Rather than a flap folding over the entry, however, it appears that the SF itself folds in toward the center of the ion pathway, blocking entry of any sodium ions into the pore. It also appears, contrary to the double-flap mechanism, that the activation gate in the inactivated form is closed rather than open, although this could be a consequence of the crystallization and packing conditions (Video 2).Flexibility at the C-terminal region of S6 thus appears to be the defining feature of the prokaryotic voltage-gated sodium channel. It may be at least partially responsible for channel opening and for the cascade of events that occur at the SF and P loops during inactivation. The presence of a flexible linking region to the CTD would enable the opening and closing to occur without uncoiling the distal coiled-coil region, which may play an important role in stabilizing the tetrameric quaternary structure of the prokaryotic sodium channels (Video 3). This feature is not present (nor necessary) in eukaryotic sodium channels, which are single polypeptide chains. Instead, they seem to have a CTD that plays an important role in fast inactivation, a function that prokaryotic sodium channels do not exhibit.Similarities and differences in the activation mechanisms of prokaryotic and eukaryotic sodium channels
Prokaryotic and eukaryotic sodium channels exhibit considerable homologies in the sequences of their transmembrane regions (Fig. 1), especially in the C-terminal ends of their S6 helices, although their SFs and CTDs are entirely different. Likewise, both exhibit transitions between activation, recovery, and slow inactivation functional states, albeit with different kinetics (as well as the absence of fast inactivation in prokaryotic channels). Therefore, it is of interest to consider if they might involve similar structural features in a common mechanism for opening and closing.New studies (Bagnéris et al., 2014) on the structure and electrophysiological consequences of binding channel blocker drugs to prokaryotic and human Nav1.1 channels have shown the parallel nature of their block by a range of ligands that bind in the hydrophobic cavity and prevent passage of ions from the extracellular to intracellular surfaces. This apparently occurs not by closing the activation gate but by the hydrophobic ligands creating a barrier to exit at the end of the SF region, and is entirely consistent with the mechanisms described above.Furthermore, a recent study was done to test whether a similar mechanism of opening and closing could be operating in human Nav1.4 and prokaryotic sodium channels (Oelstrom et al., 2014). Channels with their fast inactivation mechanism disabled could be functionally held in either the open or closed state. An accessibility labeling technique was used to probe sites in S6 of domain IV (Fig. 1). Residues C terminal to the proposed activation gate were accessible to labeling from the intracellular side in both the open and closed states, whereas residues above this were only accessible in the open state. In the open state, the channel could be labeled up to the ring of hydrophobic residues immediately preceding the equivalent to residue T206 in the prokaryotic channels, again consistent with a mechanism involving a hinge mechanism in the middle of S6. These results strongly support the type of mechanism for opening and closing presented above, with a flexible hinge region that opens the pore up to the intracellular compartment in the open state, but not in the closed state.However, it is important to note that significant differences in the activation/inactivation and selectivity mechanisms do exist between prokaryotic and eukaryotic channels (see, for example, Ahern, 2013; Goldschen-Ohm et al., 2013; Finol-Urdaneta et al., 2014, for detailed discussions). These arise in large part because of the asymmetry of the pseudotetrameric eukaryotic structures as opposed to the simpler homotetramers that comprise the prokaryotic channels. They result in asynchronous movements of VSs, asymmetric ion-binding sites, and novel loops between the different domains that have specialized roles, for example in fast inactivation, which are not seen in the prokaryotic channels. Nevertheless, the availability of several crystal forms of prokaryotic voltage-gated sodium channels in different conformational states has vastly increased our knowledge of the structure–function relationships for these channels, and provides new insight into their mechanisms of opening, closing, and inactivation (Fig. 5). These, in turn, may inform our understanding of both the structure and function of sodium channels in general. The new model proposed, based on the detailed structures of prokaryotic channels in crystal environments, will, however, await confirmation by further functional experiments on channels in their biological context, as well as structural studies of eukaryotic channels. 相似文献14.
μ-Conotoxins are peptide blockers of voltage-gated sodium channels (sodium channels), inhibiting tetrodotoxin-sensitive neuronal (Na(v) 1.2) and skeletal (Na(v) 1.4) subtypes with highest affinity. Structure-activity relationship studies of μ-conotoxins SIIIA, TIIIA, and KIIIA have shown that it is mainly the C-terminal part of the three-loop peptide that is involved in binding to the sodium channel. In this study, we characterize the effect of N- and C-terminal extensions of μ-conotoxins SIIIA, SIIIB, and TIIIA on their potency and selectivity for neuronal versus muscle sodium channels. Interestingly, extending the N- or C-terminal of the peptide by introducing neutral, positive, and/or negatively charged residues, the selectivity of the native peptide can be altered from neuronal to skeletal and the other way around. The results from this study provide further insight into the binding profile of μ-conotoxins at voltage-gated sodium channels, revealing that binding interactions outside the cysteine-stablilized loops can contribute to μ-conotoxin affinity and sodium channel selectivity. 相似文献
15.
16.
Cao-Hoang L Dumont F Marechal PA Le-Thanh M Gervais P 《Applied microbiology and biotechnology》2008,77(6):1379-1387
The effects of slow chilling (2°C min−1) and rapid chilling (2,000°C min−1) were investigated on the survival and membrane fluidity of Escherichia coli, of Bacillus subtilis, and of Saccharomyces cerevisiae. Cell death was found to be dependent on the physiological state of cell cultures and on the rate of temperature downshift.
Slow temperature decrease allowed cell stabilization, whereas the rapid chilling induced an immediate loss of viability of
up to more than 90 and 70% for the exponentially growing cells of E. coli and B. subtilis, respectively. To relate the results of viability with changes in membrane physical state, membrane anisotropy variation
was monitored during thermal stress using the fluorescence probe 1,6-diphenyl-1,3,5-hexatriene. No variation in the membrane
fluidity of all the three microorganisms was found after the slow chilling. It is interesting to note that fluorescence measurements
showed an irreversible rigidification of the membrane of exponentially growing cells of E. coli and B. subtilis after the instantaneous cold shock, which was not observed with S. cerevisiae. This irreversible effect of the rapid cold shock on the membrane correlated well with high rates of cell inactivation. Thus,
membrane alteration seems to be the principal cause of the cold shock injury. 相似文献
17.
The alpha subunit of the purified voltage-sensitive sodium channel from rat brain is rapidly phosphorylated to the extent of 3-4 mol phosphate/mol by purified protein kinase C. The alpha subunit of the native sodium channel in synaptosomal membranes is also phosphorylated by added protein kinase C as assessed by specific immunoprecipitation and polyacrylamide gel electrophoresis of labeled membranes. Our results suggest coordinate regulation of sodium channel phosphorylation state by cAMP-dependent and calcium/phospholipid-dependent protein kinases. 相似文献
18.
Summary Incubation of Kluyveromyces bulgaricus in sodium and potassium salts led to in vivo activation of -galactosidase. The activation reaction was relatively slow since, at 37°C, it took 30 min to come to completion. The reaction was irreversible and was favoured by high salt concentrations with chlorides proving to be more efficient than phosphates. After incubation in KCl, the final activity obtained was 1.49 U/mg dry yeast and this represented a 10-fold increase in activity compared with the control value measured in ammonium phosphate. Hydrolysis of onitrophenyl--galactoside (ONPG) was insensitive to inhibitors of the transport systems and energy metabolism. There results suggest that K. bulgaricus resting cells take up substrates and ions that readily influence -galactosidase activity. 相似文献
19.
We have characterized the effects of prepulse hyperpolarization and extracellular Mg(2+) on the ionic and gating currents of the Drosophila ether-à-go-go K(+) channel (eag). Hyperpolarizing prepulses significantly slowed channel opening elicited by a subsequent depolarization, revealing rate-limiting transitions for activation of the ionic currents. Extracellular Mg(2+) dramatically slowed activation of eag ionic currents evoked with or without prepulse hyperpolarization and regulated the kinetics of channel opening from a nearby closed state(s). These results suggest that Mg(2+) modulates voltage-dependent gating and pore opening in eag channels. To investigate the mechanism of this modulation, eag gating currents were recorded using the cut-open oocyte voltage clamp. Prepulse hyperpolarization and extracellular Mg(2+) slowed the time course of ON gating currents. These kinetic changes resembled the results at the ionic current level, but were much smaller in magnitude, suggesting that prepulse hyperpolarization and Mg(2+) modulate gating transitions that occur slowly and/or move relatively little gating charge. To determine whether quantitatively different effects on ionic and gating currents could be obtained from a sequential activation pathway, computer simulations were performed. Simulations using a sequential model for activation reproduced the key features of eag ionic and gating currents and their modulation by prepulse hyperpolarization and extracellular Mg(2+). We have also identified mutations in the S3-S4 loop that modify or eliminate the regulation of eag gating by prepulse hyperpolarization and Mg(2+), indicating an important role for this region in the voltage-dependent activation of eag. 相似文献
20.
Natacha Kremer Julia Schwartzman René Augustin Lawrence Zhou Edward G. Ruby Stéphane Hourdez Margaret J. McFall-Ngai 《Proceedings. Biological sciences / The Royal Society》2014,281(1785)
We identified and sequenced from the squid Euprymna scolopes two isoforms of haemocyanin that share the common structural/physiological characteristics of haemocyanin from a closely related cephalopod, Sepia officinalis, including a pronounced Bohr effect. We examined the potential roles for haemocyanin in the animal''s symbiosis with the luminous bacterium Vibrio fischeri. Our data demonstrate that, as in other cephalopods, the haemocyanin is primarily synthesized in the gills. It transits through the general circulation into other tissues and is exported into crypt spaces that support the bacterial partner, which requires oxygen for its bioluminescence. We showed that the gradient of pH between the circulating haemolymph and the matrix of the crypt spaces in adult squid favours offloading of oxygen from the haemocyanin to the symbionts. Haemocyanin is also localized to the apical surfaces and associated mucus of a juvenile-specific epithelium on which the symbionts gather, and where their specificity is determined during the recruitment into the association. The haemocyanin has an antimicrobial activity, which may be involved in this enrichment of V. fischeri during symbiont initiation. Taken together, these data provide evidence that the haemocyanin plays a role in shaping two stages of the squid–vibrio partnership. 相似文献