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1.
Mutations in the cytoplasmic tail (CT) of voltage gated sodium channels cause a spectrum of inherited diseases of cellular excitability, yet to date only one mutation in the CT of the human skeletal muscle voltage gated sodium channel (hNa V1.4 F1705I) has been linked to cold aggravated myotonia. The functional effects of altered regulation of hNa V1.4 F1705I are incompletely understood. The location of the hNa V1.4 F1705I in the CT prompted us to examine the role of Ca 2+ and calmodulin (CaM) regulation in the manifestations of myotonia. To study Na channel related mechanisms of myotonia we exploited the differences in rat and human Na V1.4 channel regulation by Ca 2+ and CaM. hNa V1.4 F1705I inactivation gating is Ca 2+-sensitive compared to wild type hNa V1.4 which is Ca 2+ insensitive and the mutant channel exhibits a depolarizing shift of the V 1/2 of inactivation with CaM over expression. In contrast the same mutation in the rNa V1.4 channel background (rNa V1.4 F1698I) eliminates Ca 2+ sensitivity of gating without affecting the CaM over expression induced hyperpolarizing shift in steady-state inactivation. The differences in the Ca 2+ sensitivity of gating between wild type and mutant human and rat Na V1.4 channels are in part mediated by a divergence in the amino acid sequence in the EF hand like (EFL) region of the CT. Thus the composition of the EFL region contributes to the species differences in Ca 2+/CaM regulation of the mutant channels that produce myotonia. The myotonia mutation F1705I slows I Na decay in a Ca 2+-sensitive fashion. The combination of the altered voltage dependence and kinetics of I Na decay contribute to the myotonic phenotype and may involve the Ca 2+-sensing apparatus in the CT of Na V1.4. 相似文献
2.
Voltage-gated sodium channels (VGSCs) are fundamental to the initiation and propagation of action potentials in excitable cells. Ca2+/calmodulin (CaM) binds to VGSC type II (NaV1.2) isoleucine and glutamine (IQ) motif. An autism-associated mutation in NaV1.2 IQ motif, Arg1902Cys (R1902C), has been reported to affect the combination between CaM and the IQ motif compared to that of the wild type IQ motif. However, the detailed properties for the Ca2+-regulated binding of CaM to NaV1.2 IQ (1901Lys-1927Lys, IQwt) and mutant IQ motif (IQR1902C) remains unclear. Here, the binding ability of CaM and CaM's constituent proteins including N- and C lobe to the IQ motif of NaV1.2 and its mutant was investigated by protein pull-down experiments. We discovered that the combination between CaM and the IQ motif was U-shaped with the highest at [Ca2+] ≈ free and the lowest at 100 nM [Ca2+]. In the IQR1902C mutant, Ca2+-dependence of CaM binding was nearly lost. Consequently, the binding of CaM to IQR1902C at 100 and 500 nM [Ca2+] was increased compared to that of IQwt. Both N- and C lobe of CaM could bind with NaV1.2 IQ motif and IQR1902C mutant, with the major effect of C lobe. Furthermore, CaMKII had no impact on the binding between CaM and NaV1.2 IQ motif. This research offers novel insight to the regulation of NaV1.2 IQwt and IQR1902C motif, an autism-associated mutation, by CaM. 相似文献
4.
Ca 2+ entry through L-type calcium channels (Ca V1.2) is critical in shaping the cardiac action potential and initiating cardiac contraction. Modulation of Ca V1.2 channel gating directly affects myocyte excitability and cardiac function. We have found that phospholemman (PLM), a member of the FXYD family and regulator of cardiac ion transport, coimmunoprecipitates with Ca V1.2 channels from guinea pig myocytes, which suggests PLM is an endogenous modulator. Cotransfection of PLM in HEK293 cells slowed Ca V1.2 current activation at voltages near the threshold for activation, slowed deactivation after long and strong depolarizing steps, enhanced the rate and magnitude of voltage-dependent inactivation (VDI), and slowed recovery from inactivation. However, Ca 2+-dependent inactivation was not affected. Consistent with slower channel closing, PLM significantly increased Ca 2+ influx via Ca V1.2 channels during the repolarization phase of a human cardiac action potential waveform. Our results support PLM as an endogenous regulator of Ca V1.2 channel gating. The enhanced VDI induced by PLM may help protect the heart under conditions such as ischemia or tachycardia where the channels are depolarized for prolonged periods of time and could induce Ca 2+ overload. The time and voltage-dependent slowed deactivation could represent a gating shift that helps maintain Ca 2+ influx during the cardiac action potential waveform plateau phase. 相似文献
5.
The Rem, Rem2, Rad, and Gem/Kir (RGK) GTPases, comprise a subfamily of small Ras-related GTP-binding proteins, and have been shown to potently inhibit high voltage-activated Ca 2+ channel current following overexpression. Although the molecular mechanisms underlying RGK-mediated Ca 2+ channel regulation remains controversial, recent studies suggest that RGK proteins inhibit Ca 2+ channel currents at the plasma membrane in part by interactions with accessory channel β subunits. In this paper, we extend our understanding of the molecular determinants required for RGK-mediated channel regulation by demonstrating a direct interaction between Rem and the proximal C-terminus of Ca V1.2 (PCT), including the CB/IQ domain known to contribute to Ca 2+/calmodulin (CaM)-mediated channel regulation. The Rem2 and Rad GTPases display similar patterns of PCT binding, suggesting that the Ca V1.2 C-terminus represents a common binding partner for all RGK proteins. In vitro Rem:PCT binding is disrupted by Ca 2+/CaM, and this effect is not due to Ca 2+/CaM binding to the Rem C-terminus. In addition, co-overexpression of CaM partially relieves Rem-mediated L-type Ca 2+ channel inhibition and slows the kinetics of Ca 2+-dependent channel inactivation. Taken together, these results suggest that the association of Rem with the PCT represents a crucial molecular determinant in RGK-mediated Ca 2+ channel regulation and that the physiological function of the RGK GTPases must be re-evaluated. Rather than serving as endogenous inhibitors of Ca 2+ channel activity, these studies indicate that RGK proteins may play a more nuanced role, regulating Ca 2+ currents via modulation of Ca 2+/CaM-mediated channel inactivation kinetics. 相似文献
6.
Voltage-gated sodium channels (Na Vs) are membrane proteins responsible for the rapid upstroke of the action potential in excitable cells. There are nine human voltage-sensitive Na V1 isoforms that, in addition to their sequence differences, differ in tissue distribution and specific function. This review focuses on isoforms Na V1.4 and Na V1.5, which are primarily expressed in skeletal and cardiac muscle cells, respectively. The determination of the structures of several eukaryotic Na Vs by single-particle cryo-electron microscopy (cryo-EM) has brought new perspective to the study of the channels. Alignment of the cryo-EM structure of the transmembrane channel pore with x-ray crystallographic structures of the cytoplasmic domains illustrates the complementary nature of the techniques and highlights the intricate cellular mechanisms that modulate these channels. Here, we review structural insights into the cytoplasmic C-terminal regulation of Na V1.4 and Na V1.5 with special attention to Ca 2+ sensing by calmodulin, implications for disease, and putative channel dimerization. 相似文献
7.
The dominant role of Ca V2 voltage-gated calcium channels for driving neurotransmitter release is broadly conserved. Given the overlapping functional properties of Ca V2 and Ca V1 channels, and less so Ca V3 channels, it is unclear why there have not been major shifts toward dependence on other Ca V channels for synaptic transmission. Here, we provide a structural and functional profile of the Ca V2 channel cloned from the early-diverging animal Trichoplax adhaerens, which lacks a nervous system but possesses single gene homologues for Ca V1–Ca V3 channels. Remarkably, the highly divergent channel possesses similar features as human Ca V2.1 and other Ca V2 channels, including high voltage–activated currents that are larger in external Ba 2+ than in Ca 2+; voltage-dependent kinetics of activation, inactivation, and deactivation; and bimodal recovery from inactivation. Altogether, the functional profile of Trichoplax Ca V2 suggests that the core features of presynaptic Ca V2 channels were established early during animal evolution, after Ca V1 and Ca V2 channels emerged via proposed gene duplication from an ancestral Ca V1/2 type channel. The Trichoplax channel was relatively insensitive to mammalian Ca V2 channel blockers ω-agatoxin-IVA and ω-conotoxin-GVIA and to metal cation blockers Cd 2+ and Ni 2+. Also absent was the capacity for voltage-dependent G-protein inhibition by co-expressed Trichoplax Gβγ subunits, which nevertheless inhibited the human Ca V2.1 channel, suggesting that this modulatory capacity evolved via changes in channel sequence/structure, and not G proteins. Last, the Trichoplax channel was immunolocalized in cells that express an endomorphin-like peptide implicated in cell signaling and locomotive behavior and other likely secretory cells, suggesting contributions to regulated exocytosis. 相似文献
8.
The function of the human voltage-gated sodium channel Na V1.5 is regulated in part by intracellular calcium signals. The ubiquitous calcium sensor protein calmodulin (CaM) is an important part of the complex calcium-sensing apparatus in Na V1.5. CaM interacts with an IQ (isoleucine-glutamine) motif in the large intracellular C-terminal domain of the channel. Using co-expression and co-purification, we have been able to isolate a CaM-IQ motif complex and to determine its high-resolution structure in absence of calcium using multi-dimensional solution NMR. Under these conditions, the Na V1.5 IQ motif interacts with the C-terminal domain (C-lobe) of CaM, with the N-terminal domain remaining free in solution. The structure reveals that the C-lobe adopts a semi-open conformation with the IQ motif bound in a narrow hydrophobic groove. Sequence similarities between voltage-gated sodium channels and voltage-gated calcium channels suggest that the structure of the CaM-Na V1.5 IQ motif complex can serve as a general model for the interaction between CaM and ion channel IQ motifs under low-calcium conditions. The structure also provides insight into the biochemical basis for disease-associated mutations that map to the IQ motif in Na V1.5. 相似文献
9.
In cardiac and skeletal myocytes, and in most neurons, the opening of voltage‐gated Na + channels (Na V channels) triggers action potentials, a process that is regulated via the interactions of the channels’ intercellular C‐termini with auxiliary proteins and/or Ca 2+. The molecular and structural details for how Ca 2+ and/or auxiliary proteins modulate Na V channel function, however, have eluded a concise mechanistic explanation and details have been shrouded for the last decade behind controversy about whether Ca 2+ acts directly upon the Na V channel or through interacting proteins, such as the Ca 2+ binding protein calmodulin (CaM). Here, we review recent advances in defining the structure of Na V intracellular C‐termini and associated proteins such as CaM or fibroblast growth factor homologous factors (FHFs) to reveal new insights into how Ca 2+ affects Na V function, and how altered Ca 2+‐dependent or FHF‐mediated regulation of Na V channels is perturbed in various disease states through mutations that disrupt CaM or FHF interaction. 相似文献
10.
Calmodulin (CaM) binding to the intracellular C-terminal tail (CTT) of the cardiac L-type Ca 2+ channel (Ca V1.2) regulates Ca 2+ entry by recognizing sites that contribute to negative feedback mechanisms for channel closing. CaM associates with Ca V1.2 under low resting [Ca 2+], but is poised to change conformation and position when intracellular [Ca 2+] rises. CaM binding Ca 2+, and the domains of CaM binding the CTT are linked thermodynamic functions. To better understand regulation, we determined the energetics of CaM domains binding to peptides representing pre-IQ sites A 1588, and C 1614 and the IQ motif studied as overlapping peptides IQ 1644 and IQ ′1650 as well as their effect on calcium binding. (Ca 2+) 4-CaM bound to all four peptides very favorably ( Kd ≤ 2 nM). Linkage analysis showed that IQ 1644-1670 bound with a Kd ~ 1 pM. In the pre-IQ region, (Ca 2+) 2-N-domain bound preferentially to A 1588, while (Ca 2+) 2-C-domain preferred C 1614. When bound to C 1614, calcium binding in the N-domain affected the tertiary conformation of the C-domain. Based on the thermodynamics, we propose a structural mechanism for calcium-dependent conformational change in which the linker between CTT sites A and C buckles to form an A-C hairpin that is bridged by calcium-saturated CaM. 相似文献
11.
Ion transport and regulation were studied in two, alternatively spliced isoforms of the Na +-Ca 2+ exchanger from Drosophila melanogaster. These exchangers, designated CALX1.1 and CALX1.2, differ by five amino acids in a region where alternative splicing also occurs in the mammalian Na +-Ca 2+ exchanger, NCX1. The CALX isoforms were expressed in Xenopus laevis oocytes and characterized electrophysiologically using the giant, excised patch clamp technique. Outward Na +-Ca 2+ exchange currents, where pipette Ca 2+
o exchanges for bath Na +
i, were examined in all cases. Although the isoforms exhibited similar transport properties with respect to their Na +
i affinities and current–voltage relationships, significant differences were observed in their Na +
i- and Ca 2+
i-dependent regulatory properties. Both isoforms underwent Na +
i-dependent inactivation, apparent as a time-dependent decrease in outward exchange current upon Na +
i application. We observed a two- to threefold difference in recovery rates from this inactive state and the extent of Na +
i-dependent inactivation was approximately twofold greater for CALX1.2 as compared with CALX1.1. Both isoforms showed regulation of Na +-Ca 2+ exchange activity by Ca 2+
i, but their responses to regulatory Ca 2+
i differed markedly. For both isoforms, the application of cytoplasmic Ca 2+
i led to a decrease in outward exchange currents. This negative regulation by Ca 2+
i is unique to Na +-Ca 2+ exchangers from Drosophila, and contrasts to the positive regulation produced by cytoplasmic Ca 2+ for all other characterized Na +-Ca 2+ exchangers. For CALX1.1, Ca 2+
i inhibited peak and steady state currents almost equally, with the extent of inhibition being ≈80%. In comparison, the effects of regulatory Ca 2+
i occurred with much higher affinity for CALX1.2, but the extent of these effects was greatly reduced (≈20–40% inhibition). For both exchangers, the effects of regulatory Ca 2+
i occurred by a direct mechanism and indirectly through effects on Na +
i-induced inactivation. Our results show that regulatory Ca 2+
i decreases Na +
i-induced inactivation of CALX1.2, whereas it stabilizes the Na +
i-induced inactive state of CALX1.1. These effects of Ca 2+
i produce striking differences in regulation between CALX isoforms. Our findings indicate that alternative splicing may play a significant role in tailoring the regulatory profile of CALX isoforms and, possibly, other Na +-Ca 2+ exchange proteins. 相似文献
12.
Voltage-gated calcium (Ca V) channels deliver Ca 2+ to trigger cellular functions ranging from cardiac muscle contraction to neurotransmitter release. The mechanism by which these channels select for Ca 2+ over other cations is thought to involve multiple Ca 2+-binding sites within the pore. Although the Ca 2+ affinity and cation preference of these sites have been extensively investigated, the effect of voltage on these sites has not received the same attention. We used a neuronal preparation enriched for N-type calcium (Ca V2.2) channels to investigate the effect of voltage on Ca 2+ flux. We found that the EC 50 for Ca 2+ permeation increases from 13 mM at 0 mV to 240 mM at 60 mV, indicating that, during permeation, Ca 2+ ions sense the electric field. These data were nicely reproduced using a three-binding-site step model. Using roscovitine to slow Ca V2.2 channel deactivation, we extended these measurements to voltages <0 mV. Permeation was minimally affected at these hyperpolarized voltages, as was predicted by the model. As an independent test of voltage effects on permeation, we examined the Ca 2+-Ba 2+ anomalous mole fraction (MF) effect, which was both concentration and voltage dependent. However, the Ca 2+-Ba 2+ anomalous MF data could not be reproduced unless we added a fourth site to our model. Thus, Ca 2+ permeation through Ca V2.2 channels may require at least four Ca 2+-binding sites. Finally, our results suggest that the high affinity of Ca 2+ for the channel helps to enhance Ca 2+ influx at depolarized voltages relative to other ions (e.g., Ba 2+ or Na +), whereas the absence of voltage effects at negative potentials prevents Ca 2+ from becoming a channel blocker. Both effects are needed to maximize Ca 2+ influx over the voltages spanned by action potentials. 相似文献
13.
The Na +-Ca 2+ exchanger (NCX) links transmembrane movements of Ca 2+ ions to the reciprocal movement of Na+ ions. It normally functions primarily as a Ca 2+ efflux mechanism in excitable tissues such as the heart, but it can also mediate Ca 2+ influx under certain conditions. Na + and Ca 2+ ions exert complex regulatory effects on NCX activity. Ca 2+ binds to two regulatory sites in the exchanger's central hydrophilic domain, and this interaction is normally essential for activation of exchange activity. High cytosolic Na + concentrations, however, can induce a constitutive activity that by-passes the need for allosteric Ca 2+ activation. Constitutive NCX activity can also be induced by high levels of phopshotidylinositol-4,5-bisphosphate (PIP 2) and by mutations affecting the regulatory calcium binding domains. In addition to promoting constitutive activity, high cytosolic Na + concentrations also induce an inactivated state of the exchanger (Na +-dependent inactivation) that becomes dominant when cytosolic pH and PIP 2 levels fall. Na+-dependent inactivation may provide a means of protecting cells from Ca 2+ overload due to NCX-mediated Ca 2+ influx during ischemia. 相似文献
14.
Calmodulin binds to IQ motifs in the α 1 subunit of Ca V1.1 and Ca V1.2, but the affinities of calmodulin for the motif and for Ca 2+ are higher when bound to Ca V1.2 IQ. The Ca V1.1 IQ and Ca V1.2 IQ sequences differ by four amino acids. We determined the structure of calmodulin bound to Ca V1.1 IQ and compared it with that of calmodulin bound to Ca V1.2 IQ. Four methionines in Ca 2+-calmodulin form a hydrophobic binding pocket for the peptide, but only one of the four nonconserved amino acids (His-1532 of Ca V1.1 and Tyr-1675 of Ca V1.2) contacts this calmodulin pocket. However, Tyr-1675 in Ca V1.2 contributes only modestly to the higher affinity of this peptide for calmodulin; the other three amino acids in Ca V1.2 contribute significantly to the difference in the Ca 2+ affinity of the bound calmodulin despite having no direct contact with calmodulin. Those residues appear to allow an interaction with calmodulin with one lobe Ca 2+-bound and one lobe Ca 2+-free. Our data also provide evidence for lobe-lobe interactions in calmodulin bound to Ca V1.2.The complexity of eukaryotic Ca 2+ signaling arises from the ability of cells to respond differently to Ca 2+ signals that vary in amplitude, duration, and location. A variety of mechanisms decode these signals to drive the appropriate physiological responses. The Ca 2+ sensor for many of these physiological responses is the Ca 2+-binding protein calmodulin (CaM). 2 The primary sequence of CaM is tightly conserved in all eukaryotes, yet it binds and regulates a broad set of target proteins in response to Ca 2+ binding. CaM has two domains that bind Ca 2+ as follows: an amino-terminal domain (N-lobe) and a carboxyl-terminal domain (C-lobe) joined via a flexible α-helix. Each lobe of CaM binds two Ca 2+ ions, and binding within each lobe is highly cooperative. The two lobes of CaM, however, have distinct Ca 2+ binding properties; the C-lobe has higher Ca 2+ affinity because of a slower rate of dissociation, whereas the N-lobe has weaker Ca 2+ affinity and faster kinetics ( 1). CaM can also bind to some target proteins in both the presence and absence of Ca 2+, and the preassociation of CaM in low Ca 2+ modulates the apparent Ca 2+ affinity of both the amino-terminal and carboxyl-terminal lobes. Differences in the Ca 2+ binding properties of the lobes and in the interaction sites of the amino- and carboxyl-terminal lobes enable CaM to decode local versus global Ca 2+ signals ( 2).Even though CaM is highly conserved, CaM target (or recognition) sites are quite heterogeneous. The ability of CaM to bind to very different targets is at least partially due to its flexibility, which allows it to assume different conformations when bound to different targets. CaM also binds to various targets in distinct Ca 2+ saturation states as follows: Ca 2+-free ( 3), Ca 2+ bound to only one of the two lobes, or fully Ca 2+-bound ( 4– 7). In addition, CaM may bind with both lobes bound to a target ( 5, 6) or with only a single lobe engaged ( 8). If a target site can bind multiple conformers of CaM, CaM may undergo several transitions that depend on Ca 2+ concentration, thereby tuning the functional response. Identification of stable intermediate states of CaM bound to individual targets will help to elucidate the steps involved in this fine-tuned control.Both Ca V1.1 and Ca V1.2 belong to the L-type family of voltage-dependent Ca 2+ channels, which bind apoCaM and Ca 2+-CaM at carboxyl-terminal recognition sites in their α 1 subunits ( 9– 14). Ca 2+ binding to CaM, bound to Ca V1.2 produces Ca 2+-dependent facilitation (CDF) ( 14). Whether Ca V1.1 undergoes CDF is not known. However, both Ca V1.2 and Ca V1.1 undergo Ca 2+- and CaM-dependent inactivation (CDI) ( 14, 15). Ca V1.1 CDI is slower and more sensitive to buffering by 1,2-bis( o-aminophenoxy)ethane- N,N,N′, N′-tetraacetic acid than Ca V1.2 CDI ( 15). Ca 2+ buffers are thought to influence CDI and/or CDF in voltage-dependent Ca 2+ channels by competing with CaM for Ca 2+ ( 16).The conformation of the carboxyl terminus of the α 1 subunit is critical for channel function and has been proposed to regulate the gating machinery of the channel ( 17, 18). Several interactions of this region include intramolecular contacts with the pore inactivation machinery and intermolecular contacts with CaM kinase II and ryanodine receptors ( 17, 19– 22). Ca 2+ regulation of Ca V1.2 may involve several motifs within this highly conserved region, including an EF hand motif and three contiguous CaM-binding sequences ( 10, 12). ApoCaM and Ca 2+-CaM-binding sites appear to overlap at the site designated as the “IQ motif” ( 9, 12, 13), which are critical for channel function at the molecular and cellular level ( 14, 23).Differences in the rate at which 1,2-bis( o-aminophenoxy)ethane- N,N,N′, N′-tetraacetic acid affects CDI of Ca V1.1 and Ca V1.2 could reflect differences in their interactions with CaM. In this study we describe the differences in CaM interactions with the IQ motifs of the Ca V1.1 and the Ca V1.2 channels in terms of crystal structure, CaM affinity, and Ca 2+ binding to CaM. We find the structures of Ca 2+-CaM-IQ complexes are similar except for a single amino acid change in the peptide that contributes to its affinity for CaM. We also find that the other three amino acids that differ in Ca V1.2 and Ca V1.1 contribute to the ability of Ca V1.2 to bind a partially Ca 2+-saturated form of CaM. 相似文献
15.
L-type Ca 2+ channels select for Ca 2+ over sodium Na + by an affinity-based mechanism. The prevailing model of Ca 2+ channel permeation describes a multi-ion pore that requires pore occupancy by at least two Ca 2+ ions to generate a Ca 2+ current. At [Ca 2+] < 1 μM, Ca 2+ channels conduct Na +. Due to the high affinity of the intrapore binding sites for Ca 2+ relative to Na +, addition of μM concentrations of Ca 2+ block Na + conductance through the channel. There is little information, however, about the potential for interaction between Na + and Ca 2+ for the second binding site in a Ca 2+ channel already occupied by one Ca 2+. The two simplest possibilities, ( a) that Na + and Ca 2+ compete for the second binding site or ( b) that full time occupancy by one Ca 2+ excludes Na + from the pore altogether, would imply considerably different mechanisms of channel permeation. We are studying permeation mechanisms in N-type Ca 2+ channels. Similar to L-type Ca 2+ channels, N-type channels conduct Na + well in the absence of external Ca 2+. Addition of 10 μM Ca 2+ inhibited Na + conductance by 95%, and addition of 1 mM Mg 2+ inhibited Na + conductance by 80%. At divalent ion concentrations of 2 mM, 120 mM Na + blocked both Ca 2+ and Ba 2+ currents. With 2 mM Ba 2+, the IC 50 for block of Ba 2+ currents by Na + was 119 mM. External Li + also blocked Ba 2+ currents in a concentration-dependent manner, with an IC 50 of 97 mM. Na + block of Ba 2+ currents was dependent on [Ba 2+]; increasing [Ba 2+] progressively reduced block with an IC 50 of 2 mM. External Na + had no effect on voltage-dependent activation or inactivation of the channel. These data suggest that at physiological concentrations, Na + and Ca 2+ compete for occupancy in a pore already occupied by a single Ca 2+. Occupancy of the pore by Na + reduced Ca 2+ channel conductance, such that in physiological solutions, Ca 2+ channel currents are between 50 and 70% of maximal. 相似文献
16.
Ca V channels are transmembrane proteins that mediate and regulate ion fluxes across cell membranes, and they are activated in response to action potentials to allow Ca 2+ influx. Since ion channels are composed of charge or polar groups, an external alternating electric field may affect the ion‐selective membrane transport and the performance of the channel. In this article, we have investigated the effect of an external GHz electric field on the dynamics of calcium ions in the selectivity filter of the Ca VAb channel. Molecular dynamics (MD) simulations and the potential of mean force (PMF) calculations were carried out, via the umbrella sampling method, to determine the free energy profile of Ca 2+ ions in the Ca VAb channels in presence and absence of an external field. Exposing Ca VAb channel to 1, 2, 3, 4, and 5 GHz electric fields increases the depth of the potential energy well and this may result in an increase in the affinity and strength of Ca 2+ ions to binding sites in the selectivity filter the channel. This increase of strength of Ca 2+ ions binding in the selectivity filter may interrupt the mechanism of Ca 2+ ion conduction, and leads to a reduction of Ca 2+ ion permeation through the Ca VAb channel. 相似文献
17.
The structural determinants of mibefradil inhibition were analyzed using wild-type and inactivation-modified Ca V1.2 (α1C) and Ca V2.3 (α1E) channels. Mibefradil inhibition of peak Ba 2+ currents was dose- and voltage-dependent. An increase of holding potentials from −80 to −100 mV significantly shifted dose-response
curves toward higher mibefradil concentrations, namely from a concentration of 108 ± 21 μ m ( n= 7) to 288 ± 17 μ m ( n= 3) for inhibition of half of the Ca v1.2 currents ( IC
50) and from IC
50= 8 ± 2 μ m ( n= 9) to 33 ± 7 μ m ( n= 4) for Ca V2.3 currents. In the presence of mibefradil, Ca V1.2 and Ca V2.3 experienced significant use-dependent inhibition (0.1 to 1 Hz) and slower recovery from inactivation suggesting mibefradil
could promote transition(s) to an absorbing inactivated state. In order to investigate the relationship between inactivation
and drug sensitivity, mibefradil inhibition was studied in inactivation-altered Ca V1.2 and Ca V2.3 mutants. Mibefradil significantly delayed the onset of channel recovery from inactivation in CEEE (Repeat I + part of
the I–II linker from Ca V1.2 in the Ca V2.3 host channel), in EC( AID)EEE (part of the I–II linker from Ca V1.2 in the Ca V2.3 host channel) as well as in Ca V1.2 E462R, and Ca V2.3 R378E (point mutation in the β-subunit binding motif) channels. Mibefradil inhibited the faster inactivating chimera EC( IS1-6)EEE with an IC
50= 7 ± 1 μ m ( n= 3), whereas the slower inactivating chimeras EC( AID)EEE and CEEE were, respectively, inhibited with IC
50= 41 ± 5 μ m ( n= 4) and IC
50= 68 ± 9 μ m ( n= 5). Dose-response curves were superimposable for the faster EC( IS1-6)EEE and Ca V2.3, whereas intermediate-inactivating channel kinetics (CEEE, Ca V1.2 E462R, and Ca V1.2 E462K) were inhibited by similar concentrations of mibefradil with IC
50≈ 55–75 μ m. The slower Ca V1.2 wild-type and Ca V1.2 Q473K channels responded to higher doses of mibefradil with IC
50≈ 100–120 μ m. Mibefradil was also found to significantly speed up the inactivation kinetics of slower channels (Ca V1.2, CEEE) with little effect on the inactivation kinetics of faster-inactivating channels (Ca V2.3). A open-channel block model for mibefradil interaction with high-voltage-activated Ca 2+ channels is discussed and shown to qualitatively account for our observations. Hence, our data agree reasonably well with
a ``receptor guarded mechanism' where fast inactivation kinetics efficiently trap mibefradil into the channel.
Received: 14 March 2001/Revised: 25 June 2001 相似文献
18.
Voltage-gated sodium channels (Na Vs) are central elements of cellular excitation. Notwithstanding advances from recent bacterial Na V (BacNa V) structures, key questions about gating and ion selectivity remain. Here, we present a closed conformation of Na VAe1p, a pore-only BacNa V derived from Na VAe1, a BacNa V from the arsenite oxidizer Alkalilimnicola ehrlichei found in Mono Lake, California, that provides insight into both fundamental properties. The structure reveals a pore domain in which the pore-lining S6 helix connects to a helical cytoplasmic tail. Electrophysiological studies of full-length BacNa Vs show that two elements defined by the Na VAe1p structure, an S6 activation gate position and the cytoplasmic tail “neck”, are central to BacNa V gating. The structure also reveals the selectivity filter ion entry site, termed the “outer ion” site. Comparison with mammalian voltage-gated calcium channel (Ca V) selectivity filters, together with functional studies, shows that this site forms a previously unknown determinant of Ca V high-affinity calcium binding. Our findings underscore commonalities between BacNa Vs and eukaryotic voltage-gated channels and provide a framework for understanding gating and ion permeation in this superfamily. 相似文献
19.
Voltage-dependent calcium channels (Ca V) enable the inward flow of calcium currents for a wide range of cells. Ca V1 and Ca V2 subtype α1 subunits form the conducting pore using four repeated membrane domains connected by intracellular linkers. The domain I-II linker connects to the membrane gate (IS6), forming an α-helix, and is bound to the Ca Vβ subunit. Previous studies indicated that this region may or may not form a continuous helix depending on the Ca V subtype, thereby modulating channel activation and inactivation properties. Here, we used small-angle x-ray scattering and ensemble modeling analysis to investigate the solution structure of these linkers, extending from the membrane domain and including the Ca Vβ-binding site, called the proximal linker (PL). The results demonstrate that the Ca V1.2 PL is more flexible than the Ca V2.2 PL, the flexibility is intrinsic and not dependent on Ca Vβ binding, and the flexibility can be most easily explained by the presence of conserved glycines. Our analysis also provides a robust example of investigating protein domains in which flexibility plays an essential role. 相似文献
20.
It is well established that syntaxin 1A (Sx1A), SNAP-25 and synaptotagmin (Syt1) either alone or in combination, modify the kinetic properties of voltage-gated Ca 2+ channels (VGCCs). The interaction interface resides mainly at the cytosolic II-III domain of the alpha1 subunit of the channels, while Sx1A interacts with the channel also via two highly conserved cysteine residues at the transmembrane domain. In the present study, we characterized Ca 2+-independent coupling of the human neuronal P/Q-type calcium channel (Ca V2.1) with Sx1A, SNAP-25, Syt1 and synaptobrevin (VAMP) in BAPTA-injected Xenopus oocytes. The co-expression of Ca V2.1 with Sx1A, SNAP-25 and Syt1, produced a multiprotein complex with distinctive kinetic properties analogous to the excitosome complexes generated by Ca V1.2, Ca V2.2, and Ca V2.3. The distinct kinetic properties of Ca V2.1 acquired by its close association with Syt1 and t-SNAREs suggest that the vesicle is tethered to the neuronal channel and to the exocytotic machinery independently of intracellular Ca 2+. To explore the relevance of these interactions to secretion we exploited a BotC1-and a BotA-sensitive secretion system developed for Xenopus oocytes not buffered by BAPTA, in which depolarization-evoked secretion is monitored by a change in membrane capacitance. The reconstituted Ca V2.1 release is consistent with the model in which the VGCC acts from within the exocytotic complex playing a signaling role in triggering release. The relevance of these results to secretion posits the role of possible rearrangements within the excitosome subsequent to Ca 2+ entry, setting the stage for the fusion of channel-tethered-vesicles upon the arrival of an action potential. 相似文献
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