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1.
Molecular dynamics and mutational analysis of a channelopathy mutation in the IIS6 helix of Ca V 1.2
Stary A Kudrnac M Beyl S Hohaus A Timin EN Wolschann P Guy HR Hering S 《Channels (Austin, Tex.)》2008,2(3):216-223
A channelopathy mutation in segment IIS6 of Ca(V)1.4 (I745T) has been shown to cause severe visual impairment by shifting the activation and inactivation curves to more hyperpolarized voltages and slowing activation and inactivation kinetics. A similar gating phenotype is caused by the corresponding mutation, I781T, in Ca(V)1.2 (midpoint of activation curve (V(0.5)) shifted to -37.7 +/- 1.2 mV). We show here that wild-type gating can partially be restored by a helix stabilizing rescue mutation N785A. V(0.5) of I781T/N785A (V(0.5) = -21.5 +/- 0.6 mV) was shifted back towards wild-type (V(0.5) = -9.9 +/- 1.1 mV). Homology models developed in our group (see accompanying article for details) were used to perform Molecular Dynamics-simulations (MD-simulations) on wild-type and mutant channels. Systematic changes in segment IIIS6 (M1187-F1194) and in helix IIS6 (N785-L786) were studied. The simulated structural changes in S6 segments of I781T/N785A were less pronounced than in I781T. A delicate balance between helix flexibility and stability enabling the formation of hydrophobic seals at the inner channel mouth appears to be important for wild-type Ca(V)1.2 gating. Our study illustrates that effects of mutations in the lower part of IIS6 may not be localized to the residue or even segment being mutated, but may affect conformations of interacting segments. 相似文献
2.
《Channels (Austin, Tex.)》2013,7(3):216-223
A channelopathy mutation in segment IIS6 of CaV1.4 (I745T) has been shown to cause severe visual impairment by shifting the activation and inactivation curves to more hyperpolarised voltages and slowing activation and inactivation kinetics. A similar gating phenotype is caused by the corresponding mutation, I781T, in CaV1.2 (midpoint of activation curve (V0.5) shifted to -37.7 ± 1.2 mV). We show here that wild type gating can partially be restored by a helix stabilising rescue mutation N785A. V0.5 of I781T/N785A (V0.5 = -21.5 ± 0.6 mV) was shifted back towards wild type (V0.5 = -9.9±1.1 mV). Homology models developed in our group (see accompanying article for details) were used to perform MD-simulations on wild-type and mutant channels. Systematic changes in segment IIIS6 (M1187 - F1194) and in helix IIS6 (N785-L786) were observed. The simulated structural changes in S6 segments of I781T/N785A were less pronounced than in I781T. A delicate balance between helix flexibility and stability enabling the formation of hydrophobic seals at the inner channel mouth appears to be important for wild type CaV1.2 gating. Our study illustrates that effects of mutations in the lower part of IIS6 may not be localized to the residue or even segment being mutated, but may affect conformations of interacting segments. 相似文献
3.
Wall-Lacelle S Hossain MI Sauvé R Blunck R Parent L 《The Journal of biological chemistry》2011,286(31):27197-27205
Mutations in distal S6 were shown to significantly alter the stability of the open state of Ca(V)2.3 (Raybaud, A., Baspinar, E. E., Dionne, F., Dodier, Y., Sauvé, R., and Parent, L. (2007) J. Biol. Chem. 282, 27944-27952). By analogy with K(V) channels, we tested the hypothesis that channel activation involves electromechanical coupling between S6 and the S4S5 linker in Ca(V)2.3. Among the 11 positions tested in the S4S5 linker of domain II, mutations of the leucine residue at position 596 were found to destabilize significantly the closed state with a -50 mV shift in the activation potential and a -20 mV shift in its charge-voltage relationship as compared with Ca(V)2.3 wt. A double mutant cycle analysis was performed by introducing pairs of glycine residues between S4S5 and S6 of Domain II. Strong coupling energies (ΔΔG(interact) > 2 kcal mol(-1)) were measured for the activation gating of 12 of 39 pairs of mutants. Leu-596 (IIS4S5) was strongly coupled with distal residues in IIS6 from Leu-699 to Asp-704. In particular, the double mutant L596G/I701G showed strong cooperativity with a ΔΔG(interact) ≈6 kcal mol(-1) suggesting that both positions contribute to the activation gating of the channel. Altogether, our results highlight the role of a leucine residue in S4S5 and provide the first series of evidence that the IIS4S5 and IIS6 regions are energetically coupled during the activation of a voltage-gated Ca(V) channel. 相似文献
4.
Michaela Kudrnac Stanislav Beyl Annette Hohaus Anna Stary Thomas Peterbauer Eugen Timin Steffen Hering 《The Journal of biological chemistry》2009,284(18):12276-12284
Voltage dependence and kinetics of CaV1.2 activation are
affected by structural changes in pore-lining S6 segments of the
α1-subunit. Significant effects are induced by either proline
or threonine substitutions in the lower third of segment IIS6 (“bundle
crossing region”), where S6 segments are likely to seal the channel in
the closed conformation (Hohaus, A., Beyl, S., Kudrnac, M., Berjukow, S.,
Timin, E. N., Marksteiner, R., Maw, M. A., and Hering, S. (2005) J. Biol.
Chem. 280, 38471–38477). Here we report that S435P in IS6 results
in a large shift of the activation curve (-25.9 ± 1.2 mV) and slower
current kinetics. Threonine substitutions at positions Leu-429 and Leu-434
induced a similar kinetic phenotype with shifted activation curves (L429T by
-6.6 ± 1.2 and L434T by -12.1 ± 1.7 mV). Inactivation curves of
all mutants were shifted to comparable extents as the activation curves.
Interdependence of IS6 and IIS6 mutations was analyzed by means of mutant
cycle analysis. Double mutations in segments IS6 and IIS6 induce either
additive (L429T/I781T, -34.1 ± 1.4 mV; L434T/I781T, -40.4 ± 1.3
mV; L429T/L779T, -12.6 ± 1.3 mV; and L434T/L779T, -22.4 ± 1.3
mV) or nonadditive shifts of the activation curves along the voltage axis
(S435P/I781T, -33.8 ± 1.4 mV). Mutant cycle analysis revealed energetic
coupling between residues Ser-435 and Ile-781, whereas other paired mutations
in segments IS6 and IIS6 had independent effects on activation gating.Ca2+ current through CaV1.2 channels initiates muscle
contraction, release of hormones and neurotransmitters, and affects
physiological processes such as vision, hearing, and gene expression
(1). Their pore-forming
α1-subunit is composed of four homologous domains formed by
six transmembrane segments (S1–S6)
(2). The signal of the
voltage-sensing machinery, consisting of multiple charged amino acids (located
in segments S4 and adjacent structures of each domain), is transmitted to the
pore region (3). Conformational
changes in pore lining S6 and adjacent segments finally lead to pore openings
(activation) and closures (inactivation).Our understanding of how CaV1.2 channels open and close is
largely based on extrapolations of structural information from potassium
channels. The crystal structures of the closed conformation of two bacterial
potassium channels (KcsA and MlotiK)
(4,
5) show a gate located at the
intracellular channel mouth formed by tightly packed S6 helices. The crystal
structure of the open conformation of Kv1.2
(6,
7) revealed a bent S6 with the
highly conserved PXP motif apparently acting as a hinge (see
8). The activation mechanism
proposed for MthK channels involves helix bending at a highly conserved
glycine at position 83 (see Ref.
9, “glycine gating
hinge” hypothesis).Compared with potassium channels, the pore of CaV is asymmetric,
and none of the four S6 segments has a putative helix-bending PXP
motif. Furthermore, the conserved glycine (corresponding to position 83 in
MthK, see Ref. 10) is only
present in segments IS6 and IIS6 (for review see Ref.
11). We have shown that
substituting proline for this glycine in IIS6 of CaV1.2 does not
significantly affect gating
(12).Zhen et al. (13)
investigated the pore lining S6 segments of CaV2.1 using the
substituted cysteine accessibility method. The accessibility of cysteines was
changed by opening and closing the channel, consistent with the gate being on
the intracellular side. The general picture of a channel gate close to the
inner channel mouth of CaV1.2 was recently supported by
pharmacological studies
(14).Substitution of hydrophilic residues in the lower third of segment IIS6 of
CaV1.2 (LAIA motif, 779–784, see Ref.
12) induces pronounced changes
in channel gating as follows: a shift in the voltage dependence of activation
accompanied by a slowing of the activation kinetics near the footstep of the
m∞(V) curve and a slowing of deactivation
at all potentials. Interestingly, these changes in channel gating resemble the
effects of proline substitution of Gly-219 in the bacterial sodium channel
from Bacillus halodurans (“Gly-219 gating hinge,” see
Ref. 15).The strongest shifts of the activation curve reported so far were observed
for proline substitutions
(12). As prolines in an
α-helix cause a rigid kink with an angle of about 26°
(16), we hypothesized that
these mutants were causing a kink in helix IIS6 similar to a bend that would
normally occur flexibly during the activation process
(12).Here we extend our previous study by systematically substituting residues
in segment IS6 of CaV1.2 by proline or the small and polar
threonine. Several functional IS6 mutants with shifted activation and
inactivation characteristics were identified (S435P, L429T, and L434T), and
the interdependence of IS6 and IIS6 mutations was analyzed. Mutant cycle
analysis revealed both mutually independent and energetically coupled
contributions of IS6 and IIS6 residues on activation gating. 相似文献
5.
Raybaud A Dodier Y Bissonnette P Simoes M Bichet DG Sauvé R Parent L 《The Journal of biological chemistry》2006,281(51):39424-39436
The putative hinge point revealed by the crystal structure of the MthK potassium channel is a glycine residue that is conserved in many ion channels. In high voltage-activated (HVA) Ca(V) channels, the mid-S6 glycine residue is only present in IS6 and IIS6, corresponding to G422 and G770 in Ca(V)1.2. Two additional glycine residues are found in the distal portion of IS6 (Gly(432) and Gly(436) in Ca(V)1.2) to form a triglycine motif unique to HVA Ca(V) channels. Lethal arrhythmias are associated with mutations of glycine residues in the human L-type Ca(2+) channel. Hence, we undertook a mutational analysis to investigate the role of S6 glycine residues in channel gating. In Ca(V)1.2, alpha-helix-breaking proline mutants (G422P and G432P) as well as the double G422A/G432A channel did not produce functional channels. The macroscopic inactivation kinetics were significantly decreased with Ca(V)1.2 wild type > G770A > G422A congruent with G436A > G432A (from the fastest to the slowest). Mutations at position Gly(432) produced mostly nonfunctional mutants. Macroscopic inactivation kinetics were markedly reduced by mutations of Gly(436) to Ala, Pro, Tyr, Glu, Arg, His, Lys, or Asp residues with stronger effects obtained with charged and polar residues. Mutations within the distal GX(3)G residues blunted Ca(2+)-dependent inactivation kinetics and prevented the increased voltage-dependent inactivation kinetics brought by positively charged residues in the I-II linker. In Ca(V)2.3, mutation of the distal glycine Gly(352) impacted significantly on the inactivation gating. Altogether, these data highlight the role of the GX(3)G motif in the voltage-dependent activation and inactivation gating of HVA Ca(V) channels with the distal glycine residue being mostly involved in the inactivation gating. 相似文献
6.
Voltage-dependent calcium channels are classified into low voltage-activated and high voltage-activated channels. We have investigated the molecular basis for this difference in voltage dependence of activation by constructing chimeras between a low voltage-activated channel (Ca(V)3.1) and a high voltage-activated channel (Ca(V)1.2), focusing on steady-state activation properties. Wild type and chimeras were expressed in oocytes, and two-electrode voltage clamp recordings were made of calcium channel currents. Replacement of domains I, III, or IV of the Ca 3.1 channel with the corresponding domain of Ca(V)1.2 led (V)to high voltage-activated channels; for these constructs the current/voltage (I/V) curves were similar to those for Ca(V)1.2 wild type. However, replacement of domain II gave only a small shift to the right of the I/V curve and modulation of the activation kinetics but did not lead to a high voltage-activating channel with an I/V curve like Ca 1.2. We also investigated the role of the voltage sensor (V)S4 by replacing the S4 segment of Ca(V)3.1 with that of Ca 1.2. For domain I, there was no shift in the I/V curve (V)as compared with Ca(V)3.1, and there were relatively small shifts to the right for domains III and IV. Taken together, these results suggest that domains I, III, and IV (rather than domain II) are apparently critical for channel opening and, therefore, contribute strongly to the difference in voltage dependence of activation between Ca 3.1 and Ca(V)1.2. However, the S4 segments in domains I, (V)III, and IV did not account for this difference in voltage dependence. 相似文献
7.
Raybaud A Baspinar EE Dionne F Dodier Y Sauvé R Parent L 《The Journal of biological chemistry》2007,282(38):27944-27952
The hydrophobic locus VAVIM is conserved in the S6 transmembrane segment of domain IV (IVS6) in Ca(V)1 and Ca(V)2 families. Herein we show that glycine substitution of the VAVIM motif in Ca(V)2.3 produced whole cell currents with inactivation kinetics that were either slower (A1719G approximately V1720G), similar (V1718G), or faster (I1721G approximately M1722G) than the wild-type channel. The fast kinetics of I1721G were observed with a approximately +10 mV shift in its voltage dependence of activation (E(0.5,act)). In contrast, the slow kinetics of A1719G and V1720G were accompanied by a significant shift of approximately -20 mV in their E(0.5,act) indicating that the relative stability of the channel closed state was decreased in these mutants. Glycine scan performed with Val (349) in IS6, Ile(701) in IIS6, and Leu(1420) in IIIS6 at positions predicted to face Val(1720) in IVS6 also produced slow inactivating currents with hyperpolarizing shifts in the activation and inactivation potentials, again pointing out a decrease in the stability of the channel closed state. Mutations to other hydrophobic residues at these positions nearly restored the channel gating. Altogether these data indicate that residues at positions equivalent to 1720 exert a critical control upon the relative stability of the channel closed and open states and more specifically, that hydrophobic residues at these positions promote the channel closed state. We discuss a three-dimensional homology model of Ca(V)2.3 based upon Kv1.2 where hydrophobic residues at positions facing Val(1720) in IS6, IIS6, and IIIS6 play a critical role in stabilizing the closed state in Ca(V)2.3. 相似文献
8.
Depil K Beyl S Stary-Weinzinger A Hohaus A Timin E Hering S 《The Journal of biological chemistry》2011,286(36):31557-31564
The Timothy syndrome mutations G402S and G406R abolish inactivation of Ca(V)1.2 and cause multiorgan dysfunction and lethal arrhythmias. To gain insights into the consequences of the G402S mutation on structure and function of the channel, we systematically mutated the corresponding Gly-432 of the rabbit channel and applied homology modeling. All mutations of Gly-432 (G432A/M/N/V/W) diminished channel inactivation. Homology modeling revealed that Gly-432 forms part of a highly conserved structure motif (G/A/G/A) of small residues in homologous positions of all four domains (Gly-432 (IS6), Ala-780 (IIS6), Gly-1193 (IIIS6), Ala-1503 (IVS6)). Corresponding mutations in domains II, III, and IV induced, in contrast, parallel shifts of activation and inactivation curves indicating a preserved coupling between both processes. Disruption between coupling of activation and inactivation was specific for mutations of Gly-432 in domain I. Mutations of Gly-432 removed inactivation irrespective of the changes in activation. In all four domains residues G/A/G/A are in close contact with larger bulky amino acids from neighboring S6 helices. These interactions apparently provide adhesion points, thereby tightly sealing the activation gate of Ca(V)1.2 in the closed state. Such a structural hypothesis is supported by changes in activation gating induced by mutations of the G/A/G/A residues. The structural implications for Ca(V)1.2 activation and inactivation gating are discussed. 相似文献
9.
Beyl S Timin EN Hohaus A Stary A Kudrnac M Guy RH Hering S 《The Journal of biological chemistry》2007,282(6):3864-3870
Voltage-gated calcium channels are in a closed conformation at rest and open temporarily when the membrane is depolarized. To gain insight into the molecular architecture of Ca(v)1.2, we probed the closed and open conformations with the charged phenylalkylamine (-)devapamil ((-)qD888). To elucidate the access pathway of (-)D888 to its binding pocket from the intracellular side, we used mutations replacing a highly conserved Ile-781 by threonine/proline in the pore-lining segment IIS6 of Ca(v)1.2 (1). The shifted channel gating of these mutants (by 30-40 mV in the hyperpolarizing direction) enabled us to evoke currents with identical kinetics at different potentials and thus investigate the effect of the membrane potentials on the drug access per se. We show here that under these conditions the development of channel block by (-)qD888 is not affected by the transmembrane voltage. Recovery from block at rest was, however, accelerated at more hyperpolarized voltages. These findings support the conclusion that Ca(v)1.2 must be opening widely to enable free access of the charged (-)D888 molecule to its binding site, whereas drug dissociation from the closed channel conformation is restricted by bulky channel gates. The functional data indicating a location of a trapped (-)D888 molecule close to the central pore region are supported by a homology model illustrating that the closed Ca(v)1.2 is able to accommodate a large cation such as (-)D888. 相似文献
10.
Mutations of nonconserved residues within the calcium channel alpha1-interaction domain inhibit beta-subunit potentiation
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Gonzalez-Gutierrez G Miranda-Laferte E Naranjo D Hidalgo P Neely A 《The Journal of general physiology》2008,132(3):383-395
Voltage-dependent calcium channels consist of a pore-forming subunit (Ca(V)alpha(1)) that includes all the molecular determinants of a voltage-gated channel, and several accessory subunits. The ancillary beta-subunit (Ca(V)beta) is a potent activator of voltage-dependent calcium channels, but the mechanisms and structural bases of this regulation remain elusive. Ca(V)beta binds reversibly to a conserved consensus sequence in Ca(V)alpha(1), the alpha(1)-interaction domain (AID), which forms an alpha-helix when complexed with Ca(V)beta. Conserved aromatic residues face to one side of the helix and strongly interact with a hydrophobic pocket on Ca(V)beta. Here, we studied the effect of mutating residues located opposite to the AID-Ca(V)beta contact surface in Ca(V)1.2. Substitution of AID-exposed residues by the corresponding amino acids present in other Ca(V)alpha(1) subunits (E462R, K465N, D469S, and Q473K) hinders Ca(V)beta's ability to increase ionic-current to charge-movement ratio (I/Q) without changing the apparent affinity for Ca(V)beta. At the single channel level, these Ca(V)1.2 mutants coexpressed with Ca(V)beta(2a) visit high open probability mode less frequently than wild-type channels. On the other hand, Ca(V)1.2 carrying either a mutation in the conserved tryptophan residue (W470S, which impairs Ca(V)beta binding), or a deletion of the whole AID sequence, does not exhibit Ca(V)beta-induced increase in I/Q. In addition, we observed a shift in the voltage dependence of activation by +12 mV in the AID-deleted channel in the absence of Ca(V)beta, suggesting a direct participation of these residues in the modulation of channel activation. Our results show that Ca(V)beta-dependent potentiation arises primarily from changes in the modal gating behavior. We envision that Ca(V)beta spatially reorients AID residues that influence the channel gate. These findings provide a new framework for understanding modulation of VDCC gating by Ca(V)beta. 相似文献
11.
Zhang JZ Yarov-Yarovoy V Scheuer T Karbat I Cohen L Gordon D Gurevitz M Catterall WA 《The Journal of biological chemistry》2011,286(38):33641-33651
Voltage-gated sodium (Na(v)) channels are the molecular targets of β-scorpion toxins, which shift the voltage dependence of activation to more negative membrane potentials by a voltage sensor-trapping mechanism. Molecular determinants of β-scorpion toxin (CssIV) binding and action on rat brain sodium channels are located in the S1-S2 (IIS1-S2) and S3-S4 (IIS3-S4) extracellular linkers of the voltage-sensing module in domain II. In IIS1-S2, mutations of two amino acid residues (Glu(779) and Pro(782)) significantly altered the toxin effect by reducing binding affinity. In IIS3-S4, six positions surrounding the key binding determinant, Gly(845), define a hot spot of high-impact residues. Two of these substitutions (A841N and L846A) reduced voltage sensor trapping. The other three substitutions (N842R, V843A, and E844N) increased voltage sensor trapping. These bidirectional effects suggest that the IIS3-S4 loop plays a primary role in determining both toxin affinity and efficacy. A high resolution molecular model constructed with the Rosetta-Membrane modeling system reveals interactions of amino acid residues in sodium channels that are crucial for toxin action with residues in CssIV that are required for its effects. In this model, the wedge-shaped CssIV inserts between the IIS1-S2 and IIS3-S4 loops of the voltage sensor, placing key amino acid residues in position to interact with binding partners in these extracellular loops. These results provide new molecular insights into the voltage sensor-trapping model of toxin action and further define the molecular requirements for the development of antagonists that can prevent or reverse toxicity of scorpion toxins. 相似文献
12.
The activation of large conductance Ca(2+)-activated (BK) potassium channels is weakly voltage dependent compared to Shaker and other voltage-gated K(+) (K(V)) channels. Yet BK and K(V) channels share many conserved charged residues in transmembrane segments S1-S4. We mutated these residues individually in mSlo1 BK channels to determine their role in voltage gating, and characterized the voltage dependence of steady-state activation (P(o)) and I(K) kinetics (tau(I(K))) over an extended voltage range in 0-50 microM [Ca(2+)](i). mSlo1 contains several positively charged arginines in S4, but only one (R213) together with residues in S2 (D153, R167) and S3 (D186) are potentially voltage sensing based on the ability of charge-altering mutations to reduce the maximal voltage dependence of P(O). The voltage dependence of P(O) and tau(I(K)) at extreme negative potentials was also reduced, implying that the closed-open conformational change and voltage sensor activation share a common source of gating charge. Although the position of charged residues in the BK and K(V) channel sequence appears conserved, the distribution of voltage-sensing residues is not. Thus the weak voltage dependence of BK channel activation does not merely reflect a lack of charge but likely differences with respect to K(V) channels in the position and movement of charged residues within the electric field. Although mutation of most sites in S1-S4 did not reduce gating charge, they often altered the equilibrium constant for voltage sensor activation. In particular, neutralization of R207 or R210 in S4 stabilizes the activated state by 3-7 kcal mol(-1), indicating a strong contribution of non-voltage-sensing residues to channel function, consistent with their participation in state-dependent salt bridge interactions. Mutations in S4 and S3 (R210E, D186A, and E180A) also unexpectedly weakened the allosteric coupling of voltage sensor activation to channel opening. The implications of our findings for BK channel voltage gating and general mechanisms of voltage sensor activation are discussed. 相似文献
13.
Pierre-Olivier Demers-Giroux Beno?te Bourdin Rémy Sauvé Lucie Parent 《The Journal of biological chemistry》2013,288(41):29281-29293
T-type CaV3 channels are important mediators of Ca2+ entry near the resting membrane potential. Little is known about the molecular mechanisms responsible for channel activation. Homology models based upon the high-resolution structure of bacterial NaV channels predict interaction between the S4-S5 helix of Domain II (IIS4-S5) and the distal S6 pore region of Domain II (IIS6) and Domain III (IIIS6). Functional intra- and inter-domain interactions were investigated with a double mutant cycle analysis. Activation gating and channel kinetics were measured for 47 single mutants and 20 pairs of mutants. Significant coupling energies (ΔΔGinteract ≥ 1.5 kcal mol−1) were measured for 4 specific pairs of mutants introduced between IIS4-S5 and IIS6 and between IIS4-S5 and IIIS6. In agreement with the computer based models, Thr-911 in IIS4-S5 was functionally coupled with Ile-1013 in IIS6 during channel activation. The interaction energy was, however, found to be stronger between Val-907 in IIS4-S5 and Ile-1013 in IIS6. In addition Val-907 was significantly coupled with Asn-1548 in IIIS6 but not with Asn-1853 in IVS6. Altogether, our results demonstrate that the S4-S5 and S6 helices from adjacent domains are energetically coupled during the activation of a low voltage-gated T-type CaV3 channel. 相似文献
14.
Cestèle S Yarov-Yarovoy V Qu Y Sampieri F Scheuer T Catterall WA 《The Journal of biological chemistry》2006,281(30):21332-21344
Voltage sensing by voltage-gated sodium channels determines the electrical excitability of cells, but the molecular mechanism is unknown. beta-Scorpion toxins bind specifically to neurotoxin receptor site 4 and induce a negative shift in the voltage dependence of activation through a voltage sensor-trapping mechanism. Kinetic analysis showed that beta-scorpion toxin binds to the resting state, and subsequently the bound toxin traps the voltage sensor in the activated state in a voltage-dependent but concentration-independent manner. The rate of voltage sensor trapping can be fit by a two-step model, in which the first step is voltage-dependent and correlates with the outward gating movement of the IIS4 segment, whereas the second step is voltage-independent and results in shifted voltage dependence of activation of the channel. Mutations of Glu(779) in extracellular loop IIS1-S2 and both Glu(837) and Leu(840) in extracellular loop IIS3-S4 reduce the binding affinity of beta-scorpion toxin. Mutations of positively charged and hydrophobic amino acid residues in the IIS4 segment do not affect beta-scorpion toxin binding but alter voltage dependence of activation and enhance beta-scorpion toxin action. Structural modeling with the Rosetta algorithm yielded a three-dimensional model of the toxin-receptor complex with the IIS4 voltage sensor at the extracellular surface. Our results provide mechanistic and structural insight into the voltage sensor-trapping mode of scorpion toxin action, define the position of the voltage sensor in the resting state of the sodium channel, and favor voltage-sensing models in which the S4 segment spans the membrane in both resting and activated states. 相似文献
15.
Yarov-Yarovoy V McPhee JC Idsvoog D Pate C Scheuer T Catterall WA 《The Journal of biological chemistry》2002,277(38):35393-35401
Alanine-scanning mutagenesis of transmembrane segments IS6 and IIS6 of the rat brain Na(v)1.2 channel alpha subunit identified mutations N418A in IS6 and L975A in IIS6 as causing strong positive shifts in the voltage dependence of activation. In contrast, mutations V424A in IS6 and L983A in IIS6 caused strong negative shifts. Most IS6 mutations opposed inactivation from closed states, but most IIS6 mutations favored such inactivation. Mutations L421C and L983A near the intracellular ends of IS6 and IIS6, respectively, exhibited significant sustained Na(+) currents at the end of 30-ms depolarizations, indicating a role for these residues in Na(+) channel fast inactivation. These residues, in combination with residues at the intracellular end of IVS6, are well situated to form an inactivation gate receptor. Mutation I409A in IS6 reduced the affinity of the local anesthetic etidocaine for the inactivated state by 6-fold, and mutations I409A and N418A reduced use-dependent block by etidocaine. No IS6 or IIS6 mutations studied affected inactivated-state affinity or use-dependent block by the neuroprotective drug sipatrigine (compound 619C89). These results suggest that the local anesthetic receptor site is formed primarily by residues in segments IIIS6 and IVS6 with the contribution of a single amino acid in segment IS6. 相似文献
16.
We have recently reported that transfer of the domain IIS6 region from rapidly inactivating R-type (alpha(1E)) calcium channels to slowly inactivating L-type (alpha(1C)) calcium channel confers rapid inactivation (Stotz, S. C., Hamid, J., Spaetgens, R. L., Jarvis, S. E., and Zamponi, G. W. (2000) J. Biol. Chem. 275, 24575-24582). Here we have identified individual amino acid residues in the IIS6 regions that are responsible for these effects. In this region, alpha(1C) and alpha(1E) channels differ in seven residues, and exchanging five of those residues individually or in combination did not significantly affect inactivation kinetics. By contrast, replacement of residues Phe-823 or Ile-829 of alpha(1C) with the corresponding alpha(1E) residues significantly accelerated inactivation rates and, when substituted concomitantly, approached the rapid inactivation kinetics of R-type channels. A systematic substitution of these residues with a series of other amino acids revealed that decreasing side chain size at position 823 accelerates inactivation, whereas a dependence of the inactivation kinetics on the degree of hydrophobicity could be observed at position 829. Although these point mutations facilitated rapid entry into the inactivated state of the channel, they had little to no effect on the rate of recovery from inactivation. This suggests that the development of and recovery from inactivation are governed by separate structural determinants. Finally, the effects of mutations that accelerated alpha(1C) inactivation could still be antagonized following coexpression of the rat beta(2a) subunit or by domain I-II linker substitutions that produce ultra slow inactivation of wild type channels, indicating that the inactivation kinetics seen with the mutants remain subject to regulation by the domain I-II linker. Overall, our results provide novel insights into a complex process underlying calcium channel inactivation. 相似文献
17.
Neutralization of gating charges in domain II of the sodium channel alpha subunit enhances voltage-sensor trapping by a beta-scorpion toxin
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Cestèle S Scheuer T Mantegazza M Rochat H Catterall WA 《The Journal of general physiology》2001,118(3):291-302
beta-Scorpion toxins shift the voltage dependence of activation of sodium channels to more negative membrane potentials, but only after a strong depolarizing prepulse to fully activate the channels. Their receptor site includes the S3-S4 loop at the extracellular end of the S4 voltage sensor in domain II of the alpha subunit. Here, we probe the role of gating charges in the IIS4 segment in beta-scorpion toxin action by mutagenesis and functional analysis of the resulting mutant sodium channels. Neutralization of the positively charged amino acid residues in the IIS4 segment by mutation to glutamine shifts the voltage dependence of channel activation to more positive membrane potentials and reduces the steepness of voltage-dependent gating, which is consistent with the presumed role of these residues as gating charges. Surprisingly, neutralization of the gating charges at the outer end of the IIS4 segment by the mutations R850Q, R850C, R853Q, and R853C markedly enhances beta-scorpion toxin action, whereas mutations R856Q, K859Q, and K862Q have no effect. In contrast to wild-type, the beta-scorpion toxin Css IV causes a negative shift of the voltage dependence of activation of mutants R853Q and R853C without a depolarizing prepulse at holding potentials from -80 to -140 mV. Reaction of mutant R853C with 2-aminoethyl methanethiosulfonate causes a positive shift of the voltage dependence of activation and restores the requirement for a depolarizing prepulse for Css IV action. Enhancement of sodium channel activation by Css IV causes large tail currents upon repolarization, indicating slowed deactivation of the IIS4 voltage sensor by the bound toxin. Our results are consistent with a voltage-sensor-trapping model in which the beta-scorpion toxin traps the IIS4 voltage sensor in its activated position as it moves outward in response to depolarization and holds it there, slowing its inward movement on deactivation and enhancing subsequent channel activation. Evidently, neutralization of R850 and R853 removes kinetic barriers to binding of the IIS4 segment by Css IV, and thereby enhances toxin-induced channel activation. 相似文献
18.
Boehning D Mak DO Foskett JK Joseph SK 《The Journal of biological chemistry》2001,276(17):13509-13512
We tested the hypothesis that key residues in a putative intraluminal loop contribute to determination of ion permeation through the intracellular Ca(2+) release channel (inositol 1,4,5-trisphosphate receptors (IP(3)Rs)) that is gated by the second messenger inositol 1,4,5-trisphosphate (IP(3)). To accomplish this, we mutated residues within the putative pore forming region of the channel and analyzed the functional properties of mutant channels using a (45)Ca(2+) flux assay and single channel electrophysiological analyses. Two IP(3)R mutations, V2548I and D2550E, retained the ability to release (45)Ca(2+) in response to IP(3). When analyzed at the single channel level; both recombinant channels had IP(3)-dependent open probabilities similar to those observed in wild-type channels. The mutation V2548I resulted in channels that exhibited a larger K(+) conductance (489 +/- 13 picosiemens (pS) for V2548I versus 364 +/- 5 pS for wild-type), but retained a Ca(2+) selectivity similar to wild-type channels (P(Ca(2+)):P(K(+)) approximately 4:1). Conversely, D2550E channels were nonselective for Ca(2+) over K(+) (P(Ca(2+)):P(K(+)) approximately 0.6:1), while the K(+) conductance was effectively unchanged (391 +/- 4 pS). These results suggest that amino acid residues Val(2548) and Asp(2550) contribute to the ion conduction pathway. We propose that the pore of IP(3)R channels has two distinct sites that control monovalent cation permeation (Val(2548)) and Ca(2+) selectivity (Asp(2550)). 相似文献
19.
Aracena P Sánchez G Donoso P Hamilton SL Hidalgo C 《The Journal of biological chemistry》2003,278(44):42927-42935
We have analyzed the effects of the endogenous redoxactive agents S-nitrosoglutathione and glutathione disulfide, and the NO donor NOR-3, on calcium release kinetics mediated by ryanodine receptor channels. Incubation of triad-enriched sarcoplasmic reticulum vesicles isolated from mammalian skeletal muscle with these three agents elicits different responses. Glutathione disulfide significantly reduces the inhibitory effect of Mg2+ without altering Ca2+ activation of release kinetics, whereas NOR-3 enhances Ca2+ activation of release kinetics without altering Mg2+ inhibition. Incubation with S-nitrosoglutathione produces both effects; it significantly enhances Ca2+ activation of release kinetics and diminishes the inhibitory effect of Mg2+ on this process. Triad incubation with [35S]nitrosoglutathione at pCa 5 promoted 35S incorporation into 2.5 cysteine residues per channel monomer; this incorporation decreased significantly at pCa 9. These findings indicate that S-nitrosoglutathione supports S-glutathionylation as well as the reported S-nitrosylation of ryanodine receptor channels (Sun, J., Xu, L., Eu, J. P., Stamler, J. S., and Meissner, G. (2003) J. Biol. Chem. 278, 8184-8189). The combined results suggest that S-glutathionylation of specific cysteine residues can modulate channel inhibition by Mg2+, whereas S-nitrosylation of different cysteines can modulate the activation of the channel by Ca2+. Possible physiological and pathological implications of the activation of skeletal Ca2+ release channels by endogenous redox species are discussed. 相似文献
20.
Ca(V)1/Ca(V)2 channels, comprised of pore-forming α(1) and auxiliary (β,α(2)δ) subunits, control diverse biological responses in excitable cells. Molecules blocking Ca(V)1/Ca(V)2 channel currents (I(Ca)) profoundly regulate physiology and have many therapeutic applications. Rad/Rem/Rem2/Gem GTPases (RGKs) strongly inhibit Ca(V)1/Ca(V)2 channels. Understanding how RGKs block I(Ca) is critical for insights into their physiological function, and may provide design principles for developing novel Ca(V)1/Ca(V)2 channel inhibitors. The RGK binding sites within Ca(V)1/Ca(V)2 channel complexes responsible for I(Ca) inhibition are ambiguous, and it is unclear whether there are mechanistic differences among distinct RGKs. All RGKs bind β subunits, but it is unknown if and how this interaction contributes to I(Ca) inhibition. We investigated the role of RGK/β interaction in Rem inhibition of recombinant Ca(V)1.2 channels, using a mutated β (β(2aTM)) selectively lacking RGK binding. Rem blocked β(2aTM)-reconstituted channels (74% inhibition) less potently than channels containing wild-type β(2a) (96% inhibition), suggesting the prevalence of both β-binding-dependent and independent modes of inhibition. Two mechanistic signatures of Rem inhibition of Ca(V)1.2 channels (decreased channel surface density and open probability), but not a third (reduced maximal gating charge), depended on Rem binding to β. We identified a novel Rem binding site in Ca(V)1.2 α(1C) N-terminus that mediated β-binding-independent inhibition. The Ca(V)2.2 α(1B) subunit lacks the Rem binding site in the N-terminus and displays a solely β-binding-dependent form of channel inhibition. Finally, we discovered an unexpected functional dichotomy amongst distinct RGKs- while Rem and Rad use both β-binding-dependent and independent mechanisms, Gem and Rem2 use only a β-binding-dependent method to inhibit Ca(V)1.2 channels. The results provide new mechanistic perspectives, and reveal unexpected variations in determinants, underlying inhibition of Ca(V)1.2/Ca(V)2.2 channels by distinct RGK GTPases. 相似文献