Probing the outer vestibule of a sodium channel voltage sensor

Yang, Naibo; George, Alfred L.; Horn, Richard

doi:10.1016/s0006-3495(97)78258-4

Cited by 74 publications

(75 citation statements)

References 23 publications

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“…The narrow region of focused membrane electrical field at the hydrophobic constriction site is highlighted by orange bars. models, the transmembrane electrical potential would drop across a short stretch of the gating pore, which was predicted from studies showing a focused electric field near the center of the VSD (44)(45)(46)(47)(48). We consider the focused electrical field to drop primarily across the HCS, which forms a hydrophobic seal of 5-6 Å thickness just above the center of the VSD (Fig.…”

Section: Conformational Changes In the Voltage-sensing Domain Duringmentioning

confidence: 99%

Structural basis for gating charge movement in the voltage sensor of a sodium channel

Yarov‐Yarovoy¹,

DeCaen²,

Westenbroek³

et al. 2011

Proc. Natl. Acad. Sci. U.S.A.

227

352

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Voltage-dependent gating of ion channels is essential for electrical signaling in excitable cells, but the structural basis for voltage sensor function is unknown. We constructed high-resolution structural models of resting, intermediate, and activated states of the voltage-sensing domain of the bacterial sodium channel NaChBac using the Rosetta modeling method, crystal structures of related channels, and experimental data showing state-dependent interactions between the gating charge-carrying arginines in the S4 segment and negatively charged residues in neighboring transmembrane segments. The resulting structural models illustrate a network of ionic and hydrogen-bonding interactions that are made sequentially by the gating charges as they move out under the influence of the electric field. The S4 segment slides 6-8 Å outward through a narrow groove formed by the S1, S2, and S3 segments, rotates ∼30°, and tilts sideways at a pivot point formed by a highly conserved hydrophobic region near the middle of the voltage sensor. The S4 segment has a 3 10 -helical conformation in the narrow inner gating pore, which allows linear movement of the gating charges across the inner one-half of the membrane. Conformational changes of the intracellular one-half of S4 during activation are rigidly coupled to lateral movement of the S4-S5 linker, which could induce movement of the S5 and S6 segments and open the intracellular gate of the pore. We confirmed the validity of these structural models by comparing with a high-resolution structure of a NaChBac homolog and showing predicted molecular interactions of hydrophobic residues in the S4 segment in disulfide-locking studies.V oltage-gated sodium (Na V ) channels are responsible for initiation and propagation of action potentials in nerve, muscle, and endocrine cells (1, 2). They are members of the structurally homologous superfamily of voltage-gated ion channel proteins that also includes voltage-gated potassium (K V ), voltage-gated calcium (Ca V ), and cyclic nucleotide-gated (CNG) channels (3). Mammalian Na V and Ca V channels consist of four homologous domains (I through IV), each containing six transmembrane segments (S1 through S6) and a membrane-reentrant pore loop between the S5 and S6 segments (1, 3). Segments S1-S4 of the channel form the voltage-sensing domain (VSD), and segments S5 and S6 and the membrane-reentrant pore loop form the pore. The bacterial Na V channel NaChBac and its relatives consist of tetramers of four identical subunits, which closely resemble one domain of vertebrate Na V and Ca V channels, but provide much simpler structures for studying the mechanism of voltage sensing (4, 5). The hallmark feature of the voltage-gated ion channels is the steep voltage dependence of activation, which derives from the voltage-driven outward movement of gating charges in response to the membrane depolarization (6, 7). The S4 transmembrane segment in the VSD has four to seven arginine residues spaced at 3-aa intervals, which serve as gating charges in the voltage-s...

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Section: Conformational Changes In the Voltage-sensing Domain Duringmentioning

confidence: 99%

Structural basis for gating charge movement in the voltage sensor of a sodium channel

Yarov‐Yarovoy¹,

DeCaen²,

Westenbroek³

et al. 2011

Proc. Natl. Acad. Sci. U.S.A.

227

352

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“…In contrast, the rate of MTSET ϩ modification of the simple thiol 2-mercaptoethanol in solution is only 12.5-fold faster than the rate with MTSES Ϫ (Table 2). To factor out the intrinsic differences in the reactivity of the two reagents, we divided the ratio of the rates of the two reagents at an introduced cysteine by the ratio of the rates for the two reagents with mercaptoethanol (Stauffer and Karlin, 1994;Cheung and Akabas, 1997;Yang et al, 1997). For ␤ 2 K213C, the ratio of ratios is ϭ 80/12.5 ϭ 6.4 and for ␤ 2 K215C, ϭ 43/12.5 ϭ 3.4.…”

Section: Mtsetmentioning

confidence: 99%

“…We can estimate the effective electrostatic potential at an introduced cysteine by using the following equation: ϭ Ϫ(1/ z MTSET Ϫ z MTSES )(RT/F )ln(), where z is the unitary charge of the MTS reagent, R is the gas constant, T is absolute temperature, F is Faraday's constant, and is the ratio of ratios calculated above (Stauffer and Karlin, 1994;Yang et al, 1997;Karlin and Akabas, 1998). The calculated negative electrostatic potentials at ␤ 2 K213C and ␤ 2 K215C in the resting state are Ϫ24 and Ϫ15 mV, respectively.…”

Section: Mtsetmentioning

confidence: 99%

Charged Residues in the α₁and β₂Pre-M1 Regions Involved in GABA_AReceptor Activation

Mercado¹,

Czajkowski²

2006

J. Neurosci.

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For Cys-loop ligand-gated ion channels (LGIC), the protein movements that couple neurotransmitter binding to channel gating are not well known. The pre-M1 region, which links the extracellular agonist-binding domain to the channel-containing transmembrane domain, is in an ideal position to transduce binding site movements to gating movements. A cluster of cationic residues in this region is observed in all LGIC subunits, and in particular, an arginine residue is absolutely conserved. We mutated charged pre-M1 residues in the GABA A receptor ␣ 1 (K219, R220, K221) and ␤ 2 (K213, K215, R216) subunits to cysteine and expressed the mutant subunits with wild-type ␤ 2 or ␣ 1 in Xenopus oocytes. Cysteine substitution of ␤ 2 R216 abolished channel gating by GABA without altering the binding of the GABA agonist [3 H]muscimol, indicating that this residue plays a key role in coupling GABA binding to gating. Tethering thiol-reactive methanethiosulfonate (MTS) reagents onto ␣ 1 K219C, ␤ 2 K213C, and ␤ 2 K215C increased maximal GABA-activated currents, suggesting that structural perturbations of the pre-M1 regions affect channel gating. GABA altered the rates of sulfhydryl modification of ␣ 1 K219C, ␤ 2 K213C, and ␤ 2 K215C, indicating that the pre-M1 regions move in response to channel activation. A positively charged MTS reagent modified ␤ 2 K213C and ␤ 2 K215C significantly faster than a negatively charged reagent, and GABA activation eliminated modification of ␤ 2 K215C by the negatively charged reagent. Overall, the data indicate that the pre-M1 region is part of the structural machinery coupling GABA binding to gating and that the transduction of binding site movements to channel movements is mediated, in part, by electrostatic interactions.

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“…Fluorescent labeling of introduced cysteine residues has been used to study conformational changes of ion channels [1,2]. However, this method is not easily applicable to proteins that contain many endogenous cysteine residues, or to the study of intracellular conformational changes.…”

Section: Introductionmentioning

confidence: 99%

The protein-labeling reagent FLASH-EDT 2 binds not only to CCXXCC motifs but also non-specifically to endogenous cysteine-rich proteins

Štroffeková

Proenza

Beam

2001

Pfl�gers Archiv European Journal of Physiology

139

108

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FLASH-EDT2--4',5'-bis(1,3,2-dithioarsolan-2-yl)fluorescein-(1,2-ethanedithiol)2--has been reported to fluoresce only after binding with high affinity to a specific tetracysteine motif (CCXXCC, "Cys4") and thus to provide a technique for labeling recombinant proteins in vivo (Griffin et al. Science 281:269-272). We have attempted to use FLASH-EDT2 as a site-specific label of the II-III loop of the dihydropyridine receptor (DHPR) in skeletal muscle. Upon expression in dysgenic myotubes (which lack endogenous alpha1s), an alpha1s mutated to contain CCRECC in the II-III loop was able to produce L-type calcium currents and to mediate skeletal-type excitation-contraction (EC) coupling, but FLASH-EDT2 labeling revealed no difference from non-transfected dysgenic myotubes. HeLa-S3 cells transfected with Cys4-containing calmodulin were significantly more fluorescent than non-transfected cells, whereas the difference between transfected and non-transfected cells was less apparent for CHO-K and HEK 293 cells. Because the fluorescence of non-transfected cells increased substantially after treatment with FLASH-EDT2, it suggested the possibility that FLASH binds to endogenous cysteine-containing proteins. This finding was confirmed in cuvette experiments in which FLASH-EDT2 fluorescence was observed after FLASH-EDT, was added to protein homogenates from myotubes or cell lines. The enhanced fluorescence was abolished by pretreatment of cells or cell homogenates with coumarine maleimide (CPM), which modifies cysteine residues covalently. Thus, enhanced FLASH fluorescence appears to occur both after binding to an introduced Cys4 motif and to endogenous, cysteine-containing proteins. Therefore, FLASH-EDT2 may be useful only for labeling those recombinant proteins that express at a very high level.

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Probing the outer vestibule of a sodium channel voltage sensor

Cited by 74 publications

References 23 publications

Structural basis for gating charge movement in the voltage sensor of a sodium channel

Structural basis for gating charge movement in the voltage sensor of a sodium channel

Charged Residues in the α₁and β₂Pre-M1 Regions Involved in GABA_AReceptor Activation

The protein-labeling reagent FLASH-EDT 2 binds not only to CCXXCC motifs but also non-specifically to endogenous cysteine-rich proteins

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Probing the outer vestibule of a sodium channel voltage sensor

Cited by 74 publications

References 23 publications

Structural basis for gating charge movement in the voltage sensor of a sodium channel

Structural basis for gating charge movement in the voltage sensor of a sodium channel

Charged Residues in the α1and β2Pre-M1 Regions Involved in GABAAReceptor Activation

The protein-labeling reagent FLASH-EDT 2 binds not only to CCXXCC motifs but also non-specifically to endogenous cysteine-rich proteins

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Charged Residues in the α₁and β₂Pre-M1 Regions Involved in GABA_AReceptor Activation