Molecular Localization of an Ion-Binding Site Within the Pore of Mammalian Sodium Channels

Backx, Peter H.; Yue, David T.; Lawrence, John H.; Marbán, Eduardo; Tomaselli, Gordon F.

doi:10.1126/science.1321496

Cited by 270 publications

(251 citation statements)

References 36 publications

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“…More recent experimental data show that the aromatic ring of Y 1p51 is engaged in -cation interactions with TTX, which stabilize TTX binding in TTX-sensitive sodium channels (22). This non-conservative aromatic residue is a well-known determinant of TTX sensitivity (23)(24)(25). For example, NaV1.2 and NaV1.4, which have, respectively, F 1p51 and Y 1p51 , are TTX-sensitive, whereas the NaV1.5 channel, which has C 1p51 , is low-sensitive to TTX.…”

Section: Analysis Of Multiple Sequencementioning

confidence: 99%

Possible Roles of Exceptionally Conserved Residues around the Selectivity Filters of Sodium and Calcium Channels

Tikhonov

Zhorov

2011

Journal of Biological Chemistry

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In the absence of x-ray structures of sodium and calcium channels their homology models are used to rationalize experimental data and design new experiments. A challenge is to model the outer-pore region that folds differently from potassium channels. Here we report a new model of the outer-pore region of the NaV1.4 channel, which suggests roles of highly conserved residues around the selectivity filter. The model takes from our previous study (Tikhonov, D. B., and Zhorov, B. S. (2005) Biophys. J. 88, 184 -197) the general disposition of the P-helices, selectivity filter residues, and the outer carboxylates, but proposes new intra-and inter-domain contacts that support structural stability of the outer pore. Glycine residues downstream from the selectivity filter are proposed to participate in knob-into-hole contacts with the P-helices and S6s. These contacts explain the adapted tetrodotoxin resistance of snakes that feed on toxic prey through valine substitution of isoleucine in the P-helix of repeat IV. Polar residues five positions upstream from the selectivity filter residues form H-bonds with the ascending-limb backbones. Exceptionally conserved tryptophans are engaged in inter-repeat H-bonds to form a ring whose -electrons would facilitate passage of ions from the outer carboxylates to the selectivity filter. The outerpore model of CaV1.2 derived from the NaV1.4 model is also stabilized by the ring of exceptionally conservative tryptophans and H-bonds between the P-helices and ascending limbs. In this model, the exceptionally conserved aspartate downstream from the selectivity-filter glutamate in repeat II facilitates passage of calcium ions to the selectivity-filter ring through the tryptophan ring. Available experimental data are discussed in view of the models.Voltage-gated ion channels are involved in the control of many physiological functions. Upon membrane depolarization, channels rapidly transit from the resting to the open state, then close to inactivated state(s), and return to the resting state upon membrane repolarization. The human genome encodes nine voltage-gated sodium (NaV1.1-NaV1.9) and ten voltage-gated calcium channels categorized as L (CaV1.1-CaV1.4), P/Q (CaV2.1), N (CaV2.2), R (CaV2.3), and T (CaV3.1-CaV3.3) types. The pore-forming ␣ 1 -subunit of calcium and sodium channels folds from a single polypeptide chain of four homologous repeats (Fig. 1A). Repeats I to IV are arranged clockwise when viewed extracellularly (1). Each repeat contains a voltage-sensing domain S1-S4, an outer helix S5, an inner helix S6, and a membrane-diving P-loop between S5 and S6. The P-loop includes a P-helix, a P-turn, and an ascending limb. Four ascending limbs, which line the outer pore, contain selectivity-filter residues in positions p50 (see Footnote 2 for residue labels). 2 Repeats I, II, III, and IV contain selectivity-filter glutamates in calcium channels (the EEEE ring) and Asp, Glu, Lys, and Ala residues in sodium channels (the DEKA ring). The ascending limbs also contain the outer carboxylates t...

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Section: Analysis Of Multiple Sequencementioning

confidence: 99%

Possible Roles of Exceptionally Conserved Residues around the Selectivity Filters of Sodium and Calcium Channels

Tikhonov

Zhorov

2011

Journal of Biological Chemistry

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“…Recently, it has been shown that the highly conserved SSl-SS2 regions of the connecting loop between the S5 and S6 transmembrane helices of K' and Na+ channels harbor critical residues that determine ion selectivity [24], conductance [5-71, as well as sensitivity to certain toxins [5,8-lo] and transition metal ions [9,10]. It is postulated that these regions are able to fold to the membrane as a /?-sheet to form a pore-lining surface [l 11.…”

Section: Introductionmentioning

confidence: 99%

Differential contribution by conserved glutamate residues to an ion‐selectivity site in the L‐type Ca²⁺ channel pore

et al. 1993

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In voltage-gated cation channels, it is thought that residues responsible for ion-selectivity are located within the pore-lining SSl-SSZ segments.In this study, we compared the ion permeation properties of mutant calcium channels in which highly conserved glutamate residues, located at analogous positions in the SS2 regions of all four motifs, were individually replaced. All of the mutants exhibited a loss of selectivity for divalent over monovalent cations. However, the permeation properties of the individual mutants varied in a position dependent manner. The results provide strong evidence that these glutamate residues, positioned at equivalent locations in the aligned sequences, play significantly different roles in forming the selectivity barrier of the calcium channel, and are probably arranged in an asymmetrical manner inside the ion-conducting pore.

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“…The residue at this position is thought to be located within the water-filled vestibule, rather than buried in the protein because it interacts strongly with the charged guanidinium toxins (11)(12)(13)(14). The site is also accessible to Cd 2ϩ and Zn 2ϩ (11)(12)(13)25), and it can form disulfide bridges with Cys introduced in other P-loops (26). We report here that Tyr͞Phe͞Cys, in addition to controlling isoform TTX sensitivity, is a critical structural feature determining QX access to its inside site of action.…”

mentioning

confidence: 99%

A critical residue for isoform difference in tetrodotoxin affinity is a molecular determinant of the external access path for local anesthetics in the cardiac sodium channel

Sunami

Glaaser

Fozzard

2000

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

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Membrane-impermeant quaternary derivatives of lidocaine (QX222 and QX314) block cardiac Na ؉ channels when applied from either side of the membrane, but they block neuronal and skeletal muscle channels poorly from the outside. To find the molecular determinants of the cardiac external QX access path, mutations of adult rat skeletal muscle ( 1) and rat heart (rH1) Na ؉ channels were studied by two-electrode voltage clamp in Xenopus oocytes. Mutating the 1 domain I P-loop Y401, which is the critical residue for isoform differences in tetrodotoxin block, to the heart sequence (Y401C) allowed outside QX222 block, but its mutation to brain type (Y401F) showed little block. 1-Y401C accelerated recovery from block by internal QX222. Block by external QX222 in 1-Y401C was diminished by chemical modification with methanethiosulfonate ethylammonium (MTSEA) to the outer vestibule or by a double mutant ( 1-Y401C͞F1579A), which altered the putative local anesthetic binding site. The reverse mutation in heart rH1-C374Y reduced outside QX314 block and slowed dissociation of internal QX222. Mutation of 1-C1572 in IVS6 to Thr, the cardiac isoform residue (C1572T), allowed external QX222 block, and accelerated recovery from internal QX222 block, as reported. Blocking efficacy of outside QX222 in 1-Y401C was more than that in 1-C1572T, and the double mutant ( 1-Y401C͞C1572T) accelerated internal QX recovery more than 1-Y401C or 1-C1572T alone. We conclude that the isoform-specific residue (Tyr͞Phe͞Cys) in the P-loop of domain I plays an important role in drug access as well as in tetrodotoxin binding. Isoform-specific residues in the IP-loop and IVS6 determine outside drug access to an internal binding site.V oltage-gated Na ϩ channels are membrane proteins responsible for generation of action potentials in nerve, skeletal muscle, and heart. The Na ϩ channel ␣-subunit consists of four homologous domains (I-IV), each containing six transmembrane segments (S1-S6) (1, 2). P-loops connecting S5 and S6 in each domain form the Na ϩ channel outer vestibule (3-7). Na ϩ channel isoforms from various tissues are highly homologous (2, 8). However, cardiac channels are relatively insensitive to tetrodotoxin (TTX) compared with brain and skeletal muscle channels (9, 10). This isoform difference is explained by an isoformspecific residue (Tyr͞Phe͞Cys) in the P-loop of domain I (IP-loop) (11-14) (Fig. 1). An aromatic residue at this position renders the channel TTX sensitive.The Na ϩ channel is also the site of action of local anesthetic antiarrhythmic drugs. Two conserved aromatic residues (Phe and Tyr) in IVS6 are involved in the local anesthetic binding site (15)(16)(17), and isoform differences in local anesthetic affinity are much less than in toxin affinity (18). In contrast, access of local anesthetic drugs differs markedly between Na ϩ channel isoforms. Quaternary membrane-impermeant derivatives of lidocaine such as QX314 and QX222 do not block nerve channels when applied to the outside (19,20), but outside QX314 blocks the cardiac chann...

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Molecular Localization of an Ion-Binding Site Within the Pore of Mammalian Sodium Channels

Cited by 270 publications

References 36 publications

Possible Roles of Exceptionally Conserved Residues around the Selectivity Filters of Sodium and Calcium Channels

Possible Roles of Exceptionally Conserved Residues around the Selectivity Filters of Sodium and Calcium Channels

Differential contribution by conserved glutamate residues to an ion‐selectivity site in the L‐type Ca²⁺ channel pore

A critical residue for isoform difference in tetrodotoxin affinity is a molecular determinant of the external access path for local anesthetics in the cardiac sodium channel

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Molecular Localization of an Ion-Binding Site Within the Pore of Mammalian Sodium Channels

Cited by 270 publications

References 36 publications

Possible Roles of Exceptionally Conserved Residues around the Selectivity Filters of Sodium and Calcium Channels

Possible Roles of Exceptionally Conserved Residues around the Selectivity Filters of Sodium and Calcium Channels

Differential contribution by conserved glutamate residues to an ion‐selectivity site in the L‐type Ca2+ channel pore

A critical residue for isoform difference in tetrodotoxin affinity is a molecular determinant of the external access path for local anesthetics in the cardiac sodium channel

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Differential contribution by conserved glutamate residues to an ion‐selectivity site in the L‐type Ca²⁺ channel pore