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Functional architecture of the inner pore of a voltage-gated Ca2+ channel.

Zhen XG, Xie C, Fitzmaurice A, Schoonover CE, Orenstein ET, Yang J - J. Gen. Physiol. (2005)

Bottom Line: However, the pattern of modification does not fit any known sequence alignment with K+ channels.These results indicate that the inner pore of VGCCs is indeed formed by the S6 segments but is different from that of K+ channels.This work provides an impetus for future studies on ion permeation, gating, and drug binding of VGCCs.

View Article: PubMed Central - PubMed

Affiliation: Department of Biological Sciences, Columbia University, New York, NY 10027, USA.

ABSTRACT
The inner pore of voltage-gated Ca2+ channels (VGCCs) is functionally important, but little is known about the architecture of this region. In K+ channels, this part of the pore is formed by the S6/M2 transmembrane segments from four symmetrically arranged subunits. The Ca2+ channel pore, however, is formed by four asymmetric domains of the same (alpha1) subunit. Here we investigated the architecture of the inner pore of P/Q-type Ca2+ channels using the substituted-cysteine accessibility method. Many positions in the S6 segments of all four repeats of the alpha1 subunit (Ca(v)2.1) were modified by internal methanethiosulfonate ethyltrimethylammonium (MTSET). However, the pattern of modification does not fit any known sequence alignment with K+ channels. In IIS6, five consecutive positions showed clear modification, suggesting a likely aqueous crevice and a loose packing between S6 and S5 segments, a notion further supported by the observation that some S5 positions were also accessible to internal MTSET. These results indicate that the inner pore of VGCCs is indeed formed by the S6 segments but is different from that of K+ channels. Interestingly some residues in IIIS6 and IVS6 whose mutations in L-type Ca2+ channels affect the binding of dihydropyridines and phenylalkylamines and are thought to face the pore appeared not to react with internal MTSET. Probing with qBBr, a rigid thiol-reactive agent with a dimension of 12 angstroms x 10 angstroms x 6 angstroms suggests that the inner pore can open to >10 angstroms. This work provides an impetus for future studies on ion permeation, gating, and drug binding of VGCCs.

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Amino acid sequences and schematic topology. (A) Amino acid sequence of S6 and S5 segments in the four repeats of the Cav2.1 subunit. Amino acid numbers are given on both sides. The numeration of residues in each repeat defined in this study is shown on the top and bottom. Position 0 presumably represents the membrane/cytoplasm interface. (B). Schematic topology of the S5 and S6 segments and the P-loop of two repeats.
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fig2: Amino acid sequences and schematic topology. (A) Amino acid sequence of S6 and S5 segments in the four repeats of the Cav2.1 subunit. Amino acid numbers are given on both sides. The numeration of residues in each repeat defined in this study is shown on the top and bottom. Position 0 presumably represents the membrane/cytoplasm interface. (B). Schematic topology of the S5 and S6 segments and the P-loop of two repeats.

Mentions: After overcoming the two technical obstacles for using SCAM, we began to identify residues that may form the inner pore, focusing on the S6 segments of the α1 subunit. We aligned the amino acid sequence of all four S6 segments based on a conserved asparagine (Fig. 2 A, top). The putative residue at the interface of the membrane and cytoplasm is defined as position 0. Residues in the membrane are numbered as 1, 2, 3, etc., from inside to outside, and residues beyond the membrane are numbered as −1, −2, −3, etc. To determine putative pore-lining residues, we replaced, one by one, a total of 87 consecutive residues in all four S6 segments with a cysteine, mainly from position 14 to position −3 in each repeat. All but five mutant α1 subunits formed functional channels in oocyte. The five mutants that failed to produce large enough Ba2+ currents are IS6-N8C, IIIS6-Y18C, IIIS6-V20C, IVS6-L7C and IVS6-N8C.


Functional architecture of the inner pore of a voltage-gated Ca2+ channel.

Zhen XG, Xie C, Fitzmaurice A, Schoonover CE, Orenstein ET, Yang J - J. Gen. Physiol. (2005)

Amino acid sequences and schematic topology. (A) Amino acid sequence of S6 and S5 segments in the four repeats of the Cav2.1 subunit. Amino acid numbers are given on both sides. The numeration of residues in each repeat defined in this study is shown on the top and bottom. Position 0 presumably represents the membrane/cytoplasm interface. (B). Schematic topology of the S5 and S6 segments and the P-loop of two repeats.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2266581&req=5

fig2: Amino acid sequences and schematic topology. (A) Amino acid sequence of S6 and S5 segments in the four repeats of the Cav2.1 subunit. Amino acid numbers are given on both sides. The numeration of residues in each repeat defined in this study is shown on the top and bottom. Position 0 presumably represents the membrane/cytoplasm interface. (B). Schematic topology of the S5 and S6 segments and the P-loop of two repeats.
Mentions: After overcoming the two technical obstacles for using SCAM, we began to identify residues that may form the inner pore, focusing on the S6 segments of the α1 subunit. We aligned the amino acid sequence of all four S6 segments based on a conserved asparagine (Fig. 2 A, top). The putative residue at the interface of the membrane and cytoplasm is defined as position 0. Residues in the membrane are numbered as 1, 2, 3, etc., from inside to outside, and residues beyond the membrane are numbered as −1, −2, −3, etc. To determine putative pore-lining residues, we replaced, one by one, a total of 87 consecutive residues in all four S6 segments with a cysteine, mainly from position 14 to position −3 in each repeat. All but five mutant α1 subunits formed functional channels in oocyte. The five mutants that failed to produce large enough Ba2+ currents are IS6-N8C, IIIS6-Y18C, IIIS6-V20C, IVS6-L7C and IVS6-N8C.

Bottom Line: However, the pattern of modification does not fit any known sequence alignment with K+ channels.These results indicate that the inner pore of VGCCs is indeed formed by the S6 segments but is different from that of K+ channels.This work provides an impetus for future studies on ion permeation, gating, and drug binding of VGCCs.

View Article: PubMed Central - PubMed

Affiliation: Department of Biological Sciences, Columbia University, New York, NY 10027, USA.

ABSTRACT
The inner pore of voltage-gated Ca2+ channels (VGCCs) is functionally important, but little is known about the architecture of this region. In K+ channels, this part of the pore is formed by the S6/M2 transmembrane segments from four symmetrically arranged subunits. The Ca2+ channel pore, however, is formed by four asymmetric domains of the same (alpha1) subunit. Here we investigated the architecture of the inner pore of P/Q-type Ca2+ channels using the substituted-cysteine accessibility method. Many positions in the S6 segments of all four repeats of the alpha1 subunit (Ca(v)2.1) were modified by internal methanethiosulfonate ethyltrimethylammonium (MTSET). However, the pattern of modification does not fit any known sequence alignment with K+ channels. In IIS6, five consecutive positions showed clear modification, suggesting a likely aqueous crevice and a loose packing between S6 and S5 segments, a notion further supported by the observation that some S5 positions were also accessible to internal MTSET. These results indicate that the inner pore of VGCCs is indeed formed by the S6 segments but is different from that of K+ channels. Interestingly some residues in IIIS6 and IVS6 whose mutations in L-type Ca2+ channels affect the binding of dihydropyridines and phenylalkylamines and are thought to face the pore appeared not to react with internal MTSET. Probing with qBBr, a rigid thiol-reactive agent with a dimension of 12 angstroms x 10 angstroms x 6 angstroms suggests that the inner pore can open to >10 angstroms. This work provides an impetus for future studies on ion permeation, gating, and drug binding of VGCCs.

Show MeSH
Related in: MedlinePlus