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Localization of the activation gate of a voltage-gated Ca2+ channel.

Xie C, Zhen XG, Yang J - J. Gen. Physiol. (2005)

Bottom Line: We found that positions above the putative membrane/cytoplasm interface, including two positions below the corresponding S6 bundle crossing in K+ channels, showed pronounced state-dependent accessibility to internal MTSET, reacting approximately 1,000-fold faster with MTSET in the open state than in the closed state.In contrast, a position at or below the putative membrane/cytoplasm interface was modified equally rapidly in both the open and closed states.Our results suggest that the S6 helices of the alpha1 subunit of VGCCs undergo conformation changes during gating and the activation gate is located at the intracellular end of the pore.

View Article: PubMed Central - PubMed

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

ABSTRACT
Ion channels open and close in response to changes in transmembrane voltage or ligand concentration. Recent studies show that K+ channels possess two gates, one at the intracellular end of the pore and the other at the selectivity filter. In this study we determined the location of the activation gate in a voltage-gated Ca2+ channel (VGCC) by examining the open/closed state dependence of the rate of modification by intracellular methanethiosulfonate ethyltrimethylammonium (MTSET) of pore-lining cysteines engineered in the S6 segments of the alpha1 subunit of P/Q type Ca2+ channels. We found that positions above the putative membrane/cytoplasm interface, including two positions below the corresponding S6 bundle crossing in K+ channels, showed pronounced state-dependent accessibility to internal MTSET, reacting approximately 1,000-fold faster with MTSET in the open state than in the closed state. In contrast, a position at or below the putative membrane/cytoplasm interface was modified equally rapidly in both the open and closed states. Our results suggest that the S6 helices of the alpha1 subunit of VGCCs undergo conformation changes during gating and the activation gate is located at the intracellular end of the pore.

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Pore-lining residues in S6. Amino acid sequence alignment of the four S6 segments in the Cav2.1 subunit and the M2/S6 segment of KcsA and Shaker K+ channels. Amino acid numbers are given on both sides. For Ca2+ channel S6 segments, the amino acid numbering defined in this study is shown on the top. Position 0 presumably represents the membrane/cytoplasm interface. Bold positions denote those that can be modified by internal MTSET. Underlined positions were studied in this work. For K+ channel M2/S6 segments, bold residues denote pore-lining positions defined either structurally or by MTS reagent accessibility. Arrow marks the M2/S6 bundle crossing.
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fig1: Pore-lining residues in S6. Amino acid sequence alignment of the four S6 segments in the Cav2.1 subunit and the M2/S6 segment of KcsA and Shaker K+ channels. Amino acid numbers are given on both sides. For Ca2+ channel S6 segments, the amino acid numbering defined in this study is shown on the top. Position 0 presumably represents the membrane/cytoplasm interface. Bold positions denote those that can be modified by internal MTSET. Underlined positions were studied in this work. For K+ channel M2/S6 segments, bold residues denote pore-lining positions defined either structurally or by MTS reagent accessibility. Arrow marks the M2/S6 bundle crossing.

Mentions: Using the substituted cysteine accessibility method, we have identified many pore-lining positions in the S6 transmembrane segment in each repeat (Zhen et al., 2005). These positions are highlighted in the amino acid sequence alignment of the four S6 segments of Cav2.1 (Fig. 1), where position 0 is presumed to lie at the membrane/cytoplasm interface and residues in the membrane are defined as 1, 2, 3, etc., from the intracellular side to the extracellular side. Fig. 1 also aligns the Cav2.1 S6 segments with the M2/S6 segment of two different types of K+ channels (adopted from Lipkind and Fozzard, 2003). According to this alignment, the S6 bundle crossing of the K+ channels is located three amino acids above the membrane/cytoplasm interface.


Localization of the activation gate of a voltage-gated Ca2+ channel.

Xie C, Zhen XG, Yang J - J. Gen. Physiol. (2005)

Pore-lining residues in S6. Amino acid sequence alignment of the four S6 segments in the Cav2.1 subunit and the M2/S6 segment of KcsA and Shaker K+ channels. Amino acid numbers are given on both sides. For Ca2+ channel S6 segments, the amino acid numbering defined in this study is shown on the top. Position 0 presumably represents the membrane/cytoplasm interface. Bold positions denote those that can be modified by internal MTSET. Underlined positions were studied in this work. For K+ channel M2/S6 segments, bold residues denote pore-lining positions defined either structurally or by MTS reagent accessibility. Arrow marks the M2/S6 bundle crossing.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2266579&req=5

fig1: Pore-lining residues in S6. Amino acid sequence alignment of the four S6 segments in the Cav2.1 subunit and the M2/S6 segment of KcsA and Shaker K+ channels. Amino acid numbers are given on both sides. For Ca2+ channel S6 segments, the amino acid numbering defined in this study is shown on the top. Position 0 presumably represents the membrane/cytoplasm interface. Bold positions denote those that can be modified by internal MTSET. Underlined positions were studied in this work. For K+ channel M2/S6 segments, bold residues denote pore-lining positions defined either structurally or by MTS reagent accessibility. Arrow marks the M2/S6 bundle crossing.
Mentions: Using the substituted cysteine accessibility method, we have identified many pore-lining positions in the S6 transmembrane segment in each repeat (Zhen et al., 2005). These positions are highlighted in the amino acid sequence alignment of the four S6 segments of Cav2.1 (Fig. 1), where position 0 is presumed to lie at the membrane/cytoplasm interface and residues in the membrane are defined as 1, 2, 3, etc., from the intracellular side to the extracellular side. Fig. 1 also aligns the Cav2.1 S6 segments with the M2/S6 segment of two different types of K+ channels (adopted from Lipkind and Fozzard, 2003). According to this alignment, the S6 bundle crossing of the K+ channels is located three amino acids above the membrane/cytoplasm interface.

Bottom Line: We found that positions above the putative membrane/cytoplasm interface, including two positions below the corresponding S6 bundle crossing in K+ channels, showed pronounced state-dependent accessibility to internal MTSET, reacting approximately 1,000-fold faster with MTSET in the open state than in the closed state.In contrast, a position at or below the putative membrane/cytoplasm interface was modified equally rapidly in both the open and closed states.Our results suggest that the S6 helices of the alpha1 subunit of VGCCs undergo conformation changes during gating and the activation gate is located at the intracellular end of the pore.

View Article: PubMed Central - PubMed

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

ABSTRACT
Ion channels open and close in response to changes in transmembrane voltage or ligand concentration. Recent studies show that K+ channels possess two gates, one at the intracellular end of the pore and the other at the selectivity filter. In this study we determined the location of the activation gate in a voltage-gated Ca2+ channel (VGCC) by examining the open/closed state dependence of the rate of modification by intracellular methanethiosulfonate ethyltrimethylammonium (MTSET) of pore-lining cysteines engineered in the S6 segments of the alpha1 subunit of P/Q type Ca2+ channels. We found that positions above the putative membrane/cytoplasm interface, including two positions below the corresponding S6 bundle crossing in K+ channels, showed pronounced state-dependent accessibility to internal MTSET, reacting approximately 1,000-fold faster with MTSET in the open state than in the closed state. In contrast, a position at or below the putative membrane/cytoplasm interface was modified equally rapidly in both the open and closed states. Our results suggest that the S6 helices of the alpha1 subunit of VGCCs undergo conformation changes during gating and the activation gate is located at the intracellular end of the pore.

Show MeSH
Related in: MedlinePlus