Side-chain charge effects and conductance determinants in the pore of ClC-0 chloride channels

Mei Fang Chen, Tsung-Yu Chen

Research output: Contribution to journalArticle

59 Citations (Scopus)

Abstract

The charge on the side chain of the internal pore residue lysine 519 (K519) of the Torpedo ClC-0 chloride (Cl-) channel affects channel conductance. Experiments that replace wild-type (WT) lysine with neutral or negatively charged residues or that modify the K519C mutant with various methane thiosulfonate (MTS) reagents show that the conductance of the channel decreases when the charge at position 519 is made more negative. This charge effect on the channel conductance diminishes in the presence of a high intracellular Cl- concentration ([Cl-]i). However, the application of high concentrations of nonpermeant ions, such as glutamate or sulfate (SO4 2-), does not change the conductance, suggesting that the electrostatic effects created by the charge at position 519 are unlikely due to a surface charge mechanism. Another pore residue, glutamate 127 (E127), plays an even more critical role in controlling channel conductance. This negatively charged residue, based on the structures of the homologous bacterial ClC channels, lies 4-5 Å from K519. Altering the charge of this residue can influence the apparent Cl- affinity as well as the saturated pore conductance in the conductance-Cl- activity curve. Amino acid residues at the selectivity filter also control the pore conductance but mutating these residues mainly affects the maximal pore conductance. These results suggest at least two different conductance determinants in the pore of ClC-0, consistent with the most recent crystal structure of the bacterial ClC channel solved to 2.5 Å, in which multiple Cl--binding sites were identified in the pore. Thus, we suggest that the occupancy of the internal Cl--binding site is directly controlled by the charged residues located at the inner pore mouth. On the other hand, the Cl--binding site at the selectivity filter controls the exit rate of Cl- and therefore determines the maximal channel conductance.

Original languageEnglish (US)
Pages (from-to)133-145
Number of pages13
JournalJournal of General Physiology
Volume122
Issue number2
DOIs
StatePublished - Aug 1 2003

Fingerprint

Chloride Channels
Chlorides
Bacterial Structures
Binding Sites
Lysine
Glutamic Acid
Torpedo
Static Electricity
Sulfates
Mouth
Ions
Amino Acids

Keywords

  • ClC-0
  • Electrostatic effect
  • Pore conductance
  • Surface charge

ASJC Scopus subject areas

  • Physiology

Cite this

Side-chain charge effects and conductance determinants in the pore of ClC-0 chloride channels. / Chen, Mei Fang; Chen, Tsung-Yu.

In: Journal of General Physiology, Vol. 122, No. 2, 01.08.2003, p. 133-145.

Research output: Contribution to journalArticle

@article{1db2fe5a74de4d1abd22b51a42e7625b,
title = "Side-chain charge effects and conductance determinants in the pore of ClC-0 chloride channels",
abstract = "The charge on the side chain of the internal pore residue lysine 519 (K519) of the Torpedo ClC-0 chloride (Cl-) channel affects channel conductance. Experiments that replace wild-type (WT) lysine with neutral or negatively charged residues or that modify the K519C mutant with various methane thiosulfonate (MTS) reagents show that the conductance of the channel decreases when the charge at position 519 is made more negative. This charge effect on the channel conductance diminishes in the presence of a high intracellular Cl- concentration ([Cl-]i). However, the application of high concentrations of nonpermeant ions, such as glutamate or sulfate (SO4 2-), does not change the conductance, suggesting that the electrostatic effects created by the charge at position 519 are unlikely due to a surface charge mechanism. Another pore residue, glutamate 127 (E127), plays an even more critical role in controlling channel conductance. This negatively charged residue, based on the structures of the homologous bacterial ClC channels, lies 4-5 {\AA} from K519. Altering the charge of this residue can influence the apparent Cl- affinity as well as the saturated pore conductance in the conductance-Cl- activity curve. Amino acid residues at the selectivity filter also control the pore conductance but mutating these residues mainly affects the maximal pore conductance. These results suggest at least two different conductance determinants in the pore of ClC-0, consistent with the most recent crystal structure of the bacterial ClC channel solved to 2.5 {\AA}, in which multiple Cl--binding sites were identified in the pore. Thus, we suggest that the occupancy of the internal Cl--binding site is directly controlled by the charged residues located at the inner pore mouth. On the other hand, the Cl--binding site at the selectivity filter controls the exit rate of Cl- and therefore determines the maximal channel conductance.",
keywords = "ClC-0, Electrostatic effect, Pore conductance, Surface charge",
author = "Chen, {Mei Fang} and Tsung-Yu Chen",
year = "2003",
month = "8",
day = "1",
doi = "10.1085/jgp.200308844",
language = "English (US)",
volume = "122",
pages = "133--145",
journal = "Journal of General Physiology",
issn = "0022-1295",
publisher = "Rockefeller University Press",
number = "2",

}

TY - JOUR

T1 - Side-chain charge effects and conductance determinants in the pore of ClC-0 chloride channels

AU - Chen, Mei Fang

AU - Chen, Tsung-Yu

PY - 2003/8/1

Y1 - 2003/8/1

N2 - The charge on the side chain of the internal pore residue lysine 519 (K519) of the Torpedo ClC-0 chloride (Cl-) channel affects channel conductance. Experiments that replace wild-type (WT) lysine with neutral or negatively charged residues or that modify the K519C mutant with various methane thiosulfonate (MTS) reagents show that the conductance of the channel decreases when the charge at position 519 is made more negative. This charge effect on the channel conductance diminishes in the presence of a high intracellular Cl- concentration ([Cl-]i). However, the application of high concentrations of nonpermeant ions, such as glutamate or sulfate (SO4 2-), does not change the conductance, suggesting that the electrostatic effects created by the charge at position 519 are unlikely due to a surface charge mechanism. Another pore residue, glutamate 127 (E127), plays an even more critical role in controlling channel conductance. This negatively charged residue, based on the structures of the homologous bacterial ClC channels, lies 4-5 Å from K519. Altering the charge of this residue can influence the apparent Cl- affinity as well as the saturated pore conductance in the conductance-Cl- activity curve. Amino acid residues at the selectivity filter also control the pore conductance but mutating these residues mainly affects the maximal pore conductance. These results suggest at least two different conductance determinants in the pore of ClC-0, consistent with the most recent crystal structure of the bacterial ClC channel solved to 2.5 Å, in which multiple Cl--binding sites were identified in the pore. Thus, we suggest that the occupancy of the internal Cl--binding site is directly controlled by the charged residues located at the inner pore mouth. On the other hand, the Cl--binding site at the selectivity filter controls the exit rate of Cl- and therefore determines the maximal channel conductance.

AB - The charge on the side chain of the internal pore residue lysine 519 (K519) of the Torpedo ClC-0 chloride (Cl-) channel affects channel conductance. Experiments that replace wild-type (WT) lysine with neutral or negatively charged residues or that modify the K519C mutant with various methane thiosulfonate (MTS) reagents show that the conductance of the channel decreases when the charge at position 519 is made more negative. This charge effect on the channel conductance diminishes in the presence of a high intracellular Cl- concentration ([Cl-]i). However, the application of high concentrations of nonpermeant ions, such as glutamate or sulfate (SO4 2-), does not change the conductance, suggesting that the electrostatic effects created by the charge at position 519 are unlikely due to a surface charge mechanism. Another pore residue, glutamate 127 (E127), plays an even more critical role in controlling channel conductance. This negatively charged residue, based on the structures of the homologous bacterial ClC channels, lies 4-5 Å from K519. Altering the charge of this residue can influence the apparent Cl- affinity as well as the saturated pore conductance in the conductance-Cl- activity curve. Amino acid residues at the selectivity filter also control the pore conductance but mutating these residues mainly affects the maximal pore conductance. These results suggest at least two different conductance determinants in the pore of ClC-0, consistent with the most recent crystal structure of the bacterial ClC channel solved to 2.5 Å, in which multiple Cl--binding sites were identified in the pore. Thus, we suggest that the occupancy of the internal Cl--binding site is directly controlled by the charged residues located at the inner pore mouth. On the other hand, the Cl--binding site at the selectivity filter controls the exit rate of Cl- and therefore determines the maximal channel conductance.

KW - ClC-0

KW - Electrostatic effect

KW - Pore conductance

KW - Surface charge

UR - http://www.scopus.com/inward/record.url?scp=0042377364&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=0042377364&partnerID=8YFLogxK

U2 - 10.1085/jgp.200308844

DO - 10.1085/jgp.200308844

M3 - Article

C2 - 12885875

AN - SCOPUS:0042377364

VL - 122

SP - 133

EP - 145

JO - Journal of General Physiology

JF - Journal of General Physiology

SN - 0022-1295

IS - 2

ER -