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Interaction between the pore and a fast gate of the cardiac sodium channel.

Townsend C, Horn R - J. Gen. Physiol. (1999)

Bottom Line: These results suggest an intimate relationship between the ion-conducting pore and the gates of the channel.A more superficial pore mutation or chemical modification of C373 reduces single channel conductance while preserving both selectivity of the pore and the modulatory effects of extracellular cations.Our results demonstrate a modulatory gating role for a region deep within the pore and suggest that the structure of the permeation pathway is largely preserved when a channel is closed.

View Article: PubMed Central - PubMed

Affiliation: Department of Physiology, Jefferson Medical College, Philadelphia, Pennsylvania 19107, USA.

ABSTRACT
Permeant ions affect a fast gating process observed in human cardiac sodium channels (Townsend, C., H.A. Hartmann, and R. Horn. 1997. J. Gen. Physiol. 110:11-21). Removal of extracellular permeant ions causes a reduction of open probability at positive membrane potentials. These results suggest an intimate relationship between the ion-conducting pore and the gates of the channel. We tested this hypothesis by three sets of manipulations designed to affect the binding of cations within the pore: application of intracellular pore blockers, mutagenesis of residues known to contribute to permeation, and chemical modification of a native cysteine residue (C373) near the extracellular mouth of the pore. The coupling between extracellular permeant ions and this fast gating process is abolished both by pore blockers and by a mutation that severely affects selectivity. A more superficial pore mutation or chemical modification of C373 reduces single channel conductance while preserving both selectivity of the pore and the modulatory effects of extracellular cations. Our results demonstrate a modulatory gating role for a region deep within the pore and suggest that the structure of the permeation pathway is largely preserved when a channel is closed.

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D1422C:F1485Q hH1a current– and open probability–voltage relations. (A) Peak current–voltage relations for  D1422C:F1485Q hH1a–transfected cells successively bathed in either Na+, Cs+, or Na+ bath solutions (n = 3). (B) Corresponding  normalized open probability versus voltage relations (see methods, n = 3 single-channel patches for each bath solution).
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Figure 7: D1422C:F1485Q hH1a current– and open probability–voltage relations. (A) Peak current–voltage relations for D1422C:F1485Q hH1a–transfected cells successively bathed in either Na+, Cs+, or Na+ bath solutions (n = 3). (B) Corresponding normalized open probability versus voltage relations (see methods, n = 3 single-channel patches for each bath solution).

Mentions: To further test for a role of the selectivity filter, we examined a more superficial pore mutant (D1422C) that affects single channel conductance, but not selectivity. This charge-neutralizing mutation reduced inward sodium conductance (25.5 vs. 34.6 pS), but not outward conductance (35.5 vs. 36.6 pS), consistent with a role of the wild-type aspartate in concentrating cations at the extracellular mouth of the pore. Fig. 7 shows that sodium replacement by cesium in the D1422C mutant has the hallmark effects observed in wild-type channels, namely a crossing of the I–V relationships and a voltage-dependent reduction of Popen at positive voltages in the absence of extracellular permeant cations. Therefore, modulation by external cations is preserved in this pore mutant.


Interaction between the pore and a fast gate of the cardiac sodium channel.

Townsend C, Horn R - J. Gen. Physiol. (1999)

D1422C:F1485Q hH1a current– and open probability–voltage relations. (A) Peak current–voltage relations for  D1422C:F1485Q hH1a–transfected cells successively bathed in either Na+, Cs+, or Na+ bath solutions (n = 3). (B) Corresponding  normalized open probability versus voltage relations (see methods, n = 3 single-channel patches for each bath solution).
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Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2223368&req=5

Figure 7: D1422C:F1485Q hH1a current– and open probability–voltage relations. (A) Peak current–voltage relations for D1422C:F1485Q hH1a–transfected cells successively bathed in either Na+, Cs+, or Na+ bath solutions (n = 3). (B) Corresponding normalized open probability versus voltage relations (see methods, n = 3 single-channel patches for each bath solution).
Mentions: To further test for a role of the selectivity filter, we examined a more superficial pore mutant (D1422C) that affects single channel conductance, but not selectivity. This charge-neutralizing mutation reduced inward sodium conductance (25.5 vs. 34.6 pS), but not outward conductance (35.5 vs. 36.6 pS), consistent with a role of the wild-type aspartate in concentrating cations at the extracellular mouth of the pore. Fig. 7 shows that sodium replacement by cesium in the D1422C mutant has the hallmark effects observed in wild-type channels, namely a crossing of the I–V relationships and a voltage-dependent reduction of Popen at positive voltages in the absence of extracellular permeant cations. Therefore, modulation by external cations is preserved in this pore mutant.

Bottom Line: These results suggest an intimate relationship between the ion-conducting pore and the gates of the channel.A more superficial pore mutation or chemical modification of C373 reduces single channel conductance while preserving both selectivity of the pore and the modulatory effects of extracellular cations.Our results demonstrate a modulatory gating role for a region deep within the pore and suggest that the structure of the permeation pathway is largely preserved when a channel is closed.

View Article: PubMed Central - PubMed

Affiliation: Department of Physiology, Jefferson Medical College, Philadelphia, Pennsylvania 19107, USA.

ABSTRACT
Permeant ions affect a fast gating process observed in human cardiac sodium channels (Townsend, C., H.A. Hartmann, and R. Horn. 1997. J. Gen. Physiol. 110:11-21). Removal of extracellular permeant ions causes a reduction of open probability at positive membrane potentials. These results suggest an intimate relationship between the ion-conducting pore and the gates of the channel. We tested this hypothesis by three sets of manipulations designed to affect the binding of cations within the pore: application of intracellular pore blockers, mutagenesis of residues known to contribute to permeation, and chemical modification of a native cysteine residue (C373) near the extracellular mouth of the pore. The coupling between extracellular permeant ions and this fast gating process is abolished both by pore blockers and by a mutation that severely affects selectivity. A more superficial pore mutation or chemical modification of C373 reduces single channel conductance while preserving both selectivity of the pore and the modulatory effects of extracellular cations. Our results demonstrate a modulatory gating role for a region deep within the pore and suggest that the structure of the permeation pathway is largely preserved when a channel is closed.

Show MeSH