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Low resistance, large dimension entrance to the inner cavity of BK channels determined by changing side-chain volume.

Geng Y, Niu X, Magleby KL - J. Gen. Physiol. (2011)

Bottom Line: MPA(-) increased currents and MTSET(+) decreased currents, with no difference between positions 321 and 324, indicating that side chains at 321/324 are accessible from the inner conduction pathway and have equivalent effects on conductance.For neutral amino acids, decreasing the size of the entrance to the inner cavity by substituting large side-chain amino acids at 321/324 decreased outward single-channel conductance, whereas increasing the size of the entrance with smaller side-chain substitutions had little effect.Substitutions had little effect on inward conductance.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Physiology and Biophysics, University of Miami Miller School of Medicine, FL 33136, USA. ygeng@-med.miami.edu

ABSTRACT
Large-conductance Ca(2+)- and voltage-activated K(+) (BK) channels have the largest conductance (250-300 pS) of all K(+)-selective channels. Yet, the contributions of the various parts of the ion conduction pathway to the conductance are not known. Here, we examine the contribution of the entrance to the inner cavity to the large conductance. Residues at E321/E324 on each of the four α subunits encircle the entrance to the inner cavity. To determine if 321/324 is accessible from the inner conduction pathway, we measured single-channel current amplitudes before and after exposure and wash of thiol reagents to the intracellular side of E321C and E324C channels. MPA(-) increased currents and MTSET(+) decreased currents, with no difference between positions 321 and 324, indicating that side chains at 321/324 are accessible from the inner conduction pathway and have equivalent effects on conductance. For neutral amino acids, decreasing the size of the entrance to the inner cavity by substituting large side-chain amino acids at 321/324 decreased outward single-channel conductance, whereas increasing the size of the entrance with smaller side-chain substitutions had little effect. Reductions in outward conductance were negated by high [K(+)](i). Substitutions had little effect on inward conductance. Fitting plots of conductance versus side-chain volume with a model consisting of one variable and one fixed resistor in series indicated an effective diameter and length of the entrance to the inner cavity for wild-type channels of 17.7 and 5.6 Å, respectively, with the resistance of the entrance ∼7% of the total resistance of the conduction pathway. The estimated dimensions are consistent with the structure of MthK, an archaeal homologue to BK channels. Our observations suggest that BK channels have a low resistance, large entrance to the inner cavity, with the entrance being as large as necessary to not limit current, but not much larger.

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The predicted effective dimensions of the entrance to the inner cavity with the two-resistor model are consistent with the crystal structure of MthK. (A) Molecular surface of a side view (extracellular at top) of the membrane-spanning portions of MthK, with front and back subunits removed, as generated with Pymol software and Protein Data Bank accession number 1LNQ (Jiang et al., 2002b). The wt glutamates at positions E92 (red) and a computer mutation glutamate at E95 (orange) are indicated by color. The green cylinder indicates the calculated effective dimensions of the entrance to the inner cavity for wt BK channels (E321/E324). The cylinder diameter is 17.7 Å (14.1 Å from Table II plus 3.6 Å for the hydrated diameter of K+), with a length of 5.62 Å. (B) Same as A, except for a bottom view looking out from the intracellular side with all four subunits. (C and D) Same as A and B, but for glycine substituted at positions E92/E95 in the structure of MthK. The green cylinders indicate the calculated effective dimensions of the entrance to the inner cavity for BK channels with glycine substitutions (E321G/E324G). The cylinder diameter is 22.1 Å (18.5 plus 3.6 Å for the hydrated diameter of K+), with a length of 5.6 Å. (E and F) Same as above, except with tryptophan substitutions at positions E92/E95 in the crystal structure of MthK. The green cylinders indicate the calculated effective dimensions of the entrance to the inner cavity for BK channels with tryptophan substations (E321W/E324W). The cylinder diameter is 9.6 Å (5.95 plus 3.6 Å for the hydrated diameter of K+), with a length of 5.62 Å. The predicted effective dimensions of the entrance to the inner cavity for wt and mutated BK channels are generally consistent with the crystal structure of wt and computer-mutated MthK channels.
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fig8: The predicted effective dimensions of the entrance to the inner cavity with the two-resistor model are consistent with the crystal structure of MthK. (A) Molecular surface of a side view (extracellular at top) of the membrane-spanning portions of MthK, with front and back subunits removed, as generated with Pymol software and Protein Data Bank accession number 1LNQ (Jiang et al., 2002b). The wt glutamates at positions E92 (red) and a computer mutation glutamate at E95 (orange) are indicated by color. The green cylinder indicates the calculated effective dimensions of the entrance to the inner cavity for wt BK channels (E321/E324). The cylinder diameter is 17.7 Å (14.1 Å from Table II plus 3.6 Å for the hydrated diameter of K+), with a length of 5.62 Å. (B) Same as A, except for a bottom view looking out from the intracellular side with all four subunits. (C and D) Same as A and B, but for glycine substituted at positions E92/E95 in the structure of MthK. The green cylinders indicate the calculated effective dimensions of the entrance to the inner cavity for BK channels with glycine substitutions (E321G/E324G). The cylinder diameter is 22.1 Å (18.5 plus 3.6 Å for the hydrated diameter of K+), with a length of 5.6 Å. (E and F) Same as above, except with tryptophan substitutions at positions E92/E95 in the crystal structure of MthK. The green cylinders indicate the calculated effective dimensions of the entrance to the inner cavity for BK channels with tryptophan substations (E321W/E324W). The cylinder diameter is 9.6 Å (5.95 plus 3.6 Å for the hydrated diameter of K+), with a length of 5.62 Å. The predicted effective dimensions of the entrance to the inner cavity for wt and mutated BK channels are generally consistent with the crystal structure of wt and computer-mutated MthK channels.

Mentions: The vertical dashed line in Fig. 6 D indicates the volume that would be occupied by the side chains of glutamate at E321/E324 for wt BK channels. Based on the side-chain volume of glutamate and the mean hydrophobic parameters for glycine substitution, the calculated dimensions of the entrance to the inner cavity for wt BK channels assuming that K+ is a point charge gives an effective diameter of 14.1 Å and a length of 5.62 Å (Table II). Assuming a hydrated diameter for K+ in the range of 3.6–6.6 Å, the effective diameter of the entrance for wt BK channels would be in the range of 17.7–20.7 Å (14.1 + 3.6–6.6 Å). Because of the complex geometry of the ion conduction pathway leading up to the entrance to the inner cavity, with a large square-shaped central pore in the gating ring of 20 Å on a side (Wu et al., 2010), four side portals between the gating ring and the transmembrane portion of the channel leading to the entrance (Zhang et al., 2009), and the likely case that the entrance to the inner cavity transitions smoothly into the inner cavity itself (see Fig. 8 A for MthK), no corrections for hydrated radius have been applied to the estimates of the length of the entrance.


Low resistance, large dimension entrance to the inner cavity of BK channels determined by changing side-chain volume.

Geng Y, Niu X, Magleby KL - J. Gen. Physiol. (2011)

The predicted effective dimensions of the entrance to the inner cavity with the two-resistor model are consistent with the crystal structure of MthK. (A) Molecular surface of a side view (extracellular at top) of the membrane-spanning portions of MthK, with front and back subunits removed, as generated with Pymol software and Protein Data Bank accession number 1LNQ (Jiang et al., 2002b). The wt glutamates at positions E92 (red) and a computer mutation glutamate at E95 (orange) are indicated by color. The green cylinder indicates the calculated effective dimensions of the entrance to the inner cavity for wt BK channels (E321/E324). The cylinder diameter is 17.7 Å (14.1 Å from Table II plus 3.6 Å for the hydrated diameter of K+), with a length of 5.62 Å. (B) Same as A, except for a bottom view looking out from the intracellular side with all four subunits. (C and D) Same as A and B, but for glycine substituted at positions E92/E95 in the structure of MthK. The green cylinders indicate the calculated effective dimensions of the entrance to the inner cavity for BK channels with glycine substitutions (E321G/E324G). The cylinder diameter is 22.1 Å (18.5 plus 3.6 Å for the hydrated diameter of K+), with a length of 5.6 Å. (E and F) Same as above, except with tryptophan substitutions at positions E92/E95 in the crystal structure of MthK. The green cylinders indicate the calculated effective dimensions of the entrance to the inner cavity for BK channels with tryptophan substations (E321W/E324W). The cylinder diameter is 9.6 Å (5.95 plus 3.6 Å for the hydrated diameter of K+), with a length of 5.62 Å. The predicted effective dimensions of the entrance to the inner cavity for wt and mutated BK channels are generally consistent with the crystal structure of wt and computer-mutated MthK channels.
© Copyright Policy - openaccess
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC3105516&req=5

fig8: The predicted effective dimensions of the entrance to the inner cavity with the two-resistor model are consistent with the crystal structure of MthK. (A) Molecular surface of a side view (extracellular at top) of the membrane-spanning portions of MthK, with front and back subunits removed, as generated with Pymol software and Protein Data Bank accession number 1LNQ (Jiang et al., 2002b). The wt glutamates at positions E92 (red) and a computer mutation glutamate at E95 (orange) are indicated by color. The green cylinder indicates the calculated effective dimensions of the entrance to the inner cavity for wt BK channels (E321/E324). The cylinder diameter is 17.7 Å (14.1 Å from Table II plus 3.6 Å for the hydrated diameter of K+), with a length of 5.62 Å. (B) Same as A, except for a bottom view looking out from the intracellular side with all four subunits. (C and D) Same as A and B, but for glycine substituted at positions E92/E95 in the structure of MthK. The green cylinders indicate the calculated effective dimensions of the entrance to the inner cavity for BK channels with glycine substitutions (E321G/E324G). The cylinder diameter is 22.1 Å (18.5 plus 3.6 Å for the hydrated diameter of K+), with a length of 5.6 Å. (E and F) Same as above, except with tryptophan substitutions at positions E92/E95 in the crystal structure of MthK. The green cylinders indicate the calculated effective dimensions of the entrance to the inner cavity for BK channels with tryptophan substations (E321W/E324W). The cylinder diameter is 9.6 Å (5.95 plus 3.6 Å for the hydrated diameter of K+), with a length of 5.62 Å. The predicted effective dimensions of the entrance to the inner cavity for wt and mutated BK channels are generally consistent with the crystal structure of wt and computer-mutated MthK channels.
Mentions: The vertical dashed line in Fig. 6 D indicates the volume that would be occupied by the side chains of glutamate at E321/E324 for wt BK channels. Based on the side-chain volume of glutamate and the mean hydrophobic parameters for glycine substitution, the calculated dimensions of the entrance to the inner cavity for wt BK channels assuming that K+ is a point charge gives an effective diameter of 14.1 Å and a length of 5.62 Å (Table II). Assuming a hydrated diameter for K+ in the range of 3.6–6.6 Å, the effective diameter of the entrance for wt BK channels would be in the range of 17.7–20.7 Å (14.1 + 3.6–6.6 Å). Because of the complex geometry of the ion conduction pathway leading up to the entrance to the inner cavity, with a large square-shaped central pore in the gating ring of 20 Å on a side (Wu et al., 2010), four side portals between the gating ring and the transmembrane portion of the channel leading to the entrance (Zhang et al., 2009), and the likely case that the entrance to the inner cavity transitions smoothly into the inner cavity itself (see Fig. 8 A for MthK), no corrections for hydrated radius have been applied to the estimates of the length of the entrance.

Bottom Line: MPA(-) increased currents and MTSET(+) decreased currents, with no difference between positions 321 and 324, indicating that side chains at 321/324 are accessible from the inner conduction pathway and have equivalent effects on conductance.For neutral amino acids, decreasing the size of the entrance to the inner cavity by substituting large side-chain amino acids at 321/324 decreased outward single-channel conductance, whereas increasing the size of the entrance with smaller side-chain substitutions had little effect.Substitutions had little effect on inward conductance.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Physiology and Biophysics, University of Miami Miller School of Medicine, FL 33136, USA. ygeng@-med.miami.edu

ABSTRACT
Large-conductance Ca(2+)- and voltage-activated K(+) (BK) channels have the largest conductance (250-300 pS) of all K(+)-selective channels. Yet, the contributions of the various parts of the ion conduction pathway to the conductance are not known. Here, we examine the contribution of the entrance to the inner cavity to the large conductance. Residues at E321/E324 on each of the four α subunits encircle the entrance to the inner cavity. To determine if 321/324 is accessible from the inner conduction pathway, we measured single-channel current amplitudes before and after exposure and wash of thiol reagents to the intracellular side of E321C and E324C channels. MPA(-) increased currents and MTSET(+) decreased currents, with no difference between positions 321 and 324, indicating that side chains at 321/324 are accessible from the inner conduction pathway and have equivalent effects on conductance. For neutral amino acids, decreasing the size of the entrance to the inner cavity by substituting large side-chain amino acids at 321/324 decreased outward single-channel conductance, whereas increasing the size of the entrance with smaller side-chain substitutions had little effect. Reductions in outward conductance were negated by high [K(+)](i). Substitutions had little effect on inward conductance. Fitting plots of conductance versus side-chain volume with a model consisting of one variable and one fixed resistor in series indicated an effective diameter and length of the entrance to the inner cavity for wild-type channels of 17.7 and 5.6 Å, respectively, with the resistance of the entrance ∼7% of the total resistance of the conduction pathway. The estimated dimensions are consistent with the structure of MthK, an archaeal homologue to BK channels. Our observations suggest that BK channels have a low resistance, large entrance to the inner cavity, with the entrance being as large as necessary to not limit current, but not much larger.

Show MeSH
Related in: MedlinePlus