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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 two-resistor model for the conduction pathway of BK channels (Fig. 1 and Appendix) predicts characteristic plots of normalized single-channel conductance versus side-chain volume in the entrance to the inner cavity. (A) Schematic side views through the midsection of an idealized entrance to the inner cavity for side chains occupying from 0 to 1.0 of the fractional volume of the entrance. (B) Plots of theoretical normalized single-channel conductance versus side-chain volume as a fraction of the entrance to the inner cavity filled with side chains. The label on each line indicates the ratio of R1/R2Gly. R2Gly is the resistance of the entrance to the inner cavity when it is empty. Each R1/R2Gly ratio results in a unique curve as side-chain volume increases. The theoretical plots in Fig. 7 B with R1/R2Gly ratios in the range of 10–20 appear consistent with the experimental data in Fig. 6. Theoretical plots calculated with Eq. A5.
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fig7: The two-resistor model for the conduction pathway of BK channels (Fig. 1 and Appendix) predicts characteristic plots of normalized single-channel conductance versus side-chain volume in the entrance to the inner cavity. (A) Schematic side views through the midsection of an idealized entrance to the inner cavity for side chains occupying from 0 to 1.0 of the fractional volume of the entrance. (B) Plots of theoretical normalized single-channel conductance versus side-chain volume as a fraction of the entrance to the inner cavity filled with side chains. The label on each line indicates the ratio of R1/R2Gly. R2Gly is the resistance of the entrance to the inner cavity when it is empty. Each R1/R2Gly ratio results in a unique curve as side-chain volume increases. The theoretical plots in Fig. 7 B with R1/R2Gly ratios in the range of 10–20 appear consistent with the experimental data in Fig. 6. Theoretical plots calculated with Eq. A5.

Mentions: This section uses equations derived in the Appendix for a two-resistor model to fit the data in Fig. 6 to obtain estimates of the resistance and effective dimensions of the entrance to the inner cavity of BK channels. The flow of K+ through the ion conduction pathway of a BK channel is assumed to be described by two resistors in series: R1 is a fixed resistor, and R2 is a variable resistor (Fig. 1 A). R1 arises from the sum of the resistance for all parts of the conduction pathway except the entrance to the inner cavity. R2 arises from the resistance of the entrance to the inner cavity. Increasing side-chain volume in the entrance (Fig. 1, B and C) increases R2. Fig. 7 A depicts idealized side views of the entrance to the inner cavity being filled with progressively larger side chains (black). For this idealization, the conductance of the entrance, g2, would decrease linearly with increasing side-chain volume, whereas the resistance of the entrance, R2, would increase superlinearly, as 1/g2. Such a two-resistor model predicts characteristic plots of single-channel conductance versus increased side-chain volume (Fig. 7 B and Appendix), with the shape of each plot depending on the ratio of R1/R2Gly, where R2Gly is the resistance of the empty entrance to the inner cavity, as is the case for glycine substitutions at positions 321/324.


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 two-resistor model for the conduction pathway of BK channels (Fig. 1 and Appendix) predicts characteristic plots of normalized single-channel conductance versus side-chain volume in the entrance to the inner cavity. (A) Schematic side views through the midsection of an idealized entrance to the inner cavity for side chains occupying from 0 to 1.0 of the fractional volume of the entrance. (B) Plots of theoretical normalized single-channel conductance versus side-chain volume as a fraction of the entrance to the inner cavity filled with side chains. The label on each line indicates the ratio of R1/R2Gly. R2Gly is the resistance of the entrance to the inner cavity when it is empty. Each R1/R2Gly ratio results in a unique curve as side-chain volume increases. The theoretical plots in Fig. 7 B with R1/R2Gly ratios in the range of 10–20 appear consistent with the experimental data in Fig. 6. Theoretical plots calculated with Eq. A5.
© Copyright Policy - openaccess
Related In: Results  -  Collection

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

fig7: The two-resistor model for the conduction pathway of BK channels (Fig. 1 and Appendix) predicts characteristic plots of normalized single-channel conductance versus side-chain volume in the entrance to the inner cavity. (A) Schematic side views through the midsection of an idealized entrance to the inner cavity for side chains occupying from 0 to 1.0 of the fractional volume of the entrance. (B) Plots of theoretical normalized single-channel conductance versus side-chain volume as a fraction of the entrance to the inner cavity filled with side chains. The label on each line indicates the ratio of R1/R2Gly. R2Gly is the resistance of the entrance to the inner cavity when it is empty. Each R1/R2Gly ratio results in a unique curve as side-chain volume increases. The theoretical plots in Fig. 7 B with R1/R2Gly ratios in the range of 10–20 appear consistent with the experimental data in Fig. 6. Theoretical plots calculated with Eq. A5.
Mentions: This section uses equations derived in the Appendix for a two-resistor model to fit the data in Fig. 6 to obtain estimates of the resistance and effective dimensions of the entrance to the inner cavity of BK channels. The flow of K+ through the ion conduction pathway of a BK channel is assumed to be described by two resistors in series: R1 is a fixed resistor, and R2 is a variable resistor (Fig. 1 A). R1 arises from the sum of the resistance for all parts of the conduction pathway except the entrance to the inner cavity. R2 arises from the resistance of the entrance to the inner cavity. Increasing side-chain volume in the entrance (Fig. 1, B and C) increases R2. Fig. 7 A depicts idealized side views of the entrance to the inner cavity being filled with progressively larger side chains (black). For this idealization, the conductance of the entrance, g2, would decrease linearly with increasing side-chain volume, whereas the resistance of the entrance, R2, would increase superlinearly, as 1/g2. Such a two-resistor model predicts characteristic plots of single-channel conductance versus increased side-chain volume (Fig. 7 B and Appendix), with the shape of each plot depending on the ratio of R1/R2Gly, where R2Gly is the resistance of the empty entrance to the inner cavity, as is the case for glycine substitutions at positions 321/324.

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