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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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Increasing side-chain volume has little effect on iout in the presence of 2.5 M K+i. (A and B) Representative records of outward currents through single channels at +200 mV with 2.5 M of intracellular K+ for hydrophobic (A) and uncharged hydrophilic (B) amino acid substitutions at positions 321/324. The dashed lines show current levels with alanine substitutions. (C and D) Plots of iout in the presence of high 2.5-M K+i versus voltage for hydrophobic (C) and uncharged hydrophilic (D) amino acid substitutions. Plots for valine and leucine substitution superimposed the plots for alanine substitution in C and are not shown. High K+i negates the effect of side-chain volume on iout (compare with Fig. 3 with 150 mM K+i), as would be expected if larger side chains reduce the size of the entrance to the inner cavity.
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fig4: Increasing side-chain volume has little effect on iout in the presence of 2.5 M K+i. (A and B) Representative records of outward currents through single channels at +200 mV with 2.5 M of intracellular K+ for hydrophobic (A) and uncharged hydrophilic (B) amino acid substitutions at positions 321/324. The dashed lines show current levels with alanine substitutions. (C and D) Plots of iout in the presence of high 2.5-M K+i versus voltage for hydrophobic (C) and uncharged hydrophilic (D) amino acid substitutions. Plots for valine and leucine substitution superimposed the plots for alanine substitution in C and are not shown. High K+i negates the effect of side-chain volume on iout (compare with Fig. 3 with 150 mM K+i), as would be expected if larger side chains reduce the size of the entrance to the inner cavity.

Mentions: Increasing side-chain volume at the entrance to the inner cavity reduces iout for both hydrophobic and uncharged hydrophilic amino acids. (A) Representative single-channel currents at +200 mV for substitution of the indicated hydrophobic amino acids at positions 321/324. Dashed lines show the mean open and closed current levels of E321A/E324A channels. Larger side-chain amino acids at positions 321/324 decrease iout. (B) Representative all-points histograms for currents like those in A for the indicated amino acid substitutions. The values on the ordinate are proportional to the frequency of observations of the current levels indicated on the abscissa. The distributions centered on 0 pA indicate the current fluctuation around the closed level, and the distributions to the right indicate the current fluctuations around the open levels. Histograms are scaled so that the peak frequencies of the open current levels are the same. The larger amino acid side chains of tyrosine and tryptophan shift the entire distributions to lower current levels compared with alanine. The excess open-channel noise compared with wt channels (Fig. 2 A) suggests that the substitutions destabilize the open state. (C) Plots of iout versus voltage indicate that the reduction of iout for the larger side chains occurs over the range of examined voltages. Substituting valine, leucine, or phenylalanine for alanine had little effect on iout, whereas substituting tyrosine or tryptophan with their larger side chains reduced iout. (D–F) Same as A–C, except for substitutions of hydrophilic uncharged amino acids. Dashed lines in D are for serine substitution. Asparagine and glutamine with their larger side chains decrease single-channel current compared with serine and threonine with their smaller side chains. For i-V plots in this and Fig. 4, some symbols have been shifted laterally 2.5 mV to avoid overlap. 150 mM K+i.


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)

Increasing side-chain volume has little effect on iout in the presence of 2.5 M K+i. (A and B) Representative records of outward currents through single channels at +200 mV with 2.5 M of intracellular K+ for hydrophobic (A) and uncharged hydrophilic (B) amino acid substitutions at positions 321/324. The dashed lines show current levels with alanine substitutions. (C and D) Plots of iout in the presence of high 2.5-M K+i versus voltage for hydrophobic (C) and uncharged hydrophilic (D) amino acid substitutions. Plots for valine and leucine substitution superimposed the plots for alanine substitution in C and are not shown. High K+i negates the effect of side-chain volume on iout (compare with Fig. 3 with 150 mM K+i), as would be expected if larger side chains reduce the size of the entrance to the inner cavity.
© Copyright Policy - openaccess
Related In: Results  -  Collection

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

fig4: Increasing side-chain volume has little effect on iout in the presence of 2.5 M K+i. (A and B) Representative records of outward currents through single channels at +200 mV with 2.5 M of intracellular K+ for hydrophobic (A) and uncharged hydrophilic (B) amino acid substitutions at positions 321/324. The dashed lines show current levels with alanine substitutions. (C and D) Plots of iout in the presence of high 2.5-M K+i versus voltage for hydrophobic (C) and uncharged hydrophilic (D) amino acid substitutions. Plots for valine and leucine substitution superimposed the plots for alanine substitution in C and are not shown. High K+i negates the effect of side-chain volume on iout (compare with Fig. 3 with 150 mM K+i), as would be expected if larger side chains reduce the size of the entrance to the inner cavity.
Mentions: Increasing side-chain volume at the entrance to the inner cavity reduces iout for both hydrophobic and uncharged hydrophilic amino acids. (A) Representative single-channel currents at +200 mV for substitution of the indicated hydrophobic amino acids at positions 321/324. Dashed lines show the mean open and closed current levels of E321A/E324A channels. Larger side-chain amino acids at positions 321/324 decrease iout. (B) Representative all-points histograms for currents like those in A for the indicated amino acid substitutions. The values on the ordinate are proportional to the frequency of observations of the current levels indicated on the abscissa. The distributions centered on 0 pA indicate the current fluctuation around the closed level, and the distributions to the right indicate the current fluctuations around the open levels. Histograms are scaled so that the peak frequencies of the open current levels are the same. The larger amino acid side chains of tyrosine and tryptophan shift the entire distributions to lower current levels compared with alanine. The excess open-channel noise compared with wt channels (Fig. 2 A) suggests that the substitutions destabilize the open state. (C) Plots of iout versus voltage indicate that the reduction of iout for the larger side chains occurs over the range of examined voltages. Substituting valine, leucine, or phenylalanine for alanine had little effect on iout, whereas substituting tyrosine or tryptophan with their larger side chains reduced iout. (D–F) Same as A–C, except for substitutions of hydrophilic uncharged amino acids. Dashed lines in D are for serine substitution. Asparagine and glutamine with their larger side chains decrease single-channel current compared with serine and threonine with their smaller side chains. For i-V plots in this and Fig. 4, some symbols have been shifted laterally 2.5 mV to avoid overlap. 150 mM K+i.

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