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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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Decreasing the size of the entrance to the inner cavity decreases single-channel conductance for the four indicated scales of hydrophobicity in a manner consistent with a two-resistor model. (A–D) Plots of single-channel conductance versus total increase in side-chain volume for the indicated amino acid substitutions at positions 321/324 on each of the four subunits. The total increase in side-chain volume compared with glycine is 8wX Å3, where wX is the increased side-chain volume from Table I for a single amino acid. The different scales of hydrophobicity used for classifications are: (A) Lodish (2008), (B) Scale #20, (C) Scale #13, and (D) Scale #18 from Mant et al. (2009), with the dividing line between hydrophobic and hydrophilic on the Mant et al. scales based on our assessment of the scale data. The data points are plotted at the same positions in each panel, with the differences among the panels being the classification of one or more of the amino acids into hydrophobic (red) or uncharged hydrophilic (green) data. Glycine is considered a special amino acid in the Lodish (2008) classification where it is plotted as gray. The dashed lines indicate predicted changes in single-channel conductance obtained by fitting the hydrophobic (red) or hydrophilic (green) data with Eqs. A1–A4 describing the two-resistor model (Fig. 1). Fitted parameters are in Table II. 150 mM K+i. The vertical dashed line in D indicates the volume that would be occupied by the side chains of glutamate at E321/E324 in wt channels.
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fig6: Decreasing the size of the entrance to the inner cavity decreases single-channel conductance for the four indicated scales of hydrophobicity in a manner consistent with a two-resistor model. (A–D) Plots of single-channel conductance versus total increase in side-chain volume for the indicated amino acid substitutions at positions 321/324 on each of the four subunits. The total increase in side-chain volume compared with glycine is 8wX Å3, where wX is the increased side-chain volume from Table I for a single amino acid. The different scales of hydrophobicity used for classifications are: (A) Lodish (2008), (B) Scale #20, (C) Scale #13, and (D) Scale #18 from Mant et al. (2009), with the dividing line between hydrophobic and hydrophilic on the Mant et al. scales based on our assessment of the scale data. The data points are plotted at the same positions in each panel, with the differences among the panels being the classification of one or more of the amino acids into hydrophobic (red) or uncharged hydrophilic (green) data. Glycine is considered a special amino acid in the Lodish (2008) classification where it is plotted as gray. The dashed lines indicate predicted changes in single-channel conductance obtained by fitting the hydrophobic (red) or hydrophilic (green) data with Eqs. A1–A4 describing the two-resistor model (Fig. 1). Fitted parameters are in Table II. 150 mM K+i. The vertical dashed line in D indicates the volume that would be occupied by the side chains of glutamate at E321/E324 in wt channels.

Mentions: Fig. 6 plots mean single-channel conductance for outward currents versus the total increase in side-chain volume compared with glycine at the entrance to the inner cavity for the 11 examined amino acid substitutions at positions 321/324, including the special amino acid glycine. Because the current–voltage plots deviate from linearity (Fig. 3, C and F), the mean single-channel conductance over the examined range of voltage was plotted for each substitution. The total increase in side-chain volume on the abscissa is eight times the volume in Table I because substitution occurs at positions 321 and 324 on each of the four subunits. The data are plotted using four representative scales out of over 100 classifying amino acid hydrophobicity (Lodish, 2008; Mant et al., 2009), where red and green symbols in Fig. 6 represent hydrophobic and hydrophilic amino acids, respectively, as defined by the different scales. Independent of the hydrophobicity scale used, single-channel conductance decreased with increasing side-chain volume at the entrance to the inner cavity. The decrease occurred progressively faster as side-chain volume increased.


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)

Decreasing the size of the entrance to the inner cavity decreases single-channel conductance for the four indicated scales of hydrophobicity in a manner consistent with a two-resistor model. (A–D) Plots of single-channel conductance versus total increase in side-chain volume for the indicated amino acid substitutions at positions 321/324 on each of the four subunits. The total increase in side-chain volume compared with glycine is 8wX Å3, where wX is the increased side-chain volume from Table I for a single amino acid. The different scales of hydrophobicity used for classifications are: (A) Lodish (2008), (B) Scale #20, (C) Scale #13, and (D) Scale #18 from Mant et al. (2009), with the dividing line between hydrophobic and hydrophilic on the Mant et al. scales based on our assessment of the scale data. The data points are plotted at the same positions in each panel, with the differences among the panels being the classification of one or more of the amino acids into hydrophobic (red) or uncharged hydrophilic (green) data. Glycine is considered a special amino acid in the Lodish (2008) classification where it is plotted as gray. The dashed lines indicate predicted changes in single-channel conductance obtained by fitting the hydrophobic (red) or hydrophilic (green) data with Eqs. A1–A4 describing the two-resistor model (Fig. 1). Fitted parameters are in Table II. 150 mM K+i. The vertical dashed line in D indicates the volume that would be occupied by the side chains of glutamate at E321/E324 in wt channels.
© Copyright Policy - openaccess
Related In: Results  -  Collection

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Show All Figures
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fig6: Decreasing the size of the entrance to the inner cavity decreases single-channel conductance for the four indicated scales of hydrophobicity in a manner consistent with a two-resistor model. (A–D) Plots of single-channel conductance versus total increase in side-chain volume for the indicated amino acid substitutions at positions 321/324 on each of the four subunits. The total increase in side-chain volume compared with glycine is 8wX Å3, where wX is the increased side-chain volume from Table I for a single amino acid. The different scales of hydrophobicity used for classifications are: (A) Lodish (2008), (B) Scale #20, (C) Scale #13, and (D) Scale #18 from Mant et al. (2009), with the dividing line between hydrophobic and hydrophilic on the Mant et al. scales based on our assessment of the scale data. The data points are plotted at the same positions in each panel, with the differences among the panels being the classification of one or more of the amino acids into hydrophobic (red) or uncharged hydrophilic (green) data. Glycine is considered a special amino acid in the Lodish (2008) classification where it is plotted as gray. The dashed lines indicate predicted changes in single-channel conductance obtained by fitting the hydrophobic (red) or hydrophilic (green) data with Eqs. A1–A4 describing the two-resistor model (Fig. 1). Fitted parameters are in Table II. 150 mM K+i. The vertical dashed line in D indicates the volume that would be occupied by the side chains of glutamate at E321/E324 in wt channels.
Mentions: Fig. 6 plots mean single-channel conductance for outward currents versus the total increase in side-chain volume compared with glycine at the entrance to the inner cavity for the 11 examined amino acid substitutions at positions 321/324, including the special amino acid glycine. Because the current–voltage plots deviate from linearity (Fig. 3, C and F), the mean single-channel conductance over the examined range of voltage was plotted for each substitution. The total increase in side-chain volume on the abscissa is eight times the volume in Table I because substitution occurs at positions 321 and 324 on each of the four subunits. The data are plotted using four representative scales out of over 100 classifying amino acid hydrophobicity (Lodish, 2008; Mant et al., 2009), where red and green symbols in Fig. 6 represent hydrophobic and hydrophilic amino acids, respectively, as defined by the different scales. Independent of the hydrophobicity scale used, single-channel conductance decreased with increasing side-chain volume at the entrance to the inner cavity. The decrease occurred progressively faster as side-chain volume increased.

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