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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 or no effect on iin. (A and B) Representative records of inward single-channel currents at −100 mV for the indicated amino acid substitutions. Dashed lines are the current levels with alanine substitution (A) and serine substitution (B). (C and D) Plots of iin versus voltage for hydrophobic (C) and uncharged hydrophilic (D) amino acid substitutions. Side-chain volume has less of an effect on iin compared with iout (compare with Fig. 3). The reduced effect of side-chain volume on iin is consistent with larger side chains reducing the size of the entrance to the inner cavity. 150 mM K+i.
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fig5: Increasing side-chain volume has little or no effect on iin. (A and B) Representative records of inward single-channel currents at −100 mV for the indicated amino acid substitutions. Dashed lines are the current levels with alanine substitution (A) and serine substitution (B). (C and D) Plots of iin versus voltage for hydrophobic (C) and uncharged hydrophilic (D) amino acid substitutions. Side-chain volume has less of an effect on iin compared with iout (compare with Fig. 3). The reduced effect of side-chain volume on iin is consistent with larger side chains reducing the size of the entrance to the inner cavity. 150 mM K+i.

Mentions: The experiments in the previous sections examined the effect of side-chain volume on iout by making the intracellular side of the membrane positive to electrostatically push K+ out through the channel. To test the effect of side-chain volume on inward single-channel current amplitudes, iin, the intracellular side of the membrane, was made negative to attract K+ inward through the channel. Replacing alanine with larger side-chain hydrophobic amino acids reduced iin a maximum of 18% for the largest side-chain tryptophan (Fig. 5, A and C) compared with a 30% reduction for iout (Fig. 3, A–C). Replacing serine with larger side-chain uncharged hydrophilic amino acids had no effect on iin (Fig. 5, B and D) compared with an ∼13% reduction on iout (Fig. 3, D and E). A larger reduction of iout compared with iin with increasing side-chain volume is consistent with larger side-chain volumes reducing the size of the entrance to the inner cavity. For outward currents, decreasing the size of the entrance to the inner cavity could directly limit how much diffusing K+ would enter the entrance to the inner cavity (be captured) to be driven through the channel by voltage. In contrast, for inward currents, decreasing the size of entrance to the inner cavity (now the exit) could lead to an increased concentration of K+ in the inner cavity, increasing the probability of K+ exiting to the intracellular solution, compensating to some extent for the smaller exit size.


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 or no effect on iin. (A and B) Representative records of inward single-channel currents at −100 mV for the indicated amino acid substitutions. Dashed lines are the current levels with alanine substitution (A) and serine substitution (B). (C and D) Plots of iin versus voltage for hydrophobic (C) and uncharged hydrophilic (D) amino acid substitutions. Side-chain volume has less of an effect on iin compared with iout (compare with Fig. 3). The reduced effect of side-chain volume on iin is consistent with larger side chains reducing the size of the entrance to the inner cavity. 150 mM K+i.
© Copyright Policy - openaccess
Related In: Results  -  Collection

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

fig5: Increasing side-chain volume has little or no effect on iin. (A and B) Representative records of inward single-channel currents at −100 mV for the indicated amino acid substitutions. Dashed lines are the current levels with alanine substitution (A) and serine substitution (B). (C and D) Plots of iin versus voltage for hydrophobic (C) and uncharged hydrophilic (D) amino acid substitutions. Side-chain volume has less of an effect on iin compared with iout (compare with Fig. 3). The reduced effect of side-chain volume on iin is consistent with larger side chains reducing the size of the entrance to the inner cavity. 150 mM K+i.
Mentions: The experiments in the previous sections examined the effect of side-chain volume on iout by making the intracellular side of the membrane positive to electrostatically push K+ out through the channel. To test the effect of side-chain volume on inward single-channel current amplitudes, iin, the intracellular side of the membrane, was made negative to attract K+ inward through the channel. Replacing alanine with larger side-chain hydrophobic amino acids reduced iin a maximum of 18% for the largest side-chain tryptophan (Fig. 5, A and C) compared with a 30% reduction for iout (Fig. 3, A–C). Replacing serine with larger side-chain uncharged hydrophilic amino acids had no effect on iin (Fig. 5, B and D) compared with an ∼13% reduction on iout (Fig. 3, D and E). A larger reduction of iout compared with iin with increasing side-chain volume is consistent with larger side-chain volumes reducing the size of the entrance to the inner cavity. For outward currents, decreasing the size of the entrance to the inner cavity could directly limit how much diffusing K+ would enter the entrance to the inner cavity (be captured) to be driven through the channel by voltage. In contrast, for inward currents, decreasing the size of entrance to the inner cavity (now the exit) could lead to an increased concentration of K+ in the inner cavity, increasing the probability of K+ exiting to the intracellular solution, compensating to some extent for the smaller exit size.

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