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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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Residues E321C/E324C at the entrance to the inner cavity are accessible to the intracellular solution. (A–C) Representative records of outward currents through single BK channels held at +180 mV before and after applications of thiol reagents to wt and mutated channels. The dashed lines show the open and closed current levels of wt channels. Current levels after intracellular application of MPA−, MBB−, or MTSET+ to wt channels followed by thorough washing (A) were unchanged, whereas application and wash to E321C (B) or E324C (C) channels changed single-channel current levels after treatment with MPA− or MTSET+. (D and E) Plots of iout versus voltage for the indicated treatments for E321C (D) and E324C (E) channels. Treatment and wash of the thiol agents to wt channels had no effect on iout and are not plotted. In this and subsequent plots, the dashed lines are cubic spline fits, and the absence of visible error bars indicates that the SEM is less than the symbol size. Data are from three to five different oocytes for each plotted symbol. Positions E321C and E324C are both accessible to the ion conduction pathway from the intracellular side with equivalent effects of thiol reagents at these positions on iout. 150 mM K+i.
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fig2: Residues E321C/E324C at the entrance to the inner cavity are accessible to the intracellular solution. (A–C) Representative records of outward currents through single BK channels held at +180 mV before and after applications of thiol reagents to wt and mutated channels. The dashed lines show the open and closed current levels of wt channels. Current levels after intracellular application of MPA−, MBB−, or MTSET+ to wt channels followed by thorough washing (A) were unchanged, whereas application and wash to E321C (B) or E324C (C) channels changed single-channel current levels after treatment with MPA− or MTSET+. (D and E) Plots of iout versus voltage for the indicated treatments for E321C (D) and E324C (E) channels. Treatment and wash of the thiol agents to wt channels had no effect on iout and are not plotted. In this and subsequent plots, the dashed lines are cubic spline fits, and the absence of visible error bars indicates that the SEM is less than the symbol size. Data are from three to five different oocytes for each plotted symbol. Positions E321C and E324C are both accessible to the ion conduction pathway from the intracellular side with equivalent effects of thiol reagents at these positions on iout. 150 mM K+i.

Mentions: Treating wt BK channels with MPA−, MBB−, or MTSET+ did not change iout (Fig. 2 A). Thus, the cysteines of wt BK channels are either not accessible to intracellular thiol reagents or thiol binding to native cysteines does not alter iout, consistent with previous observations of no effect of intracellular thiol reagents on iout for native BK channels (Wang et al., 1997). In contrast, the treatment of E321C channels with MPA− increased iout compared with E321C alone (Fig. 2, B and D). This observation suggests that MPA− specifically binds to the cysteine at position 321 because, as described above, MPA− application to wt BK channels had no effect. The increased iout with MPA− treatment likely arises because the negative charge on MPA− bound to E321C increases the local K+ concentration in the inner cavity of the E321C channels (Brelidze et al., 2003; Nimigean et al., 2003).


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)

Residues E321C/E324C at the entrance to the inner cavity are accessible to the intracellular solution. (A–C) Representative records of outward currents through single BK channels held at +180 mV before and after applications of thiol reagents to wt and mutated channels. The dashed lines show the open and closed current levels of wt channels. Current levels after intracellular application of MPA−, MBB−, or MTSET+ to wt channels followed by thorough washing (A) were unchanged, whereas application and wash to E321C (B) or E324C (C) channels changed single-channel current levels after treatment with MPA− or MTSET+. (D and E) Plots of iout versus voltage for the indicated treatments for E321C (D) and E324C (E) channels. Treatment and wash of the thiol agents to wt channels had no effect on iout and are not plotted. In this and subsequent plots, the dashed lines are cubic spline fits, and the absence of visible error bars indicates that the SEM is less than the symbol size. Data are from three to five different oocytes for each plotted symbol. Positions E321C and E324C are both accessible to the ion conduction pathway from the intracellular side with equivalent effects of thiol reagents at these positions on iout. 150 mM K+i.
© Copyright Policy - openaccess
Related In: Results  -  Collection

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

fig2: Residues E321C/E324C at the entrance to the inner cavity are accessible to the intracellular solution. (A–C) Representative records of outward currents through single BK channels held at +180 mV before and after applications of thiol reagents to wt and mutated channels. The dashed lines show the open and closed current levels of wt channels. Current levels after intracellular application of MPA−, MBB−, or MTSET+ to wt channels followed by thorough washing (A) were unchanged, whereas application and wash to E321C (B) or E324C (C) channels changed single-channel current levels after treatment with MPA− or MTSET+. (D and E) Plots of iout versus voltage for the indicated treatments for E321C (D) and E324C (E) channels. Treatment and wash of the thiol agents to wt channels had no effect on iout and are not plotted. In this and subsequent plots, the dashed lines are cubic spline fits, and the absence of visible error bars indicates that the SEM is less than the symbol size. Data are from three to five different oocytes for each plotted symbol. Positions E321C and E324C are both accessible to the ion conduction pathway from the intracellular side with equivalent effects of thiol reagents at these positions on iout. 150 mM K+i.
Mentions: Treating wt BK channels with MPA−, MBB−, or MTSET+ did not change iout (Fig. 2 A). Thus, the cysteines of wt BK channels are either not accessible to intracellular thiol reagents or thiol binding to native cysteines does not alter iout, consistent with previous observations of no effect of intracellular thiol reagents on iout for native BK channels (Wang et al., 1997). In contrast, the treatment of E321C channels with MPA− increased iout compared with E321C alone (Fig. 2, B and D). This observation suggests that MPA− specifically binds to the cysteine at position 321 because, as described above, MPA− application to wt BK channels had no effect. The increased iout with MPA− treatment likely arises because the negative charge on MPA− bound to E321C increases the local K+ concentration in the inner cavity of the E321C channels (Brelidze et al., 2003; Nimigean et al., 2003).

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