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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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Schematic drawing of the ion conduction pathway of a BK channel and its representation by a two-resistor model. (A) Schematic side view of a section through the transmembrane segments (S0–S6) of a BK channel, with the front and back subunits removed. Arrows show direction of K+ movement for outward currents. The two-resistor model is depicted to the right. Not depicted are the intracellular gating ring with a large square central pore that is 20 Å on a side (Wu et al., 2010) and the side portals (Zhang et al., 2009) between the gating ring and the transmembrane portion of the channel. For outward current, K+ diffuses from the cytoplasm through the central pore of the gating ring and through the side portals (1) to the entrance to the inner cavity, through the entrance (2), through the inner cavity (3), through the selectivity filter (4), and through the shallow extracellular cavity (5) to the extracellular solution. For the two-resistor model, R2 is a variable resistor representing the resistance of the entrance to the inner cavity (2), and R1 is a fixed resistor representing the resistance of the remaining segments (1+3+4+5) of the ion conduction pathway. (B and C) Schematic diagrams of the entrance to the inner cavity for small (B) and large (C) side-chain substitutions at positions 321/324. The effective diameter (D) and length (L) of the entrance to the inner cavity are indicated. Increasing the volume of the side chains at positions 321/324 decreases the effective diameter, increasing the resistance of R2.
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fig1: Schematic drawing of the ion conduction pathway of a BK channel and its representation by a two-resistor model. (A) Schematic side view of a section through the transmembrane segments (S0–S6) of a BK channel, with the front and back subunits removed. Arrows show direction of K+ movement for outward currents. The two-resistor model is depicted to the right. Not depicted are the intracellular gating ring with a large square central pore that is 20 Å on a side (Wu et al., 2010) and the side portals (Zhang et al., 2009) between the gating ring and the transmembrane portion of the channel. For outward current, K+ diffuses from the cytoplasm through the central pore of the gating ring and through the side portals (1) to the entrance to the inner cavity, through the entrance (2), through the inner cavity (3), through the selectivity filter (4), and through the shallow extracellular cavity (5) to the extracellular solution. For the two-resistor model, R2 is a variable resistor representing the resistance of the entrance to the inner cavity (2), and R1 is a fixed resistor representing the resistance of the remaining segments (1+3+4+5) of the ion conduction pathway. (B and C) Schematic diagrams of the entrance to the inner cavity for small (B) and large (C) side-chain substitutions at positions 321/324. The effective diameter (D) and length (L) of the entrance to the inner cavity are indicated. Increasing the volume of the side chains at positions 321/324 decreases the effective diameter, increasing the resistance of R2.

Mentions: The purpose of our study is to determine the resistance and dimensions of the entrance to the inner cavity of BK channels to examine to what extent the entrance contributes to the high conductance of the channel. The experimental approach is depicted in Fig. 1, which presents a hypothetical midsection through a BK channel showing the membrane-spanning portions of two of the four α subunits that form the central conducting pore, where the membrane-spanning regions of the α subunits are formed from segments S0–S6 (Adelman et al., 1992; Butler et al., 1993; Meera et al., 1997). Not included in the diagram is the large intracellular gating ring of the channel, which is comprised of eight RCK domains (regulators of the conductance of K+), two per subunit (Jiang et al., 2002a,b; Wang and Sigworth, 2009; Wu et al., 2010).


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)

Schematic drawing of the ion conduction pathway of a BK channel and its representation by a two-resistor model. (A) Schematic side view of a section through the transmembrane segments (S0–S6) of a BK channel, with the front and back subunits removed. Arrows show direction of K+ movement for outward currents. The two-resistor model is depicted to the right. Not depicted are the intracellular gating ring with a large square central pore that is 20 Å on a side (Wu et al., 2010) and the side portals (Zhang et al., 2009) between the gating ring and the transmembrane portion of the channel. For outward current, K+ diffuses from the cytoplasm through the central pore of the gating ring and through the side portals (1) to the entrance to the inner cavity, through the entrance (2), through the inner cavity (3), through the selectivity filter (4), and through the shallow extracellular cavity (5) to the extracellular solution. For the two-resistor model, R2 is a variable resistor representing the resistance of the entrance to the inner cavity (2), and R1 is a fixed resistor representing the resistance of the remaining segments (1+3+4+5) of the ion conduction pathway. (B and C) Schematic diagrams of the entrance to the inner cavity for small (B) and large (C) side-chain substitutions at positions 321/324. The effective diameter (D) and length (L) of the entrance to the inner cavity are indicated. Increasing the volume of the side chains at positions 321/324 decreases the effective diameter, increasing the resistance of R2.
© Copyright Policy - openaccess
Related In: Results  -  Collection

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

fig1: Schematic drawing of the ion conduction pathway of a BK channel and its representation by a two-resistor model. (A) Schematic side view of a section through the transmembrane segments (S0–S6) of a BK channel, with the front and back subunits removed. Arrows show direction of K+ movement for outward currents. The two-resistor model is depicted to the right. Not depicted are the intracellular gating ring with a large square central pore that is 20 Å on a side (Wu et al., 2010) and the side portals (Zhang et al., 2009) between the gating ring and the transmembrane portion of the channel. For outward current, K+ diffuses from the cytoplasm through the central pore of the gating ring and through the side portals (1) to the entrance to the inner cavity, through the entrance (2), through the inner cavity (3), through the selectivity filter (4), and through the shallow extracellular cavity (5) to the extracellular solution. For the two-resistor model, R2 is a variable resistor representing the resistance of the entrance to the inner cavity (2), and R1 is a fixed resistor representing the resistance of the remaining segments (1+3+4+5) of the ion conduction pathway. (B and C) Schematic diagrams of the entrance to the inner cavity for small (B) and large (C) side-chain substitutions at positions 321/324. The effective diameter (D) and length (L) of the entrance to the inner cavity are indicated. Increasing the volume of the side chains at positions 321/324 decreases the effective diameter, increasing the resistance of R2.
Mentions: The purpose of our study is to determine the resistance and dimensions of the entrance to the inner cavity of BK channels to examine to what extent the entrance contributes to the high conductance of the channel. The experimental approach is depicted in Fig. 1, which presents a hypothetical midsection through a BK channel showing the membrane-spanning portions of two of the four α subunits that form the central conducting pore, where the membrane-spanning regions of the α subunits are formed from segments S0–S6 (Adelman et al., 1992; Butler et al., 1993; Meera et al., 1997). Not included in the diagram is the large intracellular gating ring of the channel, which is comprised of eight RCK domains (regulators of the conductance of K+), two per subunit (Jiang et al., 2002a,b; Wang and Sigworth, 2009; Wu et al., 2010).

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