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N-terminal inactivation domains of beta subunits are protected from trypsin digestion by binding within the antechamber of BK channels.

Zhang Z, Zeng XH, Xia XM, Lingle CJ - J. Gen. Physiol. (2009)

Bottom Line: Other results suggest that, even when channels are closed, an inactivation domain can also be protected from digestion by trypsin when bound within the antechamber.Together, these results confirm the idea that beta2 N termini can occupy the BK channel antechamber by interaction at some site distinct from the BK central cavity.These results indicate that inactivation domains have sites of binding in addition to those directly involved in inactivation.

View Article: PubMed Central - PubMed

Affiliation: Department of Anesthesiology, Washington University School of Medicine, St. Louis, MO 63110, USA.

ABSTRACT
N termini of auxiliary beta subunits that produce inactivation of large-conductance Ca(2+)-activated K(+) (BK) channels reach their pore-blocking position by first passing through side portals into an antechamber separating the BK pore module and the large C-terminal cytosolic domain. Previous work indicated that the beta2 subunit inactivation domain is protected from digestion by trypsin when bound in the inactivated conformation. Other results suggest that, even when channels are closed, an inactivation domain can also be protected from digestion by trypsin when bound within the antechamber. Here, we provide additional tests of this model and examine its applicability to other beta subunit N termini. First, we show that specific mutations in the beta2 inactivation segment can speed up digestion by trypsin under closed-channel conditions, supporting the idea that the beta2 N terminus is protected by binding within the antechamber. Second, we show that cytosolic channel blockers distinguish between protection mediated by inactivation and protection under closed-channel conditions, implicating two distinct sites of protection. Together, these results confirm the idea that beta2 N termini can occupy the BK channel antechamber by interaction at some site distinct from the BK central cavity. In contrast, the beta 3a N terminus is digested over 10-fold more quickly than the beta2 N terminus. Analysis of factors that contribute to differences in digestion rates suggests that binding of an N terminus within the antechamber constrains the trypsin accessibility of digestible basic residues, even when such residues are positioned outside the antechamber. Our analysis indicates that up to two N termini may simultaneously be protected from digestion. These results indicate that inactivation domains have sites of binding in addition to those directly involved in inactivation.

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Cartoons summarizing the idea of antechamber occupancy and lateral access of β2 N termini to the BK channel pore. (A) The pathway for access of the β2 N-terminal inactivation domain to the BK channel central cavity is schematized. N termini must enter the central cavity by passing through the side portals separating the BK channel pore domain from the cytosolic domains involved in Ca2+ binding. The lateral distance from the center of the pore to the position where the N terminal attaches to the β2 subunit TM1 domain is estimated to be ∼45–60 Å (Zhang et al., 2006). Each ball in the schematized N terminus represents an amino acid, with red indicating basic residues and blue indicating the FIW hydrophobic triplet essential for inactivation. (B) Cartoons schematically summarize proposed configurations of β2 N termini during gating and inactivation. Each channel contains four β2 subunits (containing a triplet of hydrophobic residues [blue] at the N terminus and two digestible basic residues, R8 and R19 [red]), each of which can potentially enter the channel antechamber (equilibrium, Ba) through side portals. The central pore is indicated by the shaded, inner circle (smaller, closed channel; larger, open channel). In this scheme, only one N terminus can occupy the antechamber at a time. Channels open in accordance with equilibrium constant L. Open channels with a β2 N terminus in the antechamber may also inactivate (equilibrium constant, Bi).
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fig1: Cartoons summarizing the idea of antechamber occupancy and lateral access of β2 N termini to the BK channel pore. (A) The pathway for access of the β2 N-terminal inactivation domain to the BK channel central cavity is schematized. N termini must enter the central cavity by passing through the side portals separating the BK channel pore domain from the cytosolic domains involved in Ca2+ binding. The lateral distance from the center of the pore to the position where the N terminal attaches to the β2 subunit TM1 domain is estimated to be ∼45–60 Å (Zhang et al., 2006). Each ball in the schematized N terminus represents an amino acid, with red indicating basic residues and blue indicating the FIW hydrophobic triplet essential for inactivation. (B) Cartoons schematically summarize proposed configurations of β2 N termini during gating and inactivation. Each channel contains four β2 subunits (containing a triplet of hydrophobic residues [blue] at the N terminus and two digestible basic residues, R8 and R19 [red]), each of which can potentially enter the channel antechamber (equilibrium, Ba) through side portals. The central pore is indicated by the shaded, inner circle (smaller, closed channel; larger, open channel). In this scheme, only one N terminus can occupy the antechamber at a time. Channels open in accordance with equilibrium constant L. Open channels with a β2 N terminus in the antechamber may also inactivate (equilibrium constant, Bi).

Mentions: Rapid inactivation of large-conductance Ca2+-activated K+ (BK) channels is mediated by N-terminal cytosolic hydrophobic peptide segments of auxiliary β subunits (Wallner et al., 1999; Xia et al., 1999; Uebele et al., 2000; Xia et al., 2000, 2003). Such peptide segments are thought to obstruct ion flux by binding within the BK channel central cavity. To access this binding site, β subunit N termini must approach the axis of the permeation pathway laterally (Fig. 1 A), passing through the so-called side portals (Gulbis et al., 2000; Kobertz et al., 2000) that separate the membrane-embedded pore module and the large cytosolic structure involved in ligand recognition (Zhang et al., 2006). BK β subunit N termini contain basic residues that can be attacked by trypsin, thereby removing β subunit–mediated inactivation. Using quantitative measurement of trypsin-mediated removal of inactivation, it has been shown that the space between the pore domain and cytosolic domain defines a volume in which the β2 N terminus is protected from digestion by trypsin, and this protected volume has been termed an antechamber (Zhang et al., 2006). The properties of removal by trypsin of β2-mediated inactivation are consistent with a model in which, even under conditions in which channels are closed, individual N termini occupy the antechamber for an appreciable fraction of time, thereby conferring some protection against digestion by trypsin (Fig. 1 B). Thus, a determinant of the time course of digestion by trypsin reflects not just the accessibility of the basic residues, but also the fraction of time a β2 N terminus resides within the protected antechamber.


N-terminal inactivation domains of beta subunits are protected from trypsin digestion by binding within the antechamber of BK channels.

Zhang Z, Zeng XH, Xia XM, Lingle CJ - J. Gen. Physiol. (2009)

Cartoons summarizing the idea of antechamber occupancy and lateral access of β2 N termini to the BK channel pore. (A) The pathway for access of the β2 N-terminal inactivation domain to the BK channel central cavity is schematized. N termini must enter the central cavity by passing through the side portals separating the BK channel pore domain from the cytosolic domains involved in Ca2+ binding. The lateral distance from the center of the pore to the position where the N terminal attaches to the β2 subunit TM1 domain is estimated to be ∼45–60 Å (Zhang et al., 2006). Each ball in the schematized N terminus represents an amino acid, with red indicating basic residues and blue indicating the FIW hydrophobic triplet essential for inactivation. (B) Cartoons schematically summarize proposed configurations of β2 N termini during gating and inactivation. Each channel contains four β2 subunits (containing a triplet of hydrophobic residues [blue] at the N terminus and two digestible basic residues, R8 and R19 [red]), each of which can potentially enter the channel antechamber (equilibrium, Ba) through side portals. The central pore is indicated by the shaded, inner circle (smaller, closed channel; larger, open channel). In this scheme, only one N terminus can occupy the antechamber at a time. Channels open in accordance with equilibrium constant L. Open channels with a β2 N terminus in the antechamber may also inactivate (equilibrium constant, Bi).
© Copyright Policy - openaccess
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC2654086&req=5

fig1: Cartoons summarizing the idea of antechamber occupancy and lateral access of β2 N termini to the BK channel pore. (A) The pathway for access of the β2 N-terminal inactivation domain to the BK channel central cavity is schematized. N termini must enter the central cavity by passing through the side portals separating the BK channel pore domain from the cytosolic domains involved in Ca2+ binding. The lateral distance from the center of the pore to the position where the N terminal attaches to the β2 subunit TM1 domain is estimated to be ∼45–60 Å (Zhang et al., 2006). Each ball in the schematized N terminus represents an amino acid, with red indicating basic residues and blue indicating the FIW hydrophobic triplet essential for inactivation. (B) Cartoons schematically summarize proposed configurations of β2 N termini during gating and inactivation. Each channel contains four β2 subunits (containing a triplet of hydrophobic residues [blue] at the N terminus and two digestible basic residues, R8 and R19 [red]), each of which can potentially enter the channel antechamber (equilibrium, Ba) through side portals. The central pore is indicated by the shaded, inner circle (smaller, closed channel; larger, open channel). In this scheme, only one N terminus can occupy the antechamber at a time. Channels open in accordance with equilibrium constant L. Open channels with a β2 N terminus in the antechamber may also inactivate (equilibrium constant, Bi).
Mentions: Rapid inactivation of large-conductance Ca2+-activated K+ (BK) channels is mediated by N-terminal cytosolic hydrophobic peptide segments of auxiliary β subunits (Wallner et al., 1999; Xia et al., 1999; Uebele et al., 2000; Xia et al., 2000, 2003). Such peptide segments are thought to obstruct ion flux by binding within the BK channel central cavity. To access this binding site, β subunit N termini must approach the axis of the permeation pathway laterally (Fig. 1 A), passing through the so-called side portals (Gulbis et al., 2000; Kobertz et al., 2000) that separate the membrane-embedded pore module and the large cytosolic structure involved in ligand recognition (Zhang et al., 2006). BK β subunit N termini contain basic residues that can be attacked by trypsin, thereby removing β subunit–mediated inactivation. Using quantitative measurement of trypsin-mediated removal of inactivation, it has been shown that the space between the pore domain and cytosolic domain defines a volume in which the β2 N terminus is protected from digestion by trypsin, and this protected volume has been termed an antechamber (Zhang et al., 2006). The properties of removal by trypsin of β2-mediated inactivation are consistent with a model in which, even under conditions in which channels are closed, individual N termini occupy the antechamber for an appreciable fraction of time, thereby conferring some protection against digestion by trypsin (Fig. 1 B). Thus, a determinant of the time course of digestion by trypsin reflects not just the accessibility of the basic residues, but also the fraction of time a β2 N terminus resides within the protected antechamber.

Bottom Line: Other results suggest that, even when channels are closed, an inactivation domain can also be protected from digestion by trypsin when bound within the antechamber.Together, these results confirm the idea that beta2 N termini can occupy the BK channel antechamber by interaction at some site distinct from the BK central cavity.These results indicate that inactivation domains have sites of binding in addition to those directly involved in inactivation.

View Article: PubMed Central - PubMed

Affiliation: Department of Anesthesiology, Washington University School of Medicine, St. Louis, MO 63110, USA.

ABSTRACT
N termini of auxiliary beta subunits that produce inactivation of large-conductance Ca(2+)-activated K(+) (BK) channels reach their pore-blocking position by first passing through side portals into an antechamber separating the BK pore module and the large C-terminal cytosolic domain. Previous work indicated that the beta2 subunit inactivation domain is protected from digestion by trypsin when bound in the inactivated conformation. Other results suggest that, even when channels are closed, an inactivation domain can also be protected from digestion by trypsin when bound within the antechamber. Here, we provide additional tests of this model and examine its applicability to other beta subunit N termini. First, we show that specific mutations in the beta2 inactivation segment can speed up digestion by trypsin under closed-channel conditions, supporting the idea that the beta2 N terminus is protected by binding within the antechamber. Second, we show that cytosolic channel blockers distinguish between protection mediated by inactivation and protection under closed-channel conditions, implicating two distinct sites of protection. Together, these results confirm the idea that beta2 N termini can occupy the BK channel antechamber by interaction at some site distinct from the BK central cavity. In contrast, the beta 3a N terminus is digested over 10-fold more quickly than the beta2 N terminus. Analysis of factors that contribute to differences in digestion rates suggests that binding of an N terminus within the antechamber constrains the trypsin accessibility of digestible basic residues, even when such residues are positioned outside the antechamber. Our analysis indicates that up to two N termini may simultaneously be protected from digestion. These results indicate that inactivation domains have sites of binding in addition to those directly involved in inactivation.

Show MeSH
Related in: MedlinePlus