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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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Comparison of removal of inactivation mediated by hβ2 and mβ3a. (A) The removal of mβ3a-mediated inactivation is plotted for four different trypsin concentrations. Fitted time constants of digestion were 5.83 ± 1.2 s (0.05 mg/ml), 10.5 ± 1.42 s (0.025 mg/ml), 23.72 ± 1.41 s (0.01 mg/ml), and 44.22 ± 3.74 s (0.005 mg/ml). (B) The effective digestion rate (min−1) is plotted as a function of trypsin concentration for both mβ3a and hβ2 (from Zhang et al., 2006). The line through the mβ3a points corresponds to a linear fit with a slope of 260 min−1/mg/ml, which assumes a molecular weight of 24 kD for trypsin corresponds to 9.97 × 104 M−1 s−1. For β2, the line corresponds to the slope through the two lowest trypsin concentrations, yielding a maximal effective rate of 6.82 × 103 M−1 s−1. (C) A log-log plot of the digestion time course for hβ2 (black circles) and mβ3a (red circles) compares the slope of the digestion process. For β2, n = 2.21 ± 0.23; for β3a, n = 3.62 ± 0.39. Note that in A, except for the time course observed with 0.05 mg/ml, the steeper slope of β3a digestion is independent of trypsin concentration. Similarly, the slope of β2 digestion is independent of trypsin concentration (not depicted).
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fig7: Comparison of removal of inactivation mediated by hβ2 and mβ3a. (A) The removal of mβ3a-mediated inactivation is plotted for four different trypsin concentrations. Fitted time constants of digestion were 5.83 ± 1.2 s (0.05 mg/ml), 10.5 ± 1.42 s (0.025 mg/ml), 23.72 ± 1.41 s (0.01 mg/ml), and 44.22 ± 3.74 s (0.005 mg/ml). (B) The effective digestion rate (min−1) is plotted as a function of trypsin concentration for both mβ3a and hβ2 (from Zhang et al., 2006). The line through the mβ3a points corresponds to a linear fit with a slope of 260 min−1/mg/ml, which assumes a molecular weight of 24 kD for trypsin corresponds to 9.97 × 104 M−1 s−1. For β2, the line corresponds to the slope through the two lowest trypsin concentrations, yielding a maximal effective rate of 6.82 × 103 M−1 s−1. (C) A log-log plot of the digestion time course for hβ2 (black circles) and mβ3a (red circles) compares the slope of the digestion process. For β2, n = 2.21 ± 0.23; for β3a, n = 3.62 ± 0.39. Note that in A, except for the time course observed with 0.05 mg/ml, the steeper slope of β3a digestion is independent of trypsin concentration. Similarly, the slope of β2 digestion is independent of trypsin concentration (not depicted).

Mentions: Time constants for digestion of mβ3a were determined at multiple trypsin concentrations (Fig. 7 A) and compared with previous estimates obtained for hβ2 (Zhang et al., 2006). For both subunits, the digestion rate was plotted as a function of [trypsin] (Fig. 7 B). For β3a, the digestion rate varied approximately linearly with [trypsin], with a slope of 9.97 × 104 M−1 s−1. For β2 (Zhang et al., 2006), the limiting slope at low concentration was 6.82 × 103 M−1 s−1, suggesting that there is a >10-fold difference between the digestion rates. Another important difference was that the power term in the fit to the removal of inactivation of mβ3a currents was clearly steeper than for β2 currents (Fig. 7 C), except with the highest tested trypsin concentration. As with β2, TBA and bbTBA had differential effects on inactivation-associated protection against β3a N-terminal digestion (Fig. 8). Whereas TBA was without effect on inactivation-associated protection (Fig. 8, A and B), bbTBA abolished the protection produced by inactivation (Fig. 8, C and D). No clear effects on closed-channel digestion were produced by either TBA or bbTBA.


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)

Comparison of removal of inactivation mediated by hβ2 and mβ3a. (A) The removal of mβ3a-mediated inactivation is plotted for four different trypsin concentrations. Fitted time constants of digestion were 5.83 ± 1.2 s (0.05 mg/ml), 10.5 ± 1.42 s (0.025 mg/ml), 23.72 ± 1.41 s (0.01 mg/ml), and 44.22 ± 3.74 s (0.005 mg/ml). (B) The effective digestion rate (min−1) is plotted as a function of trypsin concentration for both mβ3a and hβ2 (from Zhang et al., 2006). The line through the mβ3a points corresponds to a linear fit with a slope of 260 min−1/mg/ml, which assumes a molecular weight of 24 kD for trypsin corresponds to 9.97 × 104 M−1 s−1. For β2, the line corresponds to the slope through the two lowest trypsin concentrations, yielding a maximal effective rate of 6.82 × 103 M−1 s−1. (C) A log-log plot of the digestion time course for hβ2 (black circles) and mβ3a (red circles) compares the slope of the digestion process. For β2, n = 2.21 ± 0.23; for β3a, n = 3.62 ± 0.39. Note that in A, except for the time course observed with 0.05 mg/ml, the steeper slope of β3a digestion is independent of trypsin concentration. Similarly, the slope of β2 digestion is independent of trypsin concentration (not depicted).
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fig7: Comparison of removal of inactivation mediated by hβ2 and mβ3a. (A) The removal of mβ3a-mediated inactivation is plotted for four different trypsin concentrations. Fitted time constants of digestion were 5.83 ± 1.2 s (0.05 mg/ml), 10.5 ± 1.42 s (0.025 mg/ml), 23.72 ± 1.41 s (0.01 mg/ml), and 44.22 ± 3.74 s (0.005 mg/ml). (B) The effective digestion rate (min−1) is plotted as a function of trypsin concentration for both mβ3a and hβ2 (from Zhang et al., 2006). The line through the mβ3a points corresponds to a linear fit with a slope of 260 min−1/mg/ml, which assumes a molecular weight of 24 kD for trypsin corresponds to 9.97 × 104 M−1 s−1. For β2, the line corresponds to the slope through the two lowest trypsin concentrations, yielding a maximal effective rate of 6.82 × 103 M−1 s−1. (C) A log-log plot of the digestion time course for hβ2 (black circles) and mβ3a (red circles) compares the slope of the digestion process. For β2, n = 2.21 ± 0.23; for β3a, n = 3.62 ± 0.39. Note that in A, except for the time course observed with 0.05 mg/ml, the steeper slope of β3a digestion is independent of trypsin concentration. Similarly, the slope of β2 digestion is independent of trypsin concentration (not depicted).
Mentions: Time constants for digestion of mβ3a were determined at multiple trypsin concentrations (Fig. 7 A) and compared with previous estimates obtained for hβ2 (Zhang et al., 2006). For both subunits, the digestion rate was plotted as a function of [trypsin] (Fig. 7 B). For β3a, the digestion rate varied approximately linearly with [trypsin], with a slope of 9.97 × 104 M−1 s−1. For β2 (Zhang et al., 2006), the limiting slope at low concentration was 6.82 × 103 M−1 s−1, suggesting that there is a >10-fold difference between the digestion rates. Another important difference was that the power term in the fit to the removal of inactivation of mβ3a currents was clearly steeper than for β2 currents (Fig. 7 C), except with the highest tested trypsin concentration. As with β2, TBA and bbTBA had differential effects on inactivation-associated protection against β3a N-terminal digestion (Fig. 8). Whereas TBA was without effect on inactivation-associated protection (Fig. 8, A and B), bbTBA abolished the protection produced by inactivation (Fig. 8, C and D). No clear effects on closed-channel digestion were produced by either TBA or bbTBA.

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