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Divalent cation sensitivity of BK channel activation supports the existence of three distinct binding sites.

Zeng XH, Xia XM, Lingle CJ - J. Gen. Physiol. (2005)

Bottom Line: Mn2+, Co2+, and Ni2+ produce little observable effect through the high affinity regulatory mechanisms, while all six divalent cations enhance activation through the low affinity mechanism defined by residue E399.The major kinetic effect of the E399-related low affinity mechanism is to slow deactivation at mM Mg2+ or Ca2+.The results support the view that three distinct divalent-cation binding sites mediate regulation of BK channels.

View Article: PubMed Central - PubMed

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

ABSTRACT
Mutational analyses have suggested that BK channels are regulated by three distinct divalent cation-dependent regulatory mechanisms arising from the cytosolic COOH terminus of the pore-forming alpha subunit. Two mechanisms account for physiological regulation of BK channels by microM Ca2+. The third may mediate physiological regulation by mM Mg2+. Mutation of five aspartate residues (5D5N) within the so-called Ca2+ bowl removes a portion of a higher affinity Ca2+ dependence, while mutation of D362A/D367A in the first RCK domain also removes some higher affinity Ca2+ dependence. Together, 5D5N and D362A/D367A remove all effects of Ca2+ up through 1 mM while E399A removes a portion of low affinity regulation by Ca2+/Mg2+. If each proposed regulatory effect involves a distinct divalent cation binding site, the divalent cation selectivity of the actual site that defines each mechanism might differ. By examination of the ability of various divalent cations to activate currents in constructs with mutationally altered regulatory mechanisms, here we show that each putative regulatory mechanism exhibits a unique sensitivity to divalent cations. Regulation mediated by the Ca2+ bowl can be activated by Ca2+ and Sr2+, while regulation defined by D362/D367 can be activated by Ca2+, Sr2+, and Cd2+. Mn2+, Co2+, and Ni2+ produce little observable effect through the high affinity regulatory mechanisms, while all six divalent cations enhance activation through the low affinity mechanism defined by residue E399. Furthermore, each type of mutation affects kinetic properties of BK channels in distinct ways. The Ca2+ bowl mainly accelerates activation of BK channels at low [Ca2+], while the D362/D367-related high affinity site influences both activation and deactivation over the range of 10-300 microM Ca2+. The major kinetic effect of the E399-related low affinity mechanism is to slow deactivation at mM Mg2+ or Ca2+. The results support the view that three distinct divalent-cation binding sites mediate regulation of BK channels.

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The ability of Mn2+ to activate BK current depends on the E399 low affinity site. (A–C) Currents resulting from Slo1 (A), E399A (B), and the triple mutation (C, 5D5N + D362AD367A + E399A) were activated as in Fig. 1. (D–F) G–V curves were generated over [Mn2+]i from 0 to 5 mM for Slo1 (n = 6), E399A (n = 5), and the triple mutation (n = 5). Open diamonds, 0 μM; filled diamonds, 100 μM; open circles, 300 μM; filled circles, 1 mM; open triangles, 2 mM; filled triangles, 5 mM. (G) Activation ΔVh is plotted versus [Mn2+]i for Slo1 (n = 6) and E399A (n = 5). (H) Activation ΔVh is plotted versus [Mn2+]i for mutants containing E399A (5D5N + E399A, n = 4; D362A/D367A + E399A, n = 6; triple mutation, n = 5). (H) Activation ΔVh is plotted versus [Mn2+]i for Slo1 and mutants not containing E399A (5D5N, n = 5; D362A/D367A, n = 7; 5D5N + D362A/D367A, n = 8).
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fig2: The ability of Mn2+ to activate BK current depends on the E399 low affinity site. (A–C) Currents resulting from Slo1 (A), E399A (B), and the triple mutation (C, 5D5N + D362AD367A + E399A) were activated as in Fig. 1. (D–F) G–V curves were generated over [Mn2+]i from 0 to 5 mM for Slo1 (n = 6), E399A (n = 5), and the triple mutation (n = 5). Open diamonds, 0 μM; filled diamonds, 100 μM; open circles, 300 μM; filled circles, 1 mM; open triangles, 2 mM; filled triangles, 5 mM. (G) Activation ΔVh is plotted versus [Mn2+]i for Slo1 (n = 6) and E399A (n = 5). (H) Activation ΔVh is plotted versus [Mn2+]i for mutants containing E399A (5D5N + E399A, n = 4; D362A/D367A + E399A, n = 6; triple mutation, n = 5). (H) Activation ΔVh is plotted versus [Mn2+]i for Slo1 and mutants not containing E399A (5D5N, n = 5; D362A/D367A, n = 7; 5D5N + D362A/D367A, n = 8).

Mentions: Mn2+ has been shown to activate muscle membrane BK channels expressed in bilayers (Oberhauser et al., 1988). We therefore tested whether the ability of Mn2+ to modulate BK channels might be selectively associated with any of the three putative regulatory sites. Fig. 2 (A–C) shows currents activated in the presence of 0 through 5 mM Mn2+ for mSlo1, E399A, and the triple mutation (5D5N+D362A/D367A+E399A). For mSlo1, Mn2+ causes a leftward shift in the G–V curves with no change in voltage dependence (Fig. 2 D). The negative Vh shift produced by 5 mM Mn2+ relative to 0 divalent is >100 mV (Fig. 2 G), while 100 μM Mn2+ produces an ∼50-mV leftward shift. Mutation of the low affinity site alone is sufficient to abolish most of the effect of Mn2+ (Fig. 2, E and G). Moreover, additional mutation of the two high affinity sites together with E399A does not produce any additional effect other than that caused by E399A alone (Fig. 2, F and H). In fact, Mn2+ loses its ability to activate current only for those constructs in which the E399A mutation is present (Fig. 2 H), while Mn2+ produces similar current activation for all constructs containing an intact E399 site (Fig. 2 I). These results show clearly that Mn2+ modulates BK channels predominantly through the low affinity site. Furthermore, although we were only able to examine Mn2+ concentrations up through 5 mM, the E399A mutation appears to completely remove the approximately −90-mV shift produced by 5 mM Mn2+, whereas E399A removes about half of the approximately −60-mV shift caused by 10 mM Mg2+ (Xia et al., 2002).


Divalent cation sensitivity of BK channel activation supports the existence of three distinct binding sites.

Zeng XH, Xia XM, Lingle CJ - J. Gen. Physiol. (2005)

The ability of Mn2+ to activate BK current depends on the E399 low affinity site. (A–C) Currents resulting from Slo1 (A), E399A (B), and the triple mutation (C, 5D5N + D362AD367A + E399A) were activated as in Fig. 1. (D–F) G–V curves were generated over [Mn2+]i from 0 to 5 mM for Slo1 (n = 6), E399A (n = 5), and the triple mutation (n = 5). Open diamonds, 0 μM; filled diamonds, 100 μM; open circles, 300 μM; filled circles, 1 mM; open triangles, 2 mM; filled triangles, 5 mM. (G) Activation ΔVh is plotted versus [Mn2+]i for Slo1 (n = 6) and E399A (n = 5). (H) Activation ΔVh is plotted versus [Mn2+]i for mutants containing E399A (5D5N + E399A, n = 4; D362A/D367A + E399A, n = 6; triple mutation, n = 5). (H) Activation ΔVh is plotted versus [Mn2+]i for Slo1 and mutants not containing E399A (5D5N, n = 5; D362A/D367A, n = 7; 5D5N + D362A/D367A, n = 8).
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fig2: The ability of Mn2+ to activate BK current depends on the E399 low affinity site. (A–C) Currents resulting from Slo1 (A), E399A (B), and the triple mutation (C, 5D5N + D362AD367A + E399A) were activated as in Fig. 1. (D–F) G–V curves were generated over [Mn2+]i from 0 to 5 mM for Slo1 (n = 6), E399A (n = 5), and the triple mutation (n = 5). Open diamonds, 0 μM; filled diamonds, 100 μM; open circles, 300 μM; filled circles, 1 mM; open triangles, 2 mM; filled triangles, 5 mM. (G) Activation ΔVh is plotted versus [Mn2+]i for Slo1 (n = 6) and E399A (n = 5). (H) Activation ΔVh is plotted versus [Mn2+]i for mutants containing E399A (5D5N + E399A, n = 4; D362A/D367A + E399A, n = 6; triple mutation, n = 5). (H) Activation ΔVh is plotted versus [Mn2+]i for Slo1 and mutants not containing E399A (5D5N, n = 5; D362A/D367A, n = 7; 5D5N + D362A/D367A, n = 8).
Mentions: Mn2+ has been shown to activate muscle membrane BK channels expressed in bilayers (Oberhauser et al., 1988). We therefore tested whether the ability of Mn2+ to modulate BK channels might be selectively associated with any of the three putative regulatory sites. Fig. 2 (A–C) shows currents activated in the presence of 0 through 5 mM Mn2+ for mSlo1, E399A, and the triple mutation (5D5N+D362A/D367A+E399A). For mSlo1, Mn2+ causes a leftward shift in the G–V curves with no change in voltage dependence (Fig. 2 D). The negative Vh shift produced by 5 mM Mn2+ relative to 0 divalent is >100 mV (Fig. 2 G), while 100 μM Mn2+ produces an ∼50-mV leftward shift. Mutation of the low affinity site alone is sufficient to abolish most of the effect of Mn2+ (Fig. 2, E and G). Moreover, additional mutation of the two high affinity sites together with E399A does not produce any additional effect other than that caused by E399A alone (Fig. 2, F and H). In fact, Mn2+ loses its ability to activate current only for those constructs in which the E399A mutation is present (Fig. 2 H), while Mn2+ produces similar current activation for all constructs containing an intact E399 site (Fig. 2 I). These results show clearly that Mn2+ modulates BK channels predominantly through the low affinity site. Furthermore, although we were only able to examine Mn2+ concentrations up through 5 mM, the E399A mutation appears to completely remove the approximately −90-mV shift produced by 5 mM Mn2+, whereas E399A removes about half of the approximately −60-mV shift caused by 10 mM Mg2+ (Xia et al., 2002).

Bottom Line: Mn2+, Co2+, and Ni2+ produce little observable effect through the high affinity regulatory mechanisms, while all six divalent cations enhance activation through the low affinity mechanism defined by residue E399.The major kinetic effect of the E399-related low affinity mechanism is to slow deactivation at mM Mg2+ or Ca2+.The results support the view that three distinct divalent-cation binding sites mediate regulation of BK channels.

View Article: PubMed Central - PubMed

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

ABSTRACT
Mutational analyses have suggested that BK channels are regulated by three distinct divalent cation-dependent regulatory mechanisms arising from the cytosolic COOH terminus of the pore-forming alpha subunit. Two mechanisms account for physiological regulation of BK channels by microM Ca2+. The third may mediate physiological regulation by mM Mg2+. Mutation of five aspartate residues (5D5N) within the so-called Ca2+ bowl removes a portion of a higher affinity Ca2+ dependence, while mutation of D362A/D367A in the first RCK domain also removes some higher affinity Ca2+ dependence. Together, 5D5N and D362A/D367A remove all effects of Ca2+ up through 1 mM while E399A removes a portion of low affinity regulation by Ca2+/Mg2+. If each proposed regulatory effect involves a distinct divalent cation binding site, the divalent cation selectivity of the actual site that defines each mechanism might differ. By examination of the ability of various divalent cations to activate currents in constructs with mutationally altered regulatory mechanisms, here we show that each putative regulatory mechanism exhibits a unique sensitivity to divalent cations. Regulation mediated by the Ca2+ bowl can be activated by Ca2+ and Sr2+, while regulation defined by D362/D367 can be activated by Ca2+, Sr2+, and Cd2+. Mn2+, Co2+, and Ni2+ produce little observable effect through the high affinity regulatory mechanisms, while all six divalent cations enhance activation through the low affinity mechanism defined by residue E399. Furthermore, each type of mutation affects kinetic properties of BK channels in distinct ways. The Ca2+ bowl mainly accelerates activation of BK channels at low [Ca2+], while the D362/D367-related high affinity site influences both activation and deactivation over the range of 10-300 microM Ca2+. The major kinetic effect of the E399-related low affinity mechanism is to slow deactivation at mM Mg2+ or Ca2+. The results support the view that three distinct divalent-cation binding sites mediate regulation of BK channels.

Show MeSH
Related in: MedlinePlus