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Allosteric regulation of BK channel gating by Ca(2+) and Mg(2+) through a nonselective, low affinity divalent cation site.

Zhang X, Solaro CR, Lingle CJ - J. Gen. Physiol. (2001)

Bottom Line: At voltages where millimolar elevations in [Ca(2+)] increase activation rates, addition of 10 mM Mg(2+) to 0 Ca(2+) produces little effect on activation time course, while markedly slowing deactivation.This suggests that Mg(2+) does not participate in Ca(2+)-dependent steps that influence current activation rate.We conclude that millimolar Mg(2+) and Ca(2+) concentrations interact with low affinity, relatively nonselective divalent cation binding sites that are distinct from higher affinity, Ca(2+)-selective binding sites that increase current activation rates.

View Article: PubMed Central - PubMed

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

ABSTRACT
The ability of membrane voltage to activate high conductance, calcium-activated (BK-type) K(+) channels is enhanced by cytosolic calcium (Ca(2+)). Activation is sensitive to a range of [Ca(2+)] that spans over four orders of magnitude. Here, we examine the activation of BK channels resulting from expression of cloned mouse Slo1 alpha subunits at [Ca(2+)] and [Mg(2+)] up to 100 mM. The half-activation voltage (V(0.5)) is steeply dependent on [Ca(2+)] in the micromolar range, but shows a tendency towards saturation over the range of 60-300 microM Ca(2+). As [Ca(2+)] is increased to millimolar levels, the V(0.5) is strongly shifted again to more negative potentials. When channels are activated by 300 microM Ca(2+), further addition of either mM Ca(2+) or mM Mg(2+) produces similar negative shifts in steady-state activation. Millimolar Mg(2+) also produces shifts of similar magnitude in the complete absence of Ca(2+). The ability of millimolar concentrations of divalent cations to shift activation is primarily correlated with a slowing of BK current deactivation. At voltages where millimolar elevations in [Ca(2+)] increase activation rates, addition of 10 mM Mg(2+) to 0 Ca(2+) produces little effect on activation time course, while markedly slowing deactivation. This suggests that Mg(2+) does not participate in Ca(2+)-dependent steps that influence current activation rate. We conclude that millimolar Mg(2+) and Ca(2+) concentrations interact with low affinity, relatively nonselective divalent cation binding sites that are distinct from higher affinity, Ca(2+)-selective binding sites that increase current activation rates. A symmetrical model with four independent higher affinity Ca(2+) binding steps, four voltage sensors, and four independent lower affinity Ca(2+)/Mg(2+) binding steps describes well the behavior of G-V curves over a range of Ca(2+) and Mg(2+). The ability of a broad range of [Ca(2+)] to produce shifts in activation of Slo1 conductance can, therefore, be accounted for by multiple types of divalent cation binding sites.

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Comparison of effects of Ca2+ and Mg2+ on primary time constant of Slo1 current relaxations. In A, activation and deactivation time constants obtained at 0, 10, and 300 μM Ca2+ are plotted as a function of potential. In B, the shift in relaxation time constant with 1 and 4 μM are compared with 0 μM Ca2+. Note the unusual slowing of activation with 1 μM Ca2+. In C, the effects of 10 and 50 mM Ca2+ are compared with 0 Ca2+, whereas, in D, the effects of 10 and 50 mM Mg2+ are compared with 0 Ca2+. In E, the effects of 10 and 50 mM Ca2+ are compared with 300 μM Ca2+, whereas, in F, the effects of 10 and 50 mM Mg2+ plus 300 μM Ca2+ are compared with 300 μM Ca2+.
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Figure 11: Comparison of effects of Ca2+ and Mg2+ on primary time constant of Slo1 current relaxations. In A, activation and deactivation time constants obtained at 0, 10, and 300 μM Ca2+ are plotted as a function of potential. In B, the shift in relaxation time constant with 1 and 4 μM are compared with 0 μM Ca2+. Note the unusual slowing of activation with 1 μM Ca2+. In C, the effects of 10 and 50 mM Ca2+ are compared with 0 Ca2+, whereas, in D, the effects of 10 and 50 mM Mg2+ are compared with 0 Ca2+. In E, the effects of 10 and 50 mM Ca2+ are compared with 300 μM Ca2+, whereas, in F, the effects of 10 and 50 mM Mg2+ plus 300 μM Ca2+ are compared with 300 μM Ca2+.

Mentions: To summarize the similarities and differences in the effects of Ca2+ and Mg2+ on kinetic aspects of Slo1 currents, effects of various [Ca2+] and [Mg2+] were compared in the same sets of patches. At any given [Ca2+] and [Mg2+], the relaxation time constant (deactivation and activation) exhibits an approximately bell-shaped dependence on voltage. In Fig. 11 A, 10 μM Ca2+ is shown to shift both activation and deactivation times constants to a somewhat similar extent compared with 0 Ca2+, whereas with 300 μM Ca2+, the effects on τd begin to diminish while effects on activation remain pronounced. In Fig. 11 B, 4 μM Ca2+ produces a leftward shift qualitatively similar to that with 10 μM, although smaller. 1 μM Ca2+ results in the unusual slowing of activation described above, producing a slowing of the principle time constant at all voltages. In Fig. 11C and Fig. D, Fig. 10 and 50 mM Ca2+ are compared with 10 and 50 mM Mg2+. 10 and 50 mM Ca2+ produce a similar leftward shift in the relaxation time constant. In contrast, with 10 mM Mg2+, there is no apparent effect on current activation, but deactivation is slowed. 50 mM Mg2+ produces some slight additional slowing in deactivation, but also results in some increase in current activation rate. In Fig. 11 E, 10 and 50 mM Ca2+ are shown to produce a substantial additional slowing of deactivation relative to 300 μM Ca2+, with only weaker effects on current activation at positive potentials. The effects of 10 and 50 mM Mg2+ when added to 300 μM Ca2+ are quite similar (Fig. 11 F), producing a substantial slowing of current deactivation, with little effect on current activation, except for a clear slowing of activation at 50 mM. Thus, these kinetic effects remain generally consistent with the effects of Ca2+ and Mg2+ on GV curves. There is a higher affinity effect of Ca2+ that influences both current activation rates and deactivation rates. There is little evidence that Mg2+ acts at this site except for a slowing of activation, when [Mg2+] is perhaps at least three orders of magnitude greater than [Ca2+]. In contrast, both Mg2+ and Ca2+ share an ability to slow deactivation at mM concentrations, while having minimal effects on limiting rates of current activation at these concentrations.


Allosteric regulation of BK channel gating by Ca(2+) and Mg(2+) through a nonselective, low affinity divalent cation site.

Zhang X, Solaro CR, Lingle CJ - J. Gen. Physiol. (2001)

Comparison of effects of Ca2+ and Mg2+ on primary time constant of Slo1 current relaxations. In A, activation and deactivation time constants obtained at 0, 10, and 300 μM Ca2+ are plotted as a function of potential. In B, the shift in relaxation time constant with 1 and 4 μM are compared with 0 μM Ca2+. Note the unusual slowing of activation with 1 μM Ca2+. In C, the effects of 10 and 50 mM Ca2+ are compared with 0 Ca2+, whereas, in D, the effects of 10 and 50 mM Mg2+ are compared with 0 Ca2+. In E, the effects of 10 and 50 mM Ca2+ are compared with 300 μM Ca2+, whereas, in F, the effects of 10 and 50 mM Mg2+ plus 300 μM Ca2+ are compared with 300 μM Ca2+.
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Figure 11: Comparison of effects of Ca2+ and Mg2+ on primary time constant of Slo1 current relaxations. In A, activation and deactivation time constants obtained at 0, 10, and 300 μM Ca2+ are plotted as a function of potential. In B, the shift in relaxation time constant with 1 and 4 μM are compared with 0 μM Ca2+. Note the unusual slowing of activation with 1 μM Ca2+. In C, the effects of 10 and 50 mM Ca2+ are compared with 0 Ca2+, whereas, in D, the effects of 10 and 50 mM Mg2+ are compared with 0 Ca2+. In E, the effects of 10 and 50 mM Ca2+ are compared with 300 μM Ca2+, whereas, in F, the effects of 10 and 50 mM Mg2+ plus 300 μM Ca2+ are compared with 300 μM Ca2+.
Mentions: To summarize the similarities and differences in the effects of Ca2+ and Mg2+ on kinetic aspects of Slo1 currents, effects of various [Ca2+] and [Mg2+] were compared in the same sets of patches. At any given [Ca2+] and [Mg2+], the relaxation time constant (deactivation and activation) exhibits an approximately bell-shaped dependence on voltage. In Fig. 11 A, 10 μM Ca2+ is shown to shift both activation and deactivation times constants to a somewhat similar extent compared with 0 Ca2+, whereas with 300 μM Ca2+, the effects on τd begin to diminish while effects on activation remain pronounced. In Fig. 11 B, 4 μM Ca2+ produces a leftward shift qualitatively similar to that with 10 μM, although smaller. 1 μM Ca2+ results in the unusual slowing of activation described above, producing a slowing of the principle time constant at all voltages. In Fig. 11C and Fig. D, Fig. 10 and 50 mM Ca2+ are compared with 10 and 50 mM Mg2+. 10 and 50 mM Ca2+ produce a similar leftward shift in the relaxation time constant. In contrast, with 10 mM Mg2+, there is no apparent effect on current activation, but deactivation is slowed. 50 mM Mg2+ produces some slight additional slowing in deactivation, but also results in some increase in current activation rate. In Fig. 11 E, 10 and 50 mM Ca2+ are shown to produce a substantial additional slowing of deactivation relative to 300 μM Ca2+, with only weaker effects on current activation at positive potentials. The effects of 10 and 50 mM Mg2+ when added to 300 μM Ca2+ are quite similar (Fig. 11 F), producing a substantial slowing of current deactivation, with little effect on current activation, except for a clear slowing of activation at 50 mM. Thus, these kinetic effects remain generally consistent with the effects of Ca2+ and Mg2+ on GV curves. There is a higher affinity effect of Ca2+ that influences both current activation rates and deactivation rates. There is little evidence that Mg2+ acts at this site except for a slowing of activation, when [Mg2+] is perhaps at least three orders of magnitude greater than [Ca2+]. In contrast, both Mg2+ and Ca2+ share an ability to slow deactivation at mM concentrations, while having minimal effects on limiting rates of current activation at these concentrations.

Bottom Line: At voltages where millimolar elevations in [Ca(2+)] increase activation rates, addition of 10 mM Mg(2+) to 0 Ca(2+) produces little effect on activation time course, while markedly slowing deactivation.This suggests that Mg(2+) does not participate in Ca(2+)-dependent steps that influence current activation rate.We conclude that millimolar Mg(2+) and Ca(2+) concentrations interact with low affinity, relatively nonselective divalent cation binding sites that are distinct from higher affinity, Ca(2+)-selective binding sites that increase current activation rates.

View Article: PubMed Central - PubMed

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

ABSTRACT
The ability of membrane voltage to activate high conductance, calcium-activated (BK-type) K(+) channels is enhanced by cytosolic calcium (Ca(2+)). Activation is sensitive to a range of [Ca(2+)] that spans over four orders of magnitude. Here, we examine the activation of BK channels resulting from expression of cloned mouse Slo1 alpha subunits at [Ca(2+)] and [Mg(2+)] up to 100 mM. The half-activation voltage (V(0.5)) is steeply dependent on [Ca(2+)] in the micromolar range, but shows a tendency towards saturation over the range of 60-300 microM Ca(2+). As [Ca(2+)] is increased to millimolar levels, the V(0.5) is strongly shifted again to more negative potentials. When channels are activated by 300 microM Ca(2+), further addition of either mM Ca(2+) or mM Mg(2+) produces similar negative shifts in steady-state activation. Millimolar Mg(2+) also produces shifts of similar magnitude in the complete absence of Ca(2+). The ability of millimolar concentrations of divalent cations to shift activation is primarily correlated with a slowing of BK current deactivation. At voltages where millimolar elevations in [Ca(2+)] increase activation rates, addition of 10 mM Mg(2+) to 0 Ca(2+) produces little effect on activation time course, while markedly slowing deactivation. This suggests that Mg(2+) does not participate in Ca(2+)-dependent steps that influence current activation rate. We conclude that millimolar Mg(2+) and Ca(2+) concentrations interact with low affinity, relatively nonselective divalent cation binding sites that are distinct from higher affinity, Ca(2+)-selective binding sites that increase current activation rates. A symmetrical model with four independent higher affinity Ca(2+) binding steps, four voltage sensors, and four independent lower affinity Ca(2+)/Mg(2+) binding steps describes well the behavior of G-V curves over a range of Ca(2+) and Mg(2+). The ability of a broad range of [Ca(2+)] to produce shifts in activation of Slo1 conductance can, therefore, be accounted for by multiple types of divalent cation binding sites.

Show MeSH
Related in: MedlinePlus