Nonselective, Low Affinity Divalent Cation Site

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 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 M 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 M 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. key words: K channels • Ca 2 and voltage-gated K channels • Slo1 channels • stochastic models • channel kinetics I N T R O D U C T I O N Most ion channels open in response to a change in a single, primary physiological parameter. In contrast, activation of Ca 2 and voltage-dependent large conductance Ca 2 -activated K (BK)* channels is complicated by the fact that two parameters govern channel opening. Membrane depolarization and binding of Ca 2 ions interact in some way to bring about an increase in channel open probability. The dependence on Ca 2 is particularly remarkable in that the ability of voltage to open BK channels can be shifted by over four log orders of [Ca 2 ] (Moczydlowski and Latorre, 1983; Meera et al., 1996; Cox et al., 1997a; Cui et al., 1997). Although the voltage dependence of BK channel gating is thought to arise from a mechanism involving voltage-sensing residues in the S4 segment (Diaz et al., 1998; Horrigan et al., 1999; Horrigan and Aldrich, 1999; Cui and Aldrich, 2000), which is similar to the voltage-dependent K channel family, the precise mechanism by which Ca 2 exerts its effects remain unknown. Some of the effects of Ca 2 may be mediated by interaction of Ca 2 with a patch of negatively charged residues, termed the “Ca 2 bowl,” located just before the S10 hydrophobic segment near the COOH terminus of the Slo subunit (Schreiber and Salkoff, 1997; Schreiber et al., 1999). Residues just upstream of the Ca 2 bowl may also participate in defining the Ca 2 -regulatory domain (Braun and Sy, 2001). However, how this Ca 2 -sensing domain may mediate its effects remains unknown. Moreover, there are several studies that indicate that Ca 2 and/or other divalent cations may also allosterically modulate BK gating (Golowasch et al., 1986; Oberhauser et al., 1988; Solaro et al., 1995; Shi and Cui, 2001a). The relationship between V 0.5 and cytosolic Ca 2 has provided a convenient descriptive tool to evaluate the Ca 2 dependence of activation of BK current, an approach first employed by Moczydlowski and Latorre (1983). In this landmark paper (Moczydlowski and Address correspondence to Chris Lingle, Department of Anesthesiology, Washington University School of Medicine, Box 8054, St. Louis, MO 63110. Fax: (314) 362-8571; E-mail: [email protected] * Abbreviations used in this paper: BK, large conductance Ca 2 -activated K channel; NMG, N -methyl glucamine; P o , open probability. on A uust 0, 2017 jgp.rress.org D ow nladed fom