Mg2+ Enhances Voltage Sensor/Gate Coupling in BK Channels

Abstract

BK (Slo1) potassium channels are activated by millimolar intracellular Mg2+ as well as micromolar Ca2+ and membrane depolarization. Mg2+ and Ca2+ act in an approximately additive manner at different binding sites to shift the conductance–voltage (GK-V) relation, suggesting that these ligands might work through functionally similar but independent mechanisms. However, we find that the mechanism of Mg2+ action is highly dependent on voltage sensor activation and therefore differs fundamentally from that of Ca2+. Evidence that Ca2+ acts independently of voltage sensor activation includes an ability to increase open probability (PO) at extreme negative voltages where voltage sensors are in the resting state; 2 μM Ca2+ increases PO more than 15-fold at −120 mV. However 10 mM Mg2+, which has an effect on the GK-V relation similar to 2 μM Ca2+, has no detectable effect on PO when voltage sensors are in the resting state. Gating currents are only slightly altered by Mg2+ when channels are closed, indicating that Mg2+ does not act merely to promote voltage sensor activation. Indeed, channel opening is facilitated in a voltage-independent manner by Mg2+ in a mutant (R210C) whose voltage sensors are constitutively activated. Thus, 10 mM Mg2+ increases PO only when voltage sensors are activated, effectively strengthening the allosteric coupling of voltage sensor activation to channel opening. Increasing Mg2+ from 10 to 100 mM, to occupy very low affinity binding sites, has additional effects on gating that more closely resemble those of Ca2+. The effects of Mg2+ on steady-state activation and IK kinetics are discussed in terms of an allosteric gating scheme and the state-dependent interactions between Mg2+ and voltage sensor that may underlie this mechanism.

Comparative effects of H+and Ca2+on large-conductance Ca2+- and voltage-gated Slo1 K+channels

“…17, 18 The Mg 2+ -sensitivity is mediated by a mechanism distinct from that underlying the activation by Ca 2+ . 17, 18, 29, 30 We found that Mg 2+ at 6 mM induces the same shift in V 0.5 as that by lowering pH i to 6.2 without Ca 2+ (Supplementary Figure 1A). However, H + at pH i = 6.2 more effectively accelerated that activation kinetics than Mg 2+ at 6 mM (Supplementary Figure 1B, C).…”

Measurements of the BKCa Channel's High-Affinity Ca2+ Binding Constants: Effects of Membrane Voltage

“…We do not yet know the answers to these questions, but our current hypothesis is that the RCK1 sites lie in close proximity to the channel ' s voltage-sensing domains, and that as a given voltage sensor moves, it alters the structure of its nearby RCK1 Ca 2+ binding site, while having no such interaction at the Ca 2+ bowl. An allosteric interaction between the BK Ca channel ' s low-affi nity Ca 2+ binding sites (those disabled by the E399N mutation) and its voltage sensors has already been fi rmly established ( Hu et al, 2001 ;Cui et al, 1997 , Yang andSachs, 1989 ;Cui et al, 1997 ;Horrigan and Ma, 2008 ), and like the high-affi nity RCK1 sites we have investigated here, these low-affi nity sites are also thought to reside in the channel ' s RCK1 domains. Alternatively, one might suppose that Ca 2+ binding is voltage dependent because Ca 2+ binds within the electric fi eld of the membrane; however, the RCK1 domains of the channel are thought to be suspended below the channel and thus they are not likely within the membrane ' s electric fi eld.…”

The Contribution of RCK Domains to Human BK Channel Allosteric Activation

“…Moreover, a physical proximity of VSD and gating ring is suggested by their mutual coordination of Mg 2ϩ (10,11,35,65). Atomic structures of the BK gating ring (27)(28)(29), have pinpointed the locations of the calcium bowl (in RCK2) and Asp-362/Asp-367 residues (in RCK1) that, in the whole channel, could face the intracellular portion of the VSD (Fig.…”