Participation of the S4 voltage sensor in the Mg ²⁺ -dependent activation of large conductance (BK) K ⁺ channels

Abstract

The S4 transmembrane segment is the primary voltage sensor in voltage-dependent ion channels. Its movement in response to changes in membrane potential leads to the opening of the activation gate, which is formed by a separate structural component, the S6 segment. Here we show in voltage-, Ca 2؉ -, and Mg 2؉ -dependent, large conductance K ؉ channels that the S4 segment participates not only in voltage-but also Mg 2؉ -dependent activation. Mutations in S4 and the S4-S5 linker alter voltagedependent activation and have little or no effect on activation by micromolar Ca 2؉ . However, a subset of these mutations in the C-terminal half of S4 and in the S4-S5 linker either reduce or abolish the Mg 2؉ sensitivity of channel gating. Cysteine residues substituted into positions R210 and R213, marking the boundary between S4 mutations that alter Mg 2؉ sensitivity and those that do not, are accessible to a modifying reagent [sodium (2-sulfonatoethyl)methane-thiosulfonate] (MTSES) from the extracellular and intracellular side of the membrane, respectively, at ؊80 mV. This implies that interactions between S4 and a cytoplasmic domain may be involved in Mg 2؉ -dependent activation. These results indicate that the voltage sensor is critical for Mg 2؉ -dependent activation and the coupling between the voltage sensor and channel gate is a converging point for voltage-and Mg 2؉ -dependent activation pathways.

Mg 2+ mediates interaction between the voltage sensor and cytosolic domain to activate BK channels

“…4 a and b), consistent with the previous finding that R213Q abolishes Mg 2ϩ sensitivity (18). Residue 213 is located in the C-terminal half of S4, is accessible to intracellular Cys-modifying reagents when the voltage sensor is in the resting state (18), and contributes to gating charge (14,15). Thus, this residue is a sensor of both membrane voltage and Mg 2ϩ binding.…”

“…1b, is that the binding of the Mg 2ϩ may alter the local electric field that is sensed by charged residues in the VSD, thereby activating the channel. This hypothesis is consistent with the fact that Mg 2ϩ shifts the voltage dependence of channel opening to more negative voltages and that the neutralization of R213 abolishes Mg 2ϩ sensitivity (18). Another line of evidence supporting this hypothesis is that Mg 2ϩ -dependent activation is sensitive to the ionic strength of intracellular solution.…”

“…Like other voltagedependent K ϩ (Kv) channels, BK channels possess a VSD where the S4 segment contains multiple Arg residues (14,15). However, in BK channels, only one of these, R213, contributes to voltage sensing (15)(16)(17)(18). Interestingly, neutralizing R213 by mutation (R213Q) not only alters voltage-dependent activation but also abolishes Mg 2ϩ -dependent activation of BK channels, revealing that the VSD contributes to Mg 2ϩ sensitivity, although the mechanism is unknown (18).…”

“…However, in BK channels, only one of these, R213, contributes to voltage sensing (15)(16)(17)(18). Interestingly, neutralizing R213 by mutation (R213Q) not only alters voltage-dependent activation but also abolishes Mg 2ϩ -dependent activation of BK channels, revealing that the VSD contributes to Mg 2ϩ sensitivity, although the mechanism is unknown (18). Here, we investigate the mechanism of Mg 2ϩ action to determine whether and how the VSD interacts with Mg 2ϩ ions that are bound to the BK channel's COOH-terminal cytosolic domain.…”

Operation of the voltage sensor of a human voltage- and Ca 2+ -activated K + channel

“…Consequently, aqueous crevices could be a dynamic, activationdependent element of the BK Ca VSD, rather than a passive morphological feature. We find this interpretation more conceptually satisfying, because it is based on significant evidence supporting the existence of VSD crevices (37)(38)(39)(40)(41)(42)(43), including in BK Ca (54).…”