A charged residue in S4 regulates coupling among the activation gate, voltage, and Ca2+ sensors in BK channels.

Coupling between the activation gate and sensors of physiological stimuli during ion channel activation is an important, but not well-understood, molecular process. One difficulty in studying sensor-gate coupling is to distinguish whether a structural perturbation alters the function of the sensor, the gate, or their coupling. BK channels are activated by membrane voltage and intracellular Ca(2+) via allosteric mechanisms with coupling among the activation gate and sensors quantitatively defined, providing an excellent model system for studying sensor-gate coupling. By studying BK channels expressed in Xenopus oocytes, here we show that mutation E219R in S4 alters channel function by two independent mechanisms: one is to change voltage sensor activation, shifting voltage dependence, and increase valence of gating charge movements; the other is to regulate coupling among the activation gate, voltage sensor, and Ca(2+) binding via electrostatic interactions with E321/E324 located in the cytosolic side of S6 in a neighboring subunit, resulting in a shift of the voltage dependence of channel opening and increased Ca(2+) sensitivity. These results suggest a structural arrangement of the inner pore of BK channels differing from that in other voltage gated channels.

Ion sensing in the RCK1 domain of BK channels.

BK-type K(+) channels are activated by voltage and intracellular Ca(2+), which is important in modulating muscle contraction, neural transmission, and circadian pacemaker output. Previous studies suggest that the cytosolic domain of BK channels contains two different Ca(2+) binding sites, but the molecular composition of one of the sites is not completely known. Here we report, by systematic mutagenesis studies, the identification of E535 as part of this Ca(2+) binding site. This site is specific for binding to Ca(2+) but not Cd(2+). Experimental results and molecular modeling based on the X-ray crystallographic structures of the BK channel cytosolic domain suggest that the binding of Ca(2+) by the side chains of E535 and the previously identified D367 changes the conformation around the binding site and turns the side chain of M513 into a hydrophobic core, providing a basis to understand how Ca(2+) binding at this site opens the activation gate of the channel that is remotely located in the membrane.

Interaction between residues in the Mg2+-binding site regulates BK channel activation.

As a unique member of the voltage-gated potassium channel family, a large conductance, voltage- and Ca(2+)-activated K(+) (BK) channel has a large cytosolic domain that serves as the Ca(2+) sensor, in addition to a membrane-spanning domain that contains the voltage-sensing (VSD) and pore-gate domains. The conformational changes of the cytosolic domain induced by Ca(2+) binding and the conformational changes of the VSD induced by membrane voltage changes trigger the opening of the pore-gate domain. Although some structural information of these individual functional domains is available, how the interactions among these domains, especially the noncovalent interactions, control the dynamic gating process of BK channels is still not clear. Previous studies discovered that intracellular Mg(2+) binds to an interdomain binding site consisting of D99 and N172 from the membrane-spanning domain and E374 and E399 from the cytosolic domain. The bound Mg(2+) at this narrow interdomain interface activates the BK channel through an electrostatic interaction with a positively charged residue in the VSD. In this study, we investigated the potential interdomain interactions between the Mg(2+)-coordination residues and their effects on channel gating. By introducing different charges to these residues, we discovered a native interdomain interaction between D99 and E374 that can affect BK channel activation. To understand the underlying mechanism of the interdomain interactions between the Mg(2+)-coordination residues, we introduced artificial electrostatic interactions between residues 172 and 399 from two different domains. We found that the interdomain interactions between these two positions not only alter the local conformations near the Mg(2+)-binding site but also change distant conformations including the pore-gate domain, thereby affecting the voltage- and Ca(2+)-dependent activation of the BK channel. These results illustrate the importance of interdomain interactions to the allosteric gating mechanisms of BK channels.

Patch clamp and perfusion techniques for studying ion channels expressed in Xenopus oocytes.

The protocol presented here is designed to study the activation of the large conductance, voltage- and Ca(2+)-activated K(+) (BK) channels. The protocol may also be used to study the structure-function relationship for other ion channels and neurotransmitter receptors. BK channels are widely expressed in different tissues and have been implicated in many physiological functions, including regulation of smooth muscle contraction, frequency tuning of inner hair cells and regulation of neurotransmitter release. BK channels are activated by membrane depolarization and by intracellular Ca(2+) and Mg(2+). Therefore, the protocol is designed to control both the membrane voltage and the intracellular solution. In this protocol, messenger RNA of BK channels is injected into Xenopus laevis oocytes (stage V-VI) followed by 2-5 days of incubation at 18°C. Membrane patches that contain single or multiple BK channels are excised with the inside-out configuration using patch clamp techniques. The intracellular side of the patch is perfused with desired solutions during recording so that the channel activation under different conditions can be examined. To summarize, the mRNA of BK channels is injected into Xenopus laevis oocytes to express channel proteins on the oocyte membrane; patch clamp techniques are used to record currents flowing through the channels under controlled voltage and intracellular solutions.

An epilepsy/dyskinesia-associated mutation enhances BK channel activation by potentiating Ca2+ sensing.

Ca(2+)-activated BK channels modulate neuronal activities, including spike frequency adaptation and synaptic transmission. Previous studies found that Ca(2+)-binding sites and the activation gate are spatially separated in the channel protein, but the mechanism by which Ca(2+) binding opens the gate over this distance remains unknown. By studying an Asp-to-Gly mutation (D434G) associated with human syndrome of generalized epilepsy and paroxysmal dyskinesia (GEPD), we show that a cytosolic motif immediately following the activation gate S6 helix, known as the AC region, mediates the allosteric coupling between Ca(2+) binding and channel opening. The GEPD mutation inside the AC region increases BK channel activity by enhancing this allosteric coupling. We found that Ca(2+) sensitivity is enhanced by increases in solution viscosity that reduce protein dynamics. The GEPD mutation alters such a response, suggesting that a less flexible AC region may be more effective in coupling Ca(2+) binding to channel opening.

Activation of Slo1 BK channels by Mg2+ coordinated between the voltage sensor and RCK1 domains.

The voltage-sensor domain (VSD) and the ligand sensor (cytoplasmic domain) of BK channels synergistically control channel activities, thereby integrating electrical and chemical signals for cell function. Studies show that intracellular Mg2+ mediates the interaction between these sensory domains to activate the channel through an electrostatic interaction with the VSD. Here we report that Mg2+ binds to a site that consists of amino acid side chains from both the VSD (Asp99 and Asn172) and the cytoplasmic domain (Glu374 and Glu399). For each Mg2+ binding site, the residues in the VSD and those in the cytoplasmic domain come from neighboring subunits. These results suggest that the VSD and the cytoplasmic domains from different subunits may interact during channel gating, and the packing of VSD or the RCK1 domain to the pore in BK channels differ from that in Kv1.2 or MthK channels.