Slo1 BK Channels Research Articles

Membrane lipids in allosteric gating of the Slo1 BK channel

Membrane lipids in allosteric gating of the Slo1 BK channel

A gating lever and molecular logic gate that couple voltage and calcium sensor activation to opening in BK potassium channels.

BK channels uniquely integrate voltage and calcium signaling in diverse cell types through allosteric activation of their K+-conducting pore by structurally distinct V and Ca2+ sensor domains. Here, we define mechanisms and interaction pathways that link V sensors to the pore by analyzing effects on allosteric coupling of point mutations in the context of Slo1 BK channel structure. A gating lever, mediated by S4/S5 segment interaction within the transmembrane domain, rotates to engage and stabilize the open conformation of the S6 inner pore helix upon V sensor activation. In addition, an indirect pathway, mediated by the carboxyl-terminal cytosolic domain (CTD) and C-linker that connects the CTD to S6, stabilizes the closed conformation when V sensors are at rest. Unexpectedly, this mechanism, which bypasses the covalent connections of C-linker to CTD and pore, also transduces Ca2+-dependent coupling in a manner that is completely nonadditive with voltage, analogous to the function of a digital logic (OR) gate.

The enantiomer pair of 24S- and 24R-hydroxycholesterol differentially alter activity of large-conductance Ca2+ -dependent K+ (slo1 BK) channel.

24S-hydroxycholesterol (HC) is most abundant oxysterols in the brain, passes through blood brain barrier, and is therefore regarded as an intermediary for brain cholesterol elimination. We reported that large-conductance Ca2+ - and voltage-activated K+ (slo1 BK) channels are suppressed by this oxysterol, which is presumably intercalated into cell membrane to access the outer surface of the channel. Such an outer approach would make it difficult to interact with the inner, ion-conducting part of the channel. The present findings showed that 24R-HC, the racemic counterpart of 24S-HC, also suppressed slo1 BK channel but in a different voltage-dependent manner. There was a difference between the effects of the two enantiomers on activation kinetics but not on deactivation kinetics. It is suggested that the chirality contributes to the efficacy of channel blockers that act from outer lipophilic parts of channels, as with those which act on the inner, ion-permeable surface.

24S-hydroxycholesterol alters activity of large-conductance Ca2+-dependent K+ (slo1 BK) channel through intercalation into plasma membrane

Oxysterols, oxidization products of cholesterol, are regarded as bioactive lipids affecting various physiological functions. However, little is known of their effects on ion channels. Using inside-out patch clamp recording, we found that naturally occurring side-chain oxidized oxysterols, 20S‑hydroxycholesterol, 22R‑hydroxycholesterol, 24S‑hydroxycholestero, 25‑hydroxycholesterol, and 27‑hydroxycholesterol, induced current reduction of large-conductance Ca2+- and voltage-activated K+ (slo1 BK) channels heterologously expressed in HEK293T cells. In contrast with side-chain oxidized oxysterols, naturally occurring ring oxidized ones, 7α‑hydroxycholesterol and 7‑ketocholesterol were without effect. By using 24S‑hydroxycholesterol (24S‑HC), the major brain oxysterol, we explored the inhibition mechanism. 24S‑HC inhibited Slo1 BK channels with an IC50 of ~2 μM, and decreased macroscopic current by ~60%. This marked current decrease was accompanied by a rightward shift in the conductance-voltage relationship and a slowed activation kinetics, with the deactivation kinetics unaltered. Furthermore, the membrane sterol scavenger γ‑cyclodextrin was found to rescue slo1 BK channels from the inhibition, implicating that 24S-HC may be intercalated into the plasma membrane to affect the channel. These findings unveil a novel physiological importance of oxysterols from a new angle that involves ion channel regulation.

ERG-28 controls BK channel trafficking in the ER to regulate synaptic function and alcohol response in C. elegans.

Voltage- and calcium-dependent BK channels regulate calcium-dependent cellular events such as neurotransmitter release by limiting calcium influx. Their plasma membrane abundance is an important factor in determining BK current and thus regulation of calcium-dependent events. In C. elegans, we show that ERG-28, an endoplasmic reticulum (ER) membrane protein, promotes the trafficking of SLO-1 BK channels from the ER to the plasma membrane by shielding them from premature degradation. In the absence of ERG-28, SLO-1 channels undergo aspartic protease DDI-1-dependent degradation, resulting in markedly reduced expression at presynaptic terminals. Loss of erg-28 suppressed phenotypic defects of slo-1 gain-of-function mutants in locomotion, neurotransmitter release, and calcium-mediated asymmetric differentiation of the AWC olfactory neuron pair, and conferred significant ethanol-resistant locomotory behavior, resembling slo-1 loss-of-function mutants, albeit to a lesser extent. Our study thus indicates that the control of BK channel trafficking is a critical regulatory mechanism for synaptic transmission and neural function.

Atomic determinants of BK channel activation by polyunsaturated fatty acids

Docosahexaenoic acid (DHA), a polyunsaturated ω-3 fatty acid enriched in oily fish, contributes to better health by affecting multiple targets. Large-conductance Ca2+- and voltage-gated Slo1 BK channels are directly activated by nanomolar levels of DHA. We investigated DHA-channel interaction by manipulating both the fatty acid structure and the channel composition through the site-directed incorporation of unnatural amino acids. Electrophysiological measurements show that the para-group of a Tyr residue near the ion conduction pathway has a critical role. To robustly activate the channel, ionization must occur readily by a fatty acid for a good efficacy, and a long nonpolar acyl tail with a Z double bond present at the halfway position for a high affinity. The results suggest that DHA and the channel form an ion-dipole bond to promote opening and demonstrate the channel druggability. DHA, a marine-derived nutraceutical, represents a promising lead compound for rational drug design and discovery.

SLO BK Potassium Channels Couple Gap Junctions to Inhibition of Calcium Signaling in Olfactory Neuron Diversification.

The C. elegans AWC olfactory neuron pair communicates to specify asymmetric subtypes AWCOFF and AWCON in a stochastic manner. Intercellular communication between AWC and other neurons in a transient NSY-5 gap junction network antagonizes voltage-activated calcium channels, UNC-2 (CaV2) and EGL-19 (CaV1), in the AWCON cell, but how calcium signaling is downregulated by NSY-5 is only partly understood. Here, we show that voltage- and calcium-activated SLO BK potassium channels mediate gap junction signaling to inhibit calcium pathways for asymmetric AWC differentiation. Activation of vertebrate SLO-1 channels causes transient membrane hyperpolarization, which makes it an important negative feedback system for calcium entry through voltage-activated calcium channels. Consistent with the physiological roles of SLO-1, our genetic results suggest that slo-1 BK channels act downstream of NSY-5 gap junctions to inhibit calcium channel-mediated signaling in the specification of AWCON. We also show for the first time that slo-2 BK channels are important for AWC asymmetry and act redundantly with slo-1 to inhibit calcium signaling. In addition, nsy-5-dependent asymmetric expression of slo-1 and slo-2 in the AWCON neuron is necessary and sufficient for AWC asymmetry. SLO-1 and SLO-2 localize close to UNC-2 and EGL-19 in AWC, suggesting a role of possible functional coupling between SLO BK channels and voltage-activated calcium channels in AWC asymmetry. Furthermore, slo-1 and slo-2 regulate the localization of synaptic markers, UNC-2 and RAB-3, in AWC neurons to control AWC asymmetry. We also identify the requirement of bkip-1, which encodes a previously identified auxiliary subunit of SLO-1, for slo-1 and slo-2 function in AWC asymmetry. Together, these results provide an unprecedented molecular link between gap junctions and calcium pathways for terminal differentiation of olfactory neurons.

Two distinct effects of PIP2 underlie auxiliary subunit-dependent modulation of Slo1 BK channels.

Phosphatidylinositol 4,5-bisphosphate (PIP2) plays a critical role in modulating the function of numerous ion channels, including large-conductance Ca(2+)- and voltage-dependent K(+) (BK, Slo1) channels. Slo1 BK channel complexes include four pore-forming Slo1 (α) subunits as well as various regulatory auxiliary subunits (β and γ) that are expressed in different tissues. We examined the molecular and biophysical mechanisms underlying the effects of brain-derived PIP2 on human Slo1 BK channel complexes with different subunit compositions that were heterologously expressed in human embryonic kidney cells. PIP2 inhibited macroscopic currents through Slo1 channels without auxiliary subunits and through Slo1 + γ1 complexes. In contrast, PIP2 markedly increased macroscopic currents through Slo1 + β1 and Slo1 + β4 channel complexes and failed to alter macroscopic currents through Slo1 + β2 and Slo1 + β2 Δ2-19 channel complexes. Results obtained at various membrane potentials and divalent cation concentrations suggest that PIP2 promotes opening of the ion conduction gate in all channel types, regardless of the specific subunit composition. However, in the absence of β subunits positioned near the voltage-sensor domains (VSDs), as in Slo1 and probably Slo1 + γ1, PIP2 augments the negative surface charge on the cytoplasmic side of the membrane, thereby shifting the voltage dependence of VSD-mediated activation in the positive direction. When β1 or β4 subunits occupy the space surrounding the VSDs, only the stimulatory effect of PIP2 is evident. The subunit compositions of native Slo1 BK channels differ in various cell types; thus, PIP2 may exert distinct tissue- and divalent cation-dependent modulatory influences.

Differential Effects of PIP2 on Slo1 BK Channels with Different Auxiliary Subunits

Phosphatidylinositol 4, 5-bisphosphate (PIP2) regulates numerous ion channels, including large-conductance Ca2+- and voltage-dependent K+ (Slo1 BK) channels (Vaithianathan et al. 2008, J Gen Physiol 132:13-28). We examined the molecular and biophysical mechanisms of the effects of PIP2 on human Slo1 BK channels with different auxiliary subunits heterologously expressed in HEK cells. In the absence of heterologously expressed auxiliary subunits, bovine brain PIP2 (10 µM) applied to the cytoplasmic side inhibits currents through Slo1 channels. This inhibition is associated with a shift in GV by 18 mV and deceleration of the activation kinetics at positive voltages. In contrast, PIP2 markedly increases currents through Slo1+β1 and hSlo1+β4 channels by shifting their GV curves by −46 mV and −34 mV, respectively. The stimulatory effect of PIP2 on Slo1+β1 channels does not require Ca2+-sensor activation or voltage-sensor activation. Currents through Slo1+β2 channels with a deletion in the N terminus of β2 to remove inactivation (Δ2-19) are not enhanced by PIP2 but neutralization of 3 negatively charged residues in the β2 Δ2-19 N terminus introduces a modest effect of PIP2. In Slo1+β1/β4, PIP2 accelerates the macroscopic activation kinetics at positive voltages and decelerates the macroscopic deactivation kinetics at negative voltages, but in Slo1+β2 Δ2-32 PIP2 has no effect on the deactivation kinetics. Measurements using chimeric β1-β2 subunits show that the second transmembrane domain and the C terminus of β1 is important for the large electrophysiological changes in Slo1+β1 channels caused by PIP2. Furthermore, Slo1 329RKK331, β1 R11, and β1 T14 are also critical to confer the effects of PIP2. Supported in part by the NIH, DFG HE 2993/8, and Shanghai Science and Technology Commission.

Critical role of large-conductance calcium- and voltage-activated potassium channels in leptin-induced neuroprotection of N-methyl-d-aspartate-exposed cortical neurons

In the present study, the neuroprotective effects of the adipokine leptin, and the molecular mechanism involved, have been studied in rat and mice cortical neurons exposed to N-methyl-d-aspartate (NMDA) in vitro. In rat cortical neurons, leptin elicited neuroprotective effects against NMDA-induced cell death, which were concentration-dependent (10–100ng/ml) and largest when the adipokine was preincubated for 2h before the neurotoxic stimulus. In both rat and mouse cortical neurons, leptin-induced neuroprotection was fully antagonized by paxilline (Pax, 0.01–1μM) and iberiotoxin (Ibtx, 1–100nM), with EC50s of 38±10nM and 5±2nM for Pax and Ibtx, respectively, close to those reported for Pax- and Ibtx-induced Ca2+- and voltage-activated K+ channels (Slo1 BK channels) blockade; the BK channel opener NS1619 (1–30μM) induced a concentration-dependent protection against NMDA-induced excitotoxicity. Moreover, cortical neurons from mice lacking one or both alleles coding for Slo1 BK channel pore-forming subunits were insensitive to leptin-induced neuroprotection. Finally, leptin exposure dose-dependently (10–100ng/ml) increased intracellular Ca2+ levels in rat cortical neurons. In conclusion, our results suggest that Slo1 BK channel activation following increases in intracellular Ca2+ levels is a critical step for leptin-induced neuroprotection in NMDA-exposed cortical neurons in vitro, thus highlighting leptin-based intervention via BK channel activation as a potential strategy to counteract neurodegenerative diseases.

A Point Mutation that Impairs the Omega-3 Fatty Acid Sensitivity of Human Slo1 BK Channels

Docosahexaenoic acid (DHA), a long-chain omega-3 fatty acid with a 22 carbon chain, directly activates large-conductance calcium- and voltage-gated Slo1 BK channels with an EC50 = ∼500 nM and lowers blood pressure when injected into anesthetized mice (Hoshi et al. 2013 Proc Natl Acad Sci U S A. 110, 4816-21). The stimulatory action of DHA is observed in excised patches and reversible, suggesting that DHA directly interacts with the channel protein. Here we examined the molecular elements within Slo1 critical for the action of DHA. Unlike the clear stimulatory effect of DHA on human Slo1 (hSlo1) channels, we found that Drosophila Slo1 (dSlo1) channels expressed in mammalian cells were not stimulated by DHA, up to 10 μM. Prompted by this finding, we generated a series of chimeric dSlo1-hSlo1 channels and identified a single residue in S6 critical for the stimulatory effect of DHA on hSlo1. With the the human-to-Drosophila mutation Y318S, the current-enhancing effect of DHA on hSlo1, hSlo1+ β1 and hSlo1+β4 channels was severely impaired. The converse Drosophila-to-human mutation S332Y, however, failed to introduce sensitivity to DHA in dSlo1. Interestingly, the hSlo1 mutation Y318S altered the hSlo1+β1 channel sensitivity to 22- and 20-carbon omega-3 fatty acids but did not affect the response to alpha-linolenic acid with a 20-carbon chain or the omega-6 fatty acid arachidonic acid with a 20-carbon chain. Y318 in hSlo1 S6 is thus a critical determinant of the channel's sensitivity to select fatty acids. Supported in part through the NIH, DFG FOR 1738, and Shanghai Science and Technology Commission.

Auxilary-Subunitdependent Modulation of SLO1 BK Channels that Underlies the Hypotensive Effectof Fish Oil

Long-chain omega-3 fatty acids are thought to offer many health-promoting effects but their molecular effectors are only beginning to be elucidated. Slo1 BK channel complexes are subject to modulation by select omega-3 fatty acids in an auxiliary subunit-dependent manner. In predominantly vascular Slo1+β1 and largely neuronal Slo1+β4 complexes, docosahexaenoic acid (DHA), an omega-3 fatty with a 22-carbon chain, reversibly increases ionic currents up to ∼20 fold by accelerating the opening transition of the ion conduction pathway in the absence of the allosteric influences of the voltage and Ca2+ sensors. The action of DHA is strikingly smaller in Slo1+ β2 (without inactivation) and in Slo1 alone, and DHA even decreases currents through Slo1+β1 (LRRC26). The stimulatory effect of DHA on Slo1+ β1 and Slo1+ β4 critically depends on two potentially interacting residues in each N terminus. The mutagenesis based on DHA-sensitive and -insensitive Slo1 channels reveals the importance of a residue in the S6 segment. The consequence of the DHA interaction with the channel involving this S6 residue is greatly amplified by the presence of β1 or β4. The β1 subunit-dependent effect of DHA underlies the blood-pressure lowering effect of the fatty acid acutely injected into anesthetized mice, and the hypotensive effect is absent in Slo1 knockout mice.

A point mutation in the human Slo1 channel that impairs its sensitivity to omega-3 docosahexaenoic acid

Long-chain polyunsaturated omega-3 fatty acids such as docosahexaenoic acid (DHA) at nanomolar concentrations reversibly activate human large-conductance Ca2+- and voltage-gated K+ (Slo1 BK) channels containing auxiliary β1 or β4 subunits in cell-free patches. Here we examined the action of DHA on the Slo1 channel without any auxiliary subunit and sought to elucidate the biophysical mechanism and the molecular determinants of the DHA sensitivity. Measurements of ionic currents through human Slo1 (hSlo1) channels reveal that the stimulatory effect of DHA does not require activation of the voltage or Ca2+ sensors. Unlike gating of the hSlo1 channel, that of the Drosophila melanogaster Slo1 (dSlo1) channel is unaltered by DHA. Our mutagenesis study based on the differential responses of human and dSlo1 channels to DHA pinpoints that Y318 near the cytoplasmic end of S6 in the hSlo1 channel is a critical determinant of the stimulatory action of DHA. The mutation Y318S in hSlo1, which replaces Y with S as found in dSlo1, greatly diminishes the channel’s response to DHA with a 22-carbon chain whether β1 or β4 is absent or present. However, the responses to α-linolenic acid, an omegea-3 fatty acid with an 18-carbon chain, and to arachidonic acid, an omega-6 fatty acid with a 20-carbon chain, remain unaffected by the mutation. Y318 in the S6 segment of hSlo1 is thus an important determinant of the electrophysiological response of the channel to DHA. Furthermore, the mutation Y318S may prove to be useful in dissecting out the complex lipid-mediated modulation of Slo1 BK channels.

Transduction of Voltage and Ca2+Signals by Slo1 BK Channels

Large-conductance Ca2+ -and voltage-gated K+ channels are activated by an increase in intracellular Ca2+ concentration and/or depolarization. The channel activation mechanism is well described by an allosteric model encompassing the gate, voltage sensors, and Ca2+ sensors, and the model is an excellent framework to understand the influences of auxiliary β and γ subunits and regulatory factors such as Mg2+. Recent advances permit elucidation of structural correlates of the biophysical mechanism.

A non-cardiomyocyte autonomous mechanism of cardioprotection involving the SLO1 BK channel

Opening of BK-type Ca2+ activated K+ channels protects the heart against ischemia-reperfusion (IR) injury. However, the location of BK channels responsible for cardioprotection is debated. Herein we confirmed that openers of the SLO1 BK channel, NS1619 and NS11021, were protective in a mouse perfused heart model of IR injury. As anticipated, deletion of the Slo1 gene blocked this protection. However, in an isolated cardiomyocyte model of IR injury, protection by NS1619 and NS11021 was insensitive to Slo1 deletion. These data suggest that protection in intact hearts occurs by a non-cardiomyocyte autonomous, SLO1-dependent, mechanism. In this regard, an in-situ assay of intrinsic cardiac neuronal function (tachycardic response to nicotine) revealed that NS1619 preserved cardiac neurons following IR injury. Furthermore, blockade of synaptic transmission by hexamethonium suppressed cardioprotection by NS1619 in intact hearts. These results suggest that opening SLO1 protects the heart during IR injury, via a mechanism that involves intrinsic cardiac neurons. Cardiac neuronal ion channels may be useful therapeutic targets for eliciting cardioprotection.

Omega-3 fatty acids lower blood pressure by directly activating large-conductance Ca 2+ -dependent K + channels

Long-chain polyunsaturated omega-3 fatty acids such as docosahexaenoic acid (DHA), found abundantly in oily fish, may have diverse health-promoting effects, potentially protecting the immune, nervous, and cardiovascular systems. However, the mechanisms underlying the purported health-promoting effects of DHA remain largely unclear, in part because molecular signaling pathways and effectors of DHA are only beginning to be revealed. In vascular smooth muscle cells, large-conductance Ca(2+)- and voltage-activated K(+) (BK) channels provide a critical vasodilatory influence. We report here that DHA with an EC50 of ∼500 nM rapidly and reversibly activates BK channels composed of the pore-forming Slo1 subunit and the auxiliary subunit β1, increasing currents by up to ∼20-fold. The DHA action is observed in cell-free patches and does not require voltage-sensor activation or Ca(2+) binding but involves destabilization of the closed conformation of the ion conduction gate. DHA lowers blood pressure in anesthetized wild-type but not in Slo1 knockout mice. DHA ethyl ester, contained in dietary supplements, fails to activate BK channels and antagonizes the stimulatory effect of DHA. Slo1 BK channels are thus receptors for long-chain omega-3 fatty acids, and these fatty acids--unlike their ethyl ester derivatives--activate the channels and lower blood pressure. This finding has practical implications for the use of omega-3 fatty acids as nutraceuticals for the general public and also for the critically ill receiving omega-3-enriched formulas.

Differential Effects of PIP2 on Slo1 BK Channels with Different β Subunits

Phosphatidylinositol 4, 5-bisphosphate (PIP2) regulates numerous ion channels, including large-conductance Ca2+- and voltage-dependent K+ (BK) channels (Vaithianathan et al. 2008, J Gen Physiol 132:13-28). We examined the molecular and biophysical mechanisms of the current-enhancing effect of PIP2 on human Slo1 BK channels with different auxiliary subunits heterologously expressed in HEK cells. In the absence of auxiliary subunits, PIP2 (10 μM) isolated from bovine brain applied to the cytoplasmic side inhibited currents through Slo1 channels recorded without Ca2+ by shifting GV curve by 17.8±1.7 mV and decelerated the activation kinetics at positive voltages. In contrast, PIP2 markedly increased currents through Slo1+β1 and Slo1+β4 channels by shifting the GV curves by −45.5±2.2 mV and −34.0±2.9 mV, respectively. PIP2 accelerated the macroscopic activation kinetics at positive voltages and decelerated the macroscopic deactivation kinetics at negative voltages. The current-enhancing effect of PIP2 on Slo1+β1 was observed at negative voltages and also in 100 μM Ca2+. For Slo1+β2 with a deletion in the β2 N terminus to remove inactivation (Δ2-32) the GV shift by PIP2 was significantly smaller (−28.9±2.3 mV) than that observed with Slo1+β1. PIP2 had no effect on the deactivation kinetics of Slo1+β2Δ2-32 channels. Measurements using chimeric β1-β2 subunits show that the second transmembrane domain and the C terminus of β1 are important for the large electrophysiological changes in Slo1+β1 channels caused by PIP2. Supported in part through the NIH, DFG HE 2993/8, and Shanghai Science and Technology Commission.

Omega-3 Fatty Acids Activate Slo1 BK Channels and Lower Blood Pressure

Long-chain omega-3 fatty acids such as docosahexaenoic acid (DHA) found in oily fish may offer various health benefits but the underling mechanisms are only poorly understood. In vascular smooth muscle cells, large-conductance Ca2+- and voltage-dependent K+ (Slo1 BK) channels provide a vasodilatory influence. We found that DHA with EC50 of ∼500 nM directly and reversibly activates BK channels composed of the pore-forming Slo1 subunit and the auxiliary subunit β1 in excised-patches, increasing currents by up to ∼20-fold. The DHA action does not require voltage-sensor activation or Ca2+ binding but depends on an electrostatic interaction within 1 or 4. DHA acutely lowers blood pressure in anesthetized wild-type but not in Slo1 knockout mice. DHA ethyl ester (DHA EE), found in dietary supplements, fails to activate BK channels and antagonizes the stimulatory effect of DHA. On an equimolar basis, the stimulatory effect DHA on Slo+1 channels was greater than that of eicosapentaenoic acid, alpha-linolenic acid, arachidonic acid, or linoleic acid. Slo1 BK channels are thus receptors for long-chain omega-3 fatty acids that, unlike their ethyl ester derivatives, activate the channels and lower blood pressure. Supported in part through the NIH, DFG FOR 1738, and Shanghai Science and Technology Commission.

Molecular mechanism of pharmacological activation of BK channels

Large-conductance voltage- and Ca(2+)-activated K(+) (Slo1 BK) channels serve numerous cellular functions, and their dysregulation is implicated in various diseases. Drugs activating BK channels therefore bear substantial therapeutic potential, but their deployment has been hindered in part because the mode of action remains obscure. Here we provide mechanistic insight into how the dehydroabietic acid derivative Cym04 activates BK channels. As a representative of NS1619-like BK openers, Cym04 reversibly left-shifts the half-activation voltage of Slo1 BK channels. Using an established allosteric BK gating model, the Cym04 effect can be simulated by a shift of the voltage sensor and the ion conduction gate equilibria toward the activated and open state, respectively. BK activation by Cym04 occurs in a splice variant-specific manner; it does not occur in such Slo1 BK channels using an alternative neuronal exon 9, which codes for the linker connecting the transmembrane segment S6 and the cytosolic RCK1 domain--the S6/RCK linker. In addition, Cym04 does not affect Slo1 BK channels with a two-residue deletion within this linker. Mutagenesis and model-based gating analysis revealed that BK openers, such as Cym04 and NS1619 but not mallotoxin, activate BK channels by functionally interacting with the S6/RCK linker, mimicking site-specific shortening of this purported passive spring, which transmits force from the cytosolic gating ring structure to open the channel's gate.

Metal-driven Operation of the Human Large-conductance Voltage- and Ca2+-dependent Potassium Channel (BK) Gating Ring Apparatus

Large-conductance voltage- and Ca(2+)-dependent K(+) (BK, also known as MaxiK) channels are homo-tetrameric proteins with a broad expression pattern that potently regulate cellular excitability and Ca(2+) homeostasis. Their activation results from the complex synergy between the transmembrane voltage sensors and a large (>300 kDa) C-terminal, cytoplasmic complex (the "gating ring"), which confers sensitivity to intracellular Ca(2+) and other ligands. However, the molecular and biophysical operation of the gating ring remains unclear. We have used spectroscopic and particle-scale optical approaches to probe the metal-sensing properties of the human BK gating ring under physiologically relevant conditions. This functional molecular sensor undergoes Ca(2+)- and Mg(2+)-dependent conformational changes at physiologically relevant concentrations, detected by time-resolved and steady-state fluorescence spectroscopy. The lack of detectable Ba(2+)-evoked structural changes defined the metal selectivity of the gating ring. Neutralization of a high-affinity Ca(2+)-binding site (the "calcium bowl") reduced the Ca(2+) and abolished the Mg(2+) dependence of structural rearrangements. In congruence with electrophysiological investigations, these findings provide biochemical evidence that the gating ring possesses an additional high-affinity Ca(2+)-binding site and that Mg(2+) can bind to the calcium bowl with less affinity than Ca(2+). Dynamic light scattering analysis revealed a reversible Ca(2+)-dependent decrease of the hydrodynamic radius of the gating ring, consistent with a more compact overall shape. These structural changes, resolved under physiologically relevant conditions, likely represent the molecular transitions that initiate the ligand-induced activation of the human BK channel.