Gating Ring Research Articles

State-dependent FRET reports calcium- and voltage-dependent gating-ring motions in BK channels

Large-conductance voltage- and calcium-dependent potassium channels (BK, "Big K+") are important controllers of cell excitability. In the BK channel, a large C-terminal intracellular region containing a "gating-ring" structure has been proposed to transduce Ca(2+) binding into channel opening. Using patch-clamp fluorometry, we have investigated the calcium and voltage dependence of conformational changes of the gating-ring region of BK channels, while simultaneously monitoring channel conductance. Fluorescence resonance energy transfer (FRET) between fluorescent protein inserts indicates that Ca(2+) binding produces structural changes of the gating ring that are much larger than those predicted by current X-ray crystal structures of isolated gating rings.

Structural Studies of the Apo and Ca2+-Bound States of the Human BK (SLO1) Channel Gating Ring in Solution

The gating ring (GR) regulates the activity of large-conductance voltage- and Ca^(2+)-activated K^+ channels (BK) by interacting with intracellular signaling molecules. To understand the operation of this biological sensor under physiological conditions, we performed Small-Angle X-ray Scattering (SAXS) analysis, at beamline 4-2 at the Stanford Synchrotron Radiation laboratory. SAXS measurements of the purified GR were performed in the absence or in the presence of 35 μM free Ca^(2+), found to be a saturating concentration in previous work. The quality of the circularly-averaged scattering data was evaluated with Guinier analysis, while the ATSAS software suite was used to derive structural information. The radius of gyration (R_g) and maximum interparticle distance (D_(max)) of the apo GR were 48.65±1.372 A and 185 A, respectively. These values are comparable to data obtained from crystal structure of GR (3NAF), where the envelope R_g, calculated with CRYSOL, is 45.55 A, and its diameter 155.6 A. Ca^(2+)-bound GR shows a decrease in R_g to 42.77±1.058 A and D_(max) to 160 A, demonstrating the structural response of GR to Ca^(2+). Low-resolution structural models of the GR were generated from the experimental scattering pattern using DAMMIN. The Ca^(2+)-bound GR revealed notable changes in both flexible and assembly interfaces of the superstructure's constituent RCK1 (Regulator of Conductance for K^+) and RCK2 domains. Since the structural changes are resolved under physiologically-relevant conditions, we speculate that they represent the molecular transitions that initiate the Ca^(2+)-induced activation of human BK channels.

Structural Basis of Gating by RCK Domains

TrkH and its homologs TrkG and KtrB belong to a superfamily of K+ transport proteins that are required for growth of bacteria in low external K+ concentrations. The crystal structure of TrkH from Vibrio parahaemolyticus showed that TrkH forms a homodimer, and each protomer resembles a K+ channel with a unique gating mechanism. TrkH assembles with TrkA, a soluble protein comprising two Regulate-Conductance-of-K+, or RCK domains, which control the gating of certain K+ channels. In K+ channels, eight RCK domains form a four-fold symmetric gating ring that matches the four-fold symmetry of the channel. A dilation of the diameter of the gating ring directly translates into expansion of the pore-lining helices of the channel and hence opening of the permeation pathway. However, the gating ring expansion mechanism in K+ channels appears incompatible with the dimeric architecture and the different gating mechanism in TrkH. In addition, although TrkH resembles a K+ channel, its channel activity has never been demonstrated. We have recorded single-channel activity from TrkH in spheroplasts consistent with two partly coupled pores. We also found that channel activity is upregulated by ATP via TrkA. To understand how channel gating is regulated, we solved two structures of the TrkA tetrameric ring, one in complex with TrkH and one in isolation, in which the ring assumes two dramatically different conformations. The structures suggest a mechanism for how ATP increases the open probability of the TrkH ion channel by inducing conformational changes in TrkA.

Functional and structural analysis of the human SLO3 pH- and voltage-gated K + channel

The activation of eukaryotic SLO K(+) channels by intracellular cues, mediated by a cytoplasmic structure called the gating ring, is central to their physiological roles. SLO3 channels are exclusively expressed in mammalian sperm, where variations of intracellular pH are critical to cellular function. Previous studies primarily focused on the mouse SLO3 orthologue and revealed that, in murine sperm, SLO3 mediates a voltage- and alkalization-activated K(+) current essential to male fertility. Here we investigate the activation of the human SLO3 channel by intracellular pH at the functional and structural level. By using electrophysiology in a heterologous system, we show that human SLO3 opens upon intracellular pH increase and that its expression and functional properties are modulated by LRRC52, a testis-specific accessory subunit. We next present the crystal structure of the human SLO3 gating ring. Comparison with the known structures of the corresponding domain from SLO1, a Ca(2+)-activated homologue, suggests that the SLO3 gating ring structure may represent an open state. Together, these results present insights into the function of a protein expected to be critical for human reproduction and provide a framework to study the mechanism of pH gating in SLO3 channels.

Crystal Structure of a Ba2+-Bound Gating Ring Reveals Elementary Steps in RCK Domain Activation

RCK domains control activity of a variety of K(+) channels and transporters through binding of cytoplasmic ligands. To gain insight toward mechanisms of RCK domain activation, we solved the structure of the RCK domain from the Ca(2+)-gated K(+) channel, MthK, bound with Ba(2+), at 3.1Å resolution. The Ba(2+)-bound RCK domain was assembled as an octameric gating ring, as observed in structures of the full-length MthK channel, and shows Ba(2+) bound at several positions. One of the Ba(2+) sites, termed C1, overlaps with a known Ca(2+)-activation site, determined by residues D184 and E210. Functionally, Ba(2+) can activate reconstituted MthK channels as observed in electrophysiological recordings, whereas Mg(2+) (up to 100mM) was ineffective. Ba(2+) activation was abolished by the mutation D184N, suggesting that Ba(2+) activates primarily through the C1 site. Our results suggest a working hypothesis for a sequence of ligand-dependent conformational changes that may underlie RCK domain activation and channel gating.

The Contribution of RCK Domains to Human BK Channel Allosteric Activation

Large conductance voltage- and Ca(2+)-activated K(+) (BK) channels are potent regulators of cellular processes including neuronal firing, synaptic transmission, cochlear hair cell tuning, insulin release, and smooth muscle tone. Their unique activation pathway relies on structurally distinct regulatory domains including one transmembrane voltage-sensing domain (VSD) and two intracellular high affinity Ca(2+)-sensing sites per subunit (located in the RCK1 and RCK2 domains). Four pairs of RCK1 and RCK2 domains form a Ca(2+)-sensing apparatus known as the "gating ring." The allosteric interplay between voltage- and Ca(2+)-sensing apparati is a fundamental mechanism of BK channel function. Using voltage-clamp fluorometry and UV photolysis of intracellular caged Ca(2+), we optically resolved VSD activation prompted by Ca(2+) binding to the gating ring. The sudden increase of intracellular Ca(2+) concentration ([Ca(2+)](i)) induced a hyperpolarizing shift in the voltage dependence of both channel opening and VSD activation, reported by a fluorophore labeling position 202, located in the upper side of the S4 transmembrane segment. The neutralization of the Ca(2+) sensor located in the RCK2 domain abolished the effect of [Ca(2+)](i) increase on the VSD rearrangements. On the other hand, the mutation of RCK1 residues involved in Ca(2+) sensing did not prevent the effect of Ca(2+) release on the VSD, revealing a functionally distinct interaction between RCK1 and RCK2 and the VSD. A statistical-mechanical model quantifies the complex thermodynamics interplay between Ca(2+) association in two distinct sites, voltage sensor activation, and BK channel opening.

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.

Calcium and Heme Induce Distinct Conformational States of the Human BK (Slo1) Channel Gating Ring

The human BK channel gating ring (GR) is the site of action of numerous signaling molecules such as Ca2+ and heme, which ultimately modulate pore open probability. However, the precise nature of these structural rearrangements remains elusive. Recently, we demonstrated that heme-induced conformational changes in the GR are not additive to Ca2+-induced structural rearrangements (Yusifov et al., BPS meeting 2010), posing a question about the extent of similarity in the mechanical operation of the GR upon Ca2+ or heme binding. To address this question, we analyzed the conformational states of apo-GR and Ca2+- or heme-bound GR by characterizing dynamic fluorescence quenching by acrylamide, a quencher of the excited state of accessible native tryptophan (Trp) residues. The time-resolved Trp fluorescence (λex=296 nm, λem=340 nm) of apo-GR and ligand-bound GR with the progressive addition of acrylamide (up to 0.4 M) was recorded. Acrylamide addition resulted in the reduction of the mean Trp fluorescence lifetime (calculated from three-exponential iterative fittings) of apo-GR (τ0=2.74±0.05ns to τ=1.5±0.1ns), Ca2+-bound-GR (τ0=2.1±0.02ns to τ=1.27±0.021ns) and heme-bound-GR (τ0=2.1±0.1ns to τ=1.0±0.05ns). The acrylamide-Trp bimolecular quenching constants (kq) were extracted from Stern-Volmer plots of τ0/τ vs. [acrylamide]. Supplementation of saturating [Ca2+] (35 μM) or [heme] (700 nM) increased kq from 8.40×108 M−1s−1 (apo-GR) to 1.19×109 M−1s−1 and 1.95×109 M−1s−1, respectively. The increased efficiency of Trp quenching by acrylamide in the ligand-bound GR implied that native Trp residues were more accessible to the quencher, supporting ligand-induced conformational changes. Since different kq values were derived from the two ligands, it is likely that Ca2+ and heme binding induced the GR to adopt different conformational states. We propose that the two ligands can modulate BK open probability through distinct intramolecular signal transduction pathways.

Evidence for Lipid-Induced Structural Rearrangements in the Human BK (Slo1) Channel Gating Ring

Phosphoinositides, in particular, phosphatidylinositol 4,5-bisphosphate (PIP2), are known regulators of numerous ion channels and have been implicated in cellular processes such modulation of the actin cytoskeleton, cell migration and vesicle trafficking. Recently, it has been shown that PIP2 modulates BK channel activity through a direct interaction with its cytoplasmic domain (Vaithianathan et al., 2008). To understand the molecular nature of this modulation, we expressed and purified the region of the human BK channel C terminus (322IIE...ALK1005) corresponding to the Gating Ring (GR) apparatus, which includes the Regulators of K+ Conductance (RCK1 and RCK2) domains, and the RCK1-S6 linker. Using steady-state and time-resolved fluorescence spectroscopy, we have investigated the effects of the soluble PIP2 form (PIP2-diC4 and PIP2-diC8) on the conformational properties of GR in physiologically-relevant conditions. These lipids quenched the Tryptophan (Trp) fluorescence of GR in a dose-dependent and saturable fashion (Khalf = 21±0.88 μM; n = 2.3±0.094), suggesting PIP2-mediated conformational changes. PIP2 did not cause appreciable changes in the fluorescence intensity of a Trp analog (NATA, 5 μM). Similarly, a solution containing the purified RCK2 domain (665HDP...ALK1005) was largely insensitive to PIP2 up to 62 μM. The fluorescence lifetime of Trp residues was reduced by the addition of PIP2-diC8 (τavg from 2.3 ns to 1.5 ns, PIP2 62 μM), revealing the dynamic nature of PIP2 quenching of the Trp residues. Moreover, PIP2-diC8 significantly reduced the fluorescence intensity of GR complexed with ANS (8-Anilinonaphthalene-1-sulfonate), suggesting that the structural rearrangements caused by PIP2 may correlate with a decrease in the surface hydrophobicity of the GR. In summary, the BK GR appears to be sensitive to micromolar PIP2. The corresponding structural changes in this sensor may represent the elementary steps of the BK channel lipid modulation pathway.

State-Dependent FRET Reports Large Gating-Ring Motions in BK Channels

Large conductance voltage- and calcium-dependent potassium channels (BK channels) are key regulators of many important physiological processes and a key feature to their physiological role is that the channel's open probability is regulated both by changes in transmembrane voltage and by intracellular calcium concentration. The voltage sensor resides within the transmembrane region of the channel, while Ca2+ binding is sensed by a large C-terminal intracellular region, where eight Regulator of Conductance for K+ (RCK) domains form a “gating ring”. Calcium binding to this region reduces the energy required to open the channel, but the exact mechanism underlying this process is still uncertain. Structural studies using isolated gating rings from prokaryotic channels and a biochemical study of the isolated gating ring from the human channel suggest that Ca2+ binding expands the gating ring. The large movement of the gating ring would physically pull and open the gate located at the pore domain. In the present study we investigate the calcium and voltage-dependence of conformational changes in the intact human BK channel by patch-clamp recordings and simultaneous measurements of fluorescence energy transfer between CFP and YFP variants of the green fluorescent protein, inserted into three sites in the BK gating ring. Depending of the site studied, different movements are detected that differ in their Ca- and V-dependence. Here we show that Ca2+ binding produces surprisingly large structural changes that, contrary to current theories, are not obligatorily coupled to the opening of the pore and are not strictly cooperative. Instead, a mechanism such as the “flip” transitions that have been identified in pentameric neurotransmitter receptor-channels is operative.

Multiple Ca2+ Binding Sites in the RCK Domain Contribute to Gating in the MthK K+ Channel

MthK is a prokaryotic Ca2+-gated K+ channel that has yielded structural insight toward mechanisms of RCK domain controlled channel gating. In MthK, a “gating ring” of eight RCK domains regulates channel activation that is steeply dependent on [Ca2+], with a Hill coefficient >8. Using electrophysiology and X-ray crystallography, we show that each RCK domain contributes to three different regulatory Ca2+ binding sites, two of which are located at the interfaces between adjacent RCK domains. Charge-neutralizing mutations of individual Ca2+-coordinating sidechains at each of the three sites decrease Ca2+-sensitivity of the channel, shifting the EC50 for Ca2+-activation by up to 8.4-fold. Combined mutations at pairs of sites decreased the Ca2+-sensitivity of mutant channels in an approximately energetically additive manner, suggesting that Ca2+ binding at each site may act independent of one another to faciliate channel opening. Finally, a combined mutation at all three sites effectively eliminates Ca2+-dependent activation of the channel through very high [Ca2+]. The additional Ca2+ binding sites, resulting in a stoichiometry of 24 Ca2+ ions per channel, is consistent with the steep relation between [Ca2+] and MthK channel activity, and crystal structures of the MthK RCK domain in the presence of Ca2+ suggest points at which subunits may physically interact to facilitate channel opening.

Open structure of the Ca2+ gating ring in the high-conductance Ca2+-activated K+ channel.

High-conductance voltage- and Ca(2+)-activated K(+) channels function in many physiological processes that link cell membrane voltage and intracellular Ca(2+) concentration, including neuronal electrical activity, skeletal and smooth muscle contraction, and hair cell tuning. Like other voltage-dependent K(+) channels, Ca(2+)-activated K(+) channels open when the cell membrane depolarizes, but in contrast to other voltage-dependent K(+) channels, they also open when intracellular Ca(2+) concentrations rise. Channel opening by Ca(2+) is made possible by a structure called the gating ring, which is located in the cytoplasm. Recent structural studies have defined the Ca(2+)-free, closed, conformation of the gating ring, but the Ca(2+)-bound, open, conformation is not yet known. Here we present the Ca(2+)-bound conformation of the gating ring. This structure shows how one layer of the gating ring, in response to the binding of Ca(2+), opens like the petals of a flower. The degree to which it opens explains how Ca(2+) binding can open the transmembrane pore. These findings present a molecular basis for Ca(2+) activation of K(+) channels and suggest new possibilities for targeting the gating ring to treat conditions such as asthma and hypertension.

Structure and function of multiple Ca 2+ -binding sites in a K + channel regulator of K + conductance (RCK) domain

Regulator of K(+) conductance (RCK) domains control the activity of a variety of K(+) transporters and channels, including the human large conductance Ca(2+)-activated K(+) channel that is important for blood pressure regulation and control of neuronal firing, and MthK, a prokaryotic Ca(2+)-gated K(+) channel that has yielded structural insight toward mechanisms of RCK domain-controlled channel gating. In MthK, a gating ring of eight RCK domains regulates channel activation by Ca(2+). Here, using electrophysiology and X-ray crystallography, we show that each RCK domain contributes to three different regulatory Ca(2+)-binding sites, two of which are located at the interfaces between adjacent RCK domains. The additional Ca(2+)-binding sites, resulting in a stoichiometry of 24 Ca(2+) ions per channel, is consistent with the steep relation between [Ca(2+)] and MthK channel activity. Comparison of Ca(2+)-bound and unliganded RCK domains suggests a physical mechanism for Ca(2+)-dependent conformational changes that underlie gating in this class of channels.

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.

A Specific Two-pore Domain Potassium Channel Blocker Defines the Structure of the TASK-1 Open Pore

Two-pore domain potassium (K2P) channels play a key role in setting the membrane potential of excitable cells. Despite their role as putative targets for drugs and general anesthetics, little is known about the structure and the drug binding site of K2P channels. We describe A1899 as a potent and highly selective blocker of the K2P channel TASK-1. As A1899 acts as an open-channel blocker and binds to residues forming the wall of the central cavity, the drug was used to further our understanding of the channel pore. Using alanine mutagenesis screens, we have identified residues in both pore loops, the M2 and M4 segments, and the halothane response element to form the drug binding site of TASK-1. Our experimental data were used to validate a K2P open-pore homology model of TASK-1, providing structural insights for future rational design of drugs targeting K2P channels.

AlGaN/GaN C-HEMT structures for dynamic stress detection

In our work, we investigated the possibility of dynamic stress detection based on the piezoelectric polarization using AlGaN/GaN circular high electron mobility transistors (C-HEMTs). In our knowledge, stress sensors in that account are introduced for the first time. The sensor structures exhibit good linearity in the piezoelectric response under dynamic stress conditions. The measurements reveal excellent stress detection sensitivity that is independent on the measured frequency range. The sensitivity of the devices can be easily increased by increase of the area of the Schottky gate ring electrode. The further increase of the sensitivity can be tuned by an optimal selection of the DC drain and gate bias.

Calcium-Dependent Operation of the Human BK Channel Gating Ring Apparatus

The large cytoplasmic C-terminal domain (CTD) of the human BK channel forms a gating ring structure composed by two tandem RCK domains serving as a signal transducer for intracellular Ca2+ and other ligands. However, the mechanism of the gating ring's operation remains unknown. We have used steady-state and time-resolved spectroscopy in combination with dynamic light scattering to the Ca-induced conformational changes of the purified CTD of human BK channel. The CTD domain of the human BK channel assembles as a tetrameric gating ring structure (MW∼310 kDa) with a hydrodynamic radius (RH) ∼10.5 nm. In the presence of 35 μM free Ca2+, RH reversibly decreases to ∼7.5 nm. The modulation of the gating ring hydrodynamic shape suggests that it undergoes Ca2+-induced conformational transitions, which we further, characterized using time-correlated single-photon counting spectroscopy. Increasing free [Ca2+] up to 35 μM shortened the average Trp fluorescence lifetime (τavg) of the wild-type gating ring from ∼2.6ns to ∼1.9ns, while the neutralization of the high-affinity Ca-binding site within RCK2 (Ca bowl, D894-898N) attenuated the effect of Ca2+. Steady-state fluorescence analysis revealed that these ligand-dependent structural rearrangements of the gating ring possess strong divalent cation selectivity. The gating ring exhibited i) high-affinity Ca2+-binding (Khalf_1 ∼0.3μM and Khalf_2 ∼4μM); ii) significantly lowered affinity for Mg2+ (Khalf ∼200μM) and iii) no Ba2+ sensitivity up to 13mM, consistent with the lack of Ba2+-dependent BK channel activation. Interestingly, Ca2+ bowl neutralization eliminated Mg2+ sensing up to 12mM. In summary, under physiologically-relevant conditions, these ligand-induced conformational transitions are strongly ion-specific and associated with changes in the hydrodynamic properties of the BK gating ring and likely represent the ligand-induced molecular events underlying channel activation.

On the Properties of the RCK1 Domain of the Human BK (SLO1) Channel

In BK channels, four RCK1 and four RCK2 domains assemble into a cytosolic ligand-sensing superstructure known as the gating ring. While electrophysiological data suggest that both RCK1 and RCK2 contain high affinity Ca2+-sensing sites, recent crystallographic data (Wu et al., 2010) has revealed Ca2+ binding only within the RCK2 domain. Does the RCK1 domain bind Ca2+? What is the role of the high-affinity Ca2+ sensing residues D362/D367 (Xia, et al 2002) located in RCK1 domain? To address these questions, we expressed and purified the region of the human BK channel C-terminus corresponding to the amino acid sequence 322IIE¼HDP667. We have probed the structure and Ca2+ sensing properties of the RCK1 domain in solution, under physiologically relevant conditions. In addition to the α/β fold shared with its bacterial counterparts and human BK RCK2 domain, the RCK1 domain preferentially self-assembles into a homo-octameric structure. We recently reported that RCK1 undergoes Ca2+-dependent conformational changes similarly to the RCK2 domain (Yusifov et al., 2008 and 2010). The neutralization of residues D362/D367 altered the secondary and quaternary structure of the RCK1 domain and prevented Ca2+-induced structural transitions in RCK1. 45Ca2+ overlay assay suggested that the neutralization of D362/367 did not abolish the Ca2+-binding activity of RCK1 in the range of 2.1-115 µM free Ca2+. While it cannot be excluded that D362/D367 are elements of a Ca2+ binding site with multiple coordinating residues, our results favor the view that D362 /D367 have a predominantly structural role. Possibly, these mutations set RCK1 domains in a conformational state that hampers the propagation of structural changes caused by ligand binding.

HIGHLIGHTS FROM THE LITERATURE

HIGHLIGHTS FROM THE LITERATURE

Membrane stretch and cytoplasmic Ca 2+ independently modulate stretch-activated BK channel activity

Large conductance Ca 2+-activated K + (BK) channels are responsible for changes in chemical and physical signals such as Ca 2+, Mg 2+ and membrane potentials. Previously, we reported that a BK channel cloned from chick heart (SAKCaC) is activated by membrane stretch. Molecular cloning and subsequent functional characterization of SAKCaC have shown that both the membrane stretch and intracellular Ca 2+ signal allosterically regulate the channel activity via the linker of the gating ring complex. Here we investigate how these two gating principles interact with each other. We found that stretch force activated SAKCaC in the absence of cytoplasmic Ca 2+. Lack of Ca 2+ bowl (a calcium binding motif) in SAKCaC diminished the Ca 2+-dependent activation, but the mechanosensitivity of channel was intact. We also found that the abrogation of STREX (a proposed mechanosensing apparatus) in SAKCaC abolished the mechanosensitivity without altering the Ca 2+ sensitivity of channels. These observations indicate that membrane stretch and intracellular Ca 2+ could independently modulate SAKCaC activity.