Ball-and-chain inactivation in a calcium-gated potassium channel.

Inactivation is the process by which ion channels terminate ion flux through their pores while the opening stimulus is still present1. In neurons, inactivation of both sodium and potassium channels is crucial for the generation of action potentials and regulation of firing frequency1,2. A cytoplasmic domain of either the channel or an accessory subunit is thought to plug the open pore to inactivate the channel via a 'ball-and-chain' mechanism3-7. Here we use cryo-electron microscopy to identify the molecular gating mechanism in calcium-activated potassium channels by obtaining structures of the MthK channel from Methanobacterium thermoautotrophicum-a purely calcium-gated and inactivating channel-in a lipid environment. In the absence of Ca2+, we obtained a single structure in a closed state, which was shown by atomistic simulations to be highly flexible in lipid bilayers at ambient temperature, with large rocking motions of the gating ring and bending of pore-lining helices. In Ca2+-bound conditions, we obtained several structures, including multiple open-inactivated conformations, further indication of a highly dynamic protein. These different channel conformations are distinguished by rocking of the gating rings with respect to the transmembrane region, indicating symmetry breakage across the channel. Furthermore, in all conformations displaying open channel pores, the N terminus of one subunit of the channel tetramer sticks into the pore and plugs it, with free energy simulations showing that this is a strong interaction. Deletion of this N terminus leads to functionally non-inactivating channels and structures of open states without a pore plug, indicating that this previously unresolved N-terminal peptide is responsible for a ball-and-chain inactivation mechanism.

Opening of Intermediate Conductance Ca2+-Activated K+ Channels in C2C12 Skeletal Muscle Cells Increases the Myotube Diameter via the Akt/Mammalian Target of Rapamycin Pathway.

The activation of potassium channels and the ensuing hyperpolarization in skeletal myoblasts are essential for myogenic differentiation. However, the effects of K+ channel opening in myoblasts on skeletal muscle mass are unclear. Our previous study revealed that pharmacological activation of intermediate conductance Ca2+-activated K+ channels (IKCa channels) increases myotube formation. In this study, we investigated the effects of 5,6-dichloro-1-ethyl-1,3-dihydro-2H-benzimidazol-2-one (DCEBIO), a Ca2+-activated K+ channel opener, on the mass of skeletal muscle. Application of DCEBIO to C2C12 cells during myogenesis increased the diameter of C2C12 myotubes in a concentration-dependent manner. This DCEBIO-induced hypertrophy was abolished by gene silencing of IKCa channels. However, it was resistant to 1 µM but sensitive to 10 µM TRAM-34, a specific IKCa channel blocker. Furthermore, DCEBIO reduced the mitochondrial membrane potential by opening IKCa channels. Therefore, DCEBIO should increase myotube mass by opening of IKCa channels distributed in mitochondria. Pharmacological studies revealed that mitochondrial reactive oxygen species (mitoROS), Akt, and mammalian target of rapamycin (mTOR) are involved in DCEBIO-induced myotube hypertrophy. An additional study demonstrated that DCEBIO-induced muscle hypertrophic effects are only observed when applied in the early stage of myogenic differentiation. In an in vitro myotube inflammatory atrophy experiment, DCEBIO attenuated the reduction of myotube diameter induced by endotoxin. Thus, we concluded that DCEBIO increases muscle mass by activating the IKCa channel/mitoROS/Akt/mTOR pathway. Our study suggests the potential of DCEBIO in the treatment of muscle wasting diseases. SIGNIFICANCE STATEMENT: Our study shows that 5,6-dichloro-1-ethyl-1,3-dihydro-2H-benzimidazol-2-one (DCEBIO), a small molecule opener of Ca2+-activated K+ channel, increased muscle diameter via the mitochondrial reactive oxygen species/Akt/mammalian target of rapamycin pathway. And DCEBIO overwhelms C2C12 myotube atrophy induced by endotoxin challenge. Our report should inform novel role of K+ channel in muscle development and novel usage of K+ channel opener such as for the treatment of muscle wasting diseases.

Dynamic regulation of ß1 subunit trafficking controls vascular contractility.

Ion channels composed of pore-forming and auxiliary subunits control physiological functions in virtually all cell types. A conventional view is that channels assemble with their auxiliary subunits before anterograde plasma membrane trafficking of the protein complex. Whether the multisubunit composition of surface channels is fixed following protein synthesis or flexible and open to acute and, potentially, rapid modulation to control activity and cellular excitability is unclear. Arterial smooth muscle cells (myocytes) express large-conductance Ca(2+)-activated potassium (BK) channel α and auxiliary ß1 subunits that are functionally significant modulators of arterial contractility. Here, we show that native BKα subunits are primarily (∼95%) plasma membrane-localized in human and rat arterial myocytes. In contrast, only a small fraction (∼10%) of total ß1 subunits are located at the cell surface. Immunofluorescence resonance energy transfer microscopy demonstrated that intracellular ß1 subunits are stored within Rab11A-postive recycling endosomes. Nitric oxide (NO), acting via cGMP-dependent protein kinase, and cAMP-dependent pathways stimulated rapid (≤1 min) anterograde trafficking of ß1 subunit-containing recycling endosomes, which increased surface ß1 almost threefold. These ß1 subunits associated with surface-resident BKα proteins, elevating channel Ca(2+) sensitivity and activity. Our data also show that rapid ß1 subunit anterograde trafficking is the primary mechanism by which NO activates myocyte BK channels and induces vasodilation. In summary, we show that rapid ß1 subunit surface trafficking controls functional BK channel activity in arterial myocytes and vascular contractility. Conceivably, regulated auxiliary subunit trafficking may control ion channel activity in a wide variety of cell types.

Functional characterization and determination of the physiological role of a calcium-dependent potassium channel from cyanobacteria.

Despite the important achievement of the high-resolution structures of several prokaryotic channels, current understanding of their physiological roles in bacteria themselves is still far from complete. We have identified a putative two transmembrane domain-containing channel, SynCaK, in the genome of the freshwater cyanobacterium Synechocystis sp. PCC 6803, a model photosynthetic organism. SynCaK displays significant sequence homology to MthK, a calcium-dependent potassium channel isolated from Methanobacterium thermoautotrophicum. Expression of SynCaK in fusion with enhanced GFP in mammalian Chinese hamster ovary cells' plasma membrane gave rise to a calcium-activated, potassium-selective activity in patch clamp experiments. In cyanobacteria, Western blotting of isolated membrane fractions located SynCaK mainly to the plasma membrane. To understand its physiological function, a SynCaK-deficient mutant of Synechocystis sp. PCC 6803, ΔSynCaK, has been obtained. Although the potassium content in the mutant organisms was comparable to that observed in the wild type, ΔSynCaK was characterized by a depolarized resting membrane potential, as determined by a potential-sensitive fluorescent probe. Growth of the mutant under various conditions revealed that lack of SynCaK does not impair growth under osmotic or salt stress and that SynCaK is not involved in the regulation of photosynthesis. Instead, its lack conferred an increased resistance to the heavy metal zinc, an environmental pollutant. A similar result was obtained using barium, a general potassium channel inhibitor that also caused depolarization. Our findings thus indicate that SynCaK is a functional channel and identify the physiological consequences of its deletion in cyanobacteria.

Operation of the voltage sensor of a human voltage- and Ca2+-activated K+ channel.

Voltage sensor domains (VSDs) are structurally and functionally conserved protein modules that consist of four transmembrane segments (S1-S4) and confer voltage sensitivity to many ion channels. Depolarization is sensed by VSD-charged residues residing in the membrane field, inducing VSD activation that facilitates channel gating. S4 is typically thought to be the principal functional component of the VSD because it carries, in most channels, a large portion of the VSD gating charge. The VSDs of large-conductance, voltage- and Ca(2+)-activated K(+) channels are peculiar in that more gating charge is carried by transmembrane segments other than S4. Considering its "decentralized" distribution of voltage-sensing residues, we probed the BK(Ca) VSD for evidence of cooperativity between charge-carrying segments S2 and S4. We achieved this by optically tracking their activation by using voltage clamp fluorometry, in channels with intact voltage sensors and charge-neutralized mutants. The results from these experiments indicate that S2 and S4 possess distinct voltage dependence, but functionally interact, such that the effective valence of one segment is affected by charge neutralization in the other. Statistical-mechanical modeling of the experimental findings using allosteric interactions demonstrates two mechanisms (mechanical coupling and dynamic focusing of the membrane electric field) that are compatible with the observed cross-segment effects of charge neutralization.

Involvement of dominant-negative spliced variants of the intermediate conductance Ca2+-activated K+ channel, K(Ca)3.1, in immune function of lymphoid cells.

The intermediate conductance Ca(2+)-activated K(+) channel (IK(Ca) channel) encoded by K(Ca)3.1 is responsible for the control of proliferation and differentiation in various types of cells. We identified novel spliced variants of K(Ca)3.1 (human (h) K(Ca)3.1b) from the human thymus, which were lacking the N-terminal domains of the original hK(Ca)3.1a as a result of alternative splicing events. hK(Ca)3.1b was significantly expressed in human lymphoid tissues. Western blot analysis showed that hK(Ca)3.1a proteins were mainly expressed in the plasma membrane fraction, whereas hK(Ca)3.1b was in the cytoplasmic fraction. We also identified a similar N terminus lacking K(Ca)3.1 variants from mice and rat lymphoid tissues (mK(Ca)3.1b and rK(Ca)3.1b). In the HEK293 heterologous expression system, the cellular distribution of cyan fluorescent protein-tagged hK(Ca)3.1a and/or YFP-tagged hK(Ca)3.1b isoforms showed that hK(Ca)3.1b suppressed the localization of hK(Ca)3.1a to the plasma membrane. In the Xenopus oocyte translation system, co-expression of hK(Ca)3.1b with hK(Ca)3.1a suppressed IK(Ca) channel activity of hK(Ca)3.1a in a dominant-negative manner. In addition, this study indicated that up-regulation of mK(Ca)3.1b in mouse thymocytes differentiated CD4(+)CD8(+) phenotype thymocytes into CD4(-)CD8(-) ones and suppressed concanavalin-A-stimulated thymocyte growth by down-regulation of mIL-2 transcripts. Anti-proliferative effects and down-regulation of mIL-2 transcripts were also observed in mK(Ca)3.1b-overexpressing mouse thymocytes. These suggest that the N-terminal domain of K(Ca)3.1 is critical for channel trafficking to the plasma membrane and that the fine-tuning of IK(Ca) channel activity modulated through alternative splicing events may be related to the control in physiological and pathophysiological conditions in T-lymphocytes.

Zn2+ activates large conductance Ca2+-activated K+ channel via an intracellular domain.

Zinc is an essential trace element and plays crucial roles in normal development, often as an integral structural component of transcription factors and enzymes. Recent evidence suggests that intracellular Zn(2+) functions as a signaling molecule, mediating a variety of important physiological phenomena. However, the immediate effectors of intracellular Zn(2+) signaling are not well known. We show here that intracellular Zn(2+) potently and reversibly activates large-conductance voltage- and Ca(2+)-activated Slo1 K(+) (BK) channels. The full effect of Zn(2+) requires His(365) in the RCK1 (regulator of conductance for K(+)) domain of the channel. Furthermore, mutation of two nearby acidic residues, Asp(367) and Glu(399), also reduced activation of the channel by Zn(2+), suggesting a possible structural arrangement for Zn(2+) binding by the aforementioned residues. Extracellular Zn(2+) activated Slo1 BK channels when coexpressed with Zn(2+)-permeable TRPM7 (transient receptor potential melastatin 7) channels. The results thus demonstrate that Slo1 BK channels represent a positive and direct effector of Zn(2+) signaling and may participate in sculpting cellular response to an increase in intracellular Zn(2+) concentration.

Multiple palmitoyltransferases are required for palmitoylation-dependent regulation of large conductance calcium- and voltage-activated potassium channels.

Palmitoylation is emerging as an important and dynamic regulator of ion channel function; however, the specificity with which the large family of acyl palmitoyltransferases (zinc finger Asp-His-His-Cys type-containing acyl palmitoyltransferase (DHHCs)) control channel palmitoylation is poorly understood. We have previously demonstrated that the alternatively spliced stress-regulated exon (STREX) variant of the intracellular C-terminal domain of the large conductance calcium- and voltage-activated potassium (BK) channels is palmitoylated and targets the STREX domain to the plasma membrane. Using a combined imaging, biochemical, and functional approach coupled with loss-of-function (small interfering RNA knockdown of endogenous DHHCs) and gain-of-function (overexpression of recombinant DHHCs) assays, we demonstrate that multiple DHHCs control palmitoylation of the C terminus of STREX channels, the association of the STREX domain with the plasma membrane, and functional channel regulation. Cysteine residues 12 and 13 within the STREX insert were the only endogenously palmitoylated residues in the entire C terminus of the STREX channel. Palmitoylation of this dicysteine motif was controlled by DHHCs 3, 5, 7, 9, and 17, although DHHC17 showed the greatest specificity for this site upon overexpression of the cognate DHHC. DHHCs that palmitoylated the channel also co-assembled with the channel in co-immunoprecipitation experiments, and knockdown of any of these DHHCs blocked regulation of the channel by protein kinase A-dependent phosphorylation. Taken together our data reveal that a subset of DHHCs controls STREX palmitoylation and function and suggest that DHHC17 may preferentially target cysteine-rich domains. Finally, our approach may prove useful in elucidating the specificity of DHHC palmitoylation of intracellular domains of other ion channels and transmembrane proteins.

Optogenetic photochemical control of designer K+ channels in mammalian neurons.

Currently available optogenetic tools, including microbial light-activated ion channels and transporters, are transforming systems neuroscience by enabling precise remote control of neuronal firing, but they tell us little about the role of indigenous ion channels in controlling neuronal function. Here, we employ a chemical-genetic strategy to engineer light sensitivity into several mammalian K(+) channels that have different gating and modulation properties. These channels provide the means for photoregulating diverse electrophysiological functions. Photosensitivity is conferred on a channel by a tethered ligand photoswitch that contains a cysteine-reactive maleimide (M), a photoisomerizable azobenzene (A), and a quaternary ammonium (Q), a K(+) channel pore blocker. Using mutagenesis, we identify the optimal extracellular cysteine attachment site where MAQ conjugation results in pore blockade when the azobenzene moiety is in the trans but not cis configuration. With this strategy, we have conferred photosensitivity on channels containing Kv1.3 subunits (which control axonal action potential repolarization), Kv3.1 subunits (which contribute to rapid-firing properties of brain neurons), Kv7.2 subunits (which underlie "M-current"), and SK2 subunits (which are Ca(2+)-activated K(+) channels that contribute to synaptic responses). These light-regulated channels may be overexpressed in genetically targeted neurons or substituted for native channels with gene knockin technology to enable precise optopharmacological manipulation of channel function.

Ion-channel modulators: more diversity than previously thought.

Ion-channel function can be modified in various ways. For example, numerous studies have shown that currents through voltage-gated ion channels are affected by pore block or modification of voltage dependence of activation/inactivation. Recent experiments performed on various ion channels show that allosteric modulation is an important mechanism for affecting channel function. For instance, in K(Ca)2 (formerly SK) channels, the prototypic "blocker" apamin prevents conduction by an allosteric mechanism, while TRPV1 channels are prevented from closing by a tarantula toxin, DkTx, through an interaction with residues located away from the selectivity filter. The recent evidence, therefore, suggests that in several ion channels, the region around the outer mouth of the pore is rich in binding sites and could be exploited therapeutically. These discoveries also suggest that the pharmacological vocabulary should be adapted to define these various actions.

The role of BKCa channels on hyperpolarization mediated by hyperosmolarity in human articular chondrocytes.

Chondrocytes, the only cell in cartilage, are subjected to hyperosmotic challenges continuously since extracellular osmolarity in articular cartilage increases in response to mechanical loads during joint movement. Hyperosmolarity can affect membrane transport, and it is possible that load modulates matrix synthesis through alterations in intracellular composition. In the present study, the effects of hyperosmotic challenges were evaluated using the whole-cell patch clamp technique, whole cell mode on freshly isolated human and bovine articular chondrocytes. In human chondrocytes, hypertonicity induced the activation of outward Ca(2+)-sensitive K(+) currents, which were inhibited by iberiotoxin and TEA-Cl. The current induced by hypertonic switching (osmolarity from 300 to 400 mOsm/l) caused cell hyperpolarization (from -39 mV to -70 mV) with a reversal potential of -96 ± 7 mV. These results suggest a role for Ca(2+)-activated K(+) channels in human articular chondrocytes, leading to hyperpolarization as a consequence of K(+) efflux through these channels. These channels could have a role in the articular chondrocyte's response to a hyperosmotic challenge and matrix metabolism regulation by load.

Relationships between structural dynamics and functional kinetics in oligomeric membrane receptors.

Recent efforts to broaden understanding of the molecular mechanisms of membrane receptors in signal transduction make use of rate-equilibrium free-energy relationships (REFERs), previously applied to chemical reactions, enzyme kinetics, and protein folding. For oligomeric membrane receptors, we distinguish between a), the Leffler parameter alpha(L), to characterize the global transition state for the interconversion between conformations; and b), the Fersht parameter, varphi(F), to assign the degree of progression of individual residue positions at the transition state. For both alpha(L) and varphi(F), insights are achieved by using harmonic energy profiles to reflect the dynamic nature of proteins, as illustrated with single-channel results reported for normal and mutant nicotinic receptors. We also describe new applications of alpha(L) based on published results. For large-conductance calcium-activated potassium channels, data are satisfactorily fit with an alpha(L) value of 0.65, in accord with REFERs. In contrast, results reported for the flip conformational state of glycine and nicotinic receptors are in disaccord with REFERs, since they yield alpha(L) values outside the usual limits of 0-1. Concerning published varphi(F) values underlying the conformational wave hypothesis for nicotinic receptors, we note that interpretations may be complicated by variations in the width of harmonic energy profiles.

Structure of the MthK RCK in complex with cadmium.

RCK is a cytoplasmic regulatory domain of calcium-gated potassium channels. Binding of Ca(2+) by RCK leads to channel activation through a series of yet unknown conformational changes. Structures of the K(+) channel, MthK, and its cytoplasmic RCK domain revealed two binding sites for Ca(2+) per dimer. We determined the crystal structure of RCK in complex with Cd(2+) at 2.2A resolution. Cd(2+) activates MthK more efficiently, and binds at the same binding sites for Ca(2+) but with reduced coordination number. Two additional binding sites for Cd(2+) are found per dimer; one on the main Rossman-fold lobe, and the other on the small lobe of RCK. Using patch-clamp experiments, we demonstrate that Cd(2+) binding to these novel sites enhances activation by Cd(2+) and not by Ca(2+). The structure reveals a large negatively charged surface patch in the proximity of the Ca(2+)/Cd(2+) binding sites, charge neutralization of which appears to promote the channel open state.

Allosteric interactions and the modular nature of the voltage- and Ca2+-activated (BK) channel.

The high conductance voltage- and Ca(2+)-activated K(+) channel is one of the most broadly expressed channels in mammals. This channel is named BK for 'big K' because of its single-channel conductance that can be as large as 250 pS in 100 mm symmetrical K(+). BK channels increase their activity by membrane depolarization or an increase in cytosolic Ca(2+). One of the key features that defines the behaviour of BK channels is that neither Ca(2+) nor voltage is strictly necessary for channel activation. This and several other observations led to the idea that both Ca(2+) and voltage increase the open probability by an allosteric mechanism. In this type of mechanism, the processes of voltage sensor displacement, Ca(2+) binding and pore opening are independent equilibria that interact allosterically with each other. These allosteric interactions in BK channels reside in the structural characteristics of the BK channel in the sense that voltage and Ca(2+) sensors and the pore need to be contained in different structures or 'modules'. Through electrophysiological, mutagenesis, biochemical and fluorescence studies these modules have been identified and, more important, some of the interactions between them have been unveiled. In this review, we have covered the main advances achieved during the last few years in the elucidation of the structure of the BK channel and how this is related with its function as an allosteric protein.

Haem can bind to and inhibit mammalian calcium-dependent Slo1 BK channels.

Haem is essential for living organisms, functioning as a crucial element in the redox-sensitive reaction centre in haemproteins. During the biogenesis of these proteins, the haem cofactor is typically incorporated enzymatically into the haem pockets of the apo-haemprotein as the functionally indispensable prosthetic group. A class of ion channel, the large-conductance calcium-dependent Slo1 BK channels, possesses a conserved haem-binding sequence motif. Here we present electrophysiological and structural evidence showing that haem directly regulates cloned human Slo1 channels and wild-type BK channels in rat brain. Both oxidized and reduced haem binds to the hSlo1 channel protein and profoundly inhibits transmembrane K+ currents by decreasing the frequency of channel opening. This direct regulation of the BK channel identifies a previously unknown role of haem as an acute signalling molecule.

Cooperative transition between open and closed conformations in potassium channels.

Potassium (K+) ion channels switch between open and closed conformations. The nature of this important transition was revealed by comparing the X-ray crystal structures of the MthK channel from Methanobacterium thermoautotrophicum, obtained in its open conformation, and the KcsA channel from Streptomyces lividans, obtained in its closed conformation. We analyzed the dynamic characteristics and energetics of these homotetrameric structures in order to study the role of the intersubunit cooperativity in this transition. For this, elastic models and in silico alanine-scanning mutagenesis were used, respectively. Reassuringly, the calculations manifested motion from the open (closed) towards the closed (open) conformation. The calculations also revealed a network of dynamically and energetically coupled residues. Interestingly, the network suggests coupling between the selectivity filter and the gate, which are located at the two ends of the channel pore. Coupling between these two regions was not observed in calculations that were conducted with the monomer, which emphasizes the importance of the intersubunit interactions within the tetrameric structure for the cooperative gating behavior of the channel.

Reactive oxygen species impair Slo1 BK channel function by altering cysteine-mediated calcium sensing.

Vascular dysfunction is a hallmark of many diseases, including coronary heart disease, stroke and diabetes. The underlying mechanisms of these disorders, which are intimately associated with inflammation and oxidative stress caused by excess reactive oxygen species (ROS), have remained elusive. Here we report that ROS are powerful inhibitors of vascular smooth muscle calcium-dependent Slo1 BK or Maxi-K potassium channels, an important physiological determinant of vascular tone. By targeting a cysteine residue near the Ca(2+) bowl of the BK alpha subunit, H(2)O(2) virtually eliminates physiological activation of the channel, with an inhibitory potency comparable to a knockout of the auxiliary subunit BK beta 1. These results reveal a molecular structural basis for the vascular dysfunction involving oxidative stress and provide a solid rationale for a potential use of BK openers in the prevention and treatment of cardiovascular disorders.

Integrating spatiotemporal features of a ligand-regulated, multi-state allosteric protein.

Dynamic protein molecules are defined by their spatiotemporal characteristics and should thus be represented by models incoporating both characteritics. Structural biology enables determination of atomic structures of individual conformational states of a given protein. Obtaining the complementary temporal information of a given time resolution, which can be directly linked to the corresponding atomic structures, requires identifying at each time point the specific conformational state adopted by the protein. Here, we examine individual regulator of conductance to K+ (RCK) domains in the regulatory module of the MthK channel by monitoring in real time the orientation of an α-helix that is conformational-state-specific. The acquired dynamic information that specifies an RCK domain's multi-state conformational changes, combined with already available corresponding atomic structures, enables us to establish an experiment-based spatiotemporal representation of an RCK domain, and to deduce a quantitative mechanistic model of the channel.

Resolution of ångström-scale protein conformational changes by analyzing fluorescence anisotropy.

Conformational changes within typical protein molecules are rapid and small, making their quantitative resolution challenging. These changes generally involve rotational motions and may thus be resolved by determining changes in the orientation of a fluorescent label that assumes a unique orientation in each conformation. Here, by analyzing fluorescence intensities collected using a polarization microscope at a rate of 50 frames per second, we follow the changes of 10-16° in the orientation of a single bifunctional rhodamine molecule attached to a regulator of conductance to K+ (RCK) domain of the MthK channel, and thus, the transitions between its three conformational states, with effective standard deviation (σ) of 2-5°. Based on available crystal structures, the position of the fluorophore's center differs by 3.4-8.1 Å among the states. Thus, the present approach allows the resolution of protein conformational changes involving ångström-scale displacements.

Energetics of ångström-scale conformational changes in an RCK domain of the MthK K+ channel.

Allosteric proteins transition among different conformational states in a ligand-dependent manner. Upon resolution of a protein's individual states, one can determine the probabilities of these states, thereby dissecting the energetic mechanisms underlying their conformational changes. Here we examine individual regulator of conductance to K+ (RCK) domains that form the regulatory module of the Ca2+-activated MthK channel. Each domain adopts multiple conformational states differing on an ångström scale. The probabilities of these different states of the domain, assessed in different Ca2+ concentrations, allowed us to fully determine a six-state model that is minimally required to account for the energetic characteristics of the Ca2+-dependent conformational changes of an RCK domain. From the energetics of this domain, we deduced, in the framework of statistical mechanics, an analytic model that quantitatively predicts the experimentally observed Ca2+ dependence of the channel's open probability.