Modeling subunit cooperativity in opening of tetrameric ion channels

Most potassium channels are tetramers of four homologous polypeptides (subunits). During channel gating, each subunit undergoes several conformational changes independent of the state of other subunits before reaching a permissive state, from which the channel can open. However, transition from the permissive states to the open state involves a concerted movement of all subunits. This cooperative transition must be included in Markov models of channel gating. Previously, it was implemented by considering all possible combinations of four subunit states in a much larger expanded model of channel states (e.g., 27,405 channel states versus 64 subunit states), which complicates modeling and is computationally intense, especially when accurate modeling requires a large number of subunit states. To overcome these complexities and retain the tetrameric molecular structure, a modeling approach was developed to incorporate the cooperative transition directly from the subunit models. In this approach, the open state is separated from the subunit models and represented by the net flux between the open state and the permissive states. Dynamic variations of the probability of state residencies computed using this direct approach and the expanded model were identical. Implementation of the direct approach is simple and its computational time is orders-of-magnitude shorter than the equivalent expanded model.     

How ATP inhibits the open K(ATP) channel

ATP-sensitive potassium (K(ATP)) channels are composed of four pore-forming Kir6.2 subunits and four regulatory SUR1 subunits. Binding of ATP to Kir6.2 leads to inhibition of channel activity. Because there are four subunits and thus four ATP-binding sites, four binding events are possible. ATP binds to both the open and closed states of the channel and produces a decrease in the mean open time, a reduction in the mean burst duration, and an increase in the frequency and duration of the interburst closed states. Here, we investigate the mechanism of interaction of ATP with the open state of the channel by analyzing the single-channel kinetics of concatenated Kir6.2 tetramers containing from zero to four mutated Kir6.2 subunits that possess an impaired ATP-binding site. We show that the ATP-dependent decrease in the mean burst duration is well described by a Monod-Wyman-Changeux model in which channel closing is produced by all four subunits acting in a single concerted step. The data are inconsistent with a Hodgkin-Huxley model (four independent steps) or a dimer model (two independent dimers). When the channel is open, ATP binds to a single ATP-binding site with a dissociation constant of 300 microM.     

Gated Access to the Pore of a P2X Receptor: STRUCTURAL IMPLICATIONS FOR CLOSED-OPEN TRANSITIONS*

P2X receptors are ligand-gated cation channels that transition from closed to open states upon binding ATP. The crystal structure of the closed zebrafish P2X4.1 receptor directly reveals that the ion-conducting pathway is formed by three transmembrane domain 2 (TM2) α-helices, each being provided by the three subunits of the trimer. However, the transitions in TM2 that accompany channel opening are incompletely understood and remain unresolved. In this study, we quantified gated access to Cd²⁺ at substituted cysteines in TM2 of P2X2 receptors in the open and closed states. Our data for the closed state are consistent with the zebrafish P2X4.1 structure, with isoleucines and threonines (Ile-332 and Thr-336) positioned one helical turn apart lining the channel wall on approach to the gate. Our data for the open state reveal gated access to deeper parts of the pore (Thr-339, Val-343, Asp-349, and Leu-353), suggesting the closed channel gate is between Thr-336 and Thr-339. We also found unexpected interactions between native Cys-348 and D349C that result in tight Cd²⁺ binding deep within the intracellular vestibule in the open state. Interpreted with a P2X2 receptor structural model of the closed state, our data suggest that the channel gate opens near Thr-336/Thr-339 and is accompanied by movement of the pore-lining regions, which narrow toward the cytosolic end of TM2 in the open state. Such transitions would relieve the barrier to ion flow and render the intracellular vestibule less splayed during channel opening in the presence of ATP.     

K channel subconductance levels result from heteromeric pore conformations

Voltage-gated K channels assemble from four identical subunits symmetrically arranged around a central permeation pathway. Each subunit harbors a voltage-sensing domain. The sigmoidal nature of the activation kinetics suggests that multiple sensors need to undergo a conformational change before the channel can open. Following activation, individual K channels alternate stochastically between two main permeation states, open and closed. This binary character of single channel behavior suggests the presence of a structure in the permeation pathway that can exist in only two conformations. However, single channel analysis of drk1 (K(v)2.1) K channels demonstrated the existence of four additional, intermediate conductance levels. These short-lived subconductance levels are visited when the channel gate moves between the closed and fully open state. We have proposed that these sublevels arise from transient heteromeric pore conformations, in which some, but not all, subunits are in the "open" state. A minimal model based on this hypothesis relates specific subconductance states with the number of activated subunits (Chapman et al., 1997). To stringently test this hypothesis, we constructed a tandem dimer that links two K channel subunits with different activation thresholds. Activation of this dimer by strong depolarizations resulted in the characteristic binary open-close behavior. However, depolarizations to membrane potentials in between the activation thresholds of the two parents elicited highly unusual single channel gating, displaying frequent visits to two subconductance levels. The voltage dependence and kinetics of the small and large sublevels associate them with the activation of one and two subunits, respectively. The data therefore support the hypothesis that subconductance levels result from heteromeric pore conformations. In this model, both sensor movement and channel opening have a subunit basis and these processes are allosterically coupled.     

The Mantel-Haenszel Procedure Revisited: Models and Generalizations

Several statistical methods have been developed for adjusting the Odds Ratio of the relation between two dichotomous variables X and Y for some confounders Z. With the exception of the Mantel-Haenszel method, commonly used methods, notably binary logistic regression, are not symmetrical in X and Y. The classical Mantel-Haenszel method however only works for confounders with a limited number of discrete strata, which limits its utility, and appears to have no basis in statistical models. Here we revisit the Mantel-Haenszel method and propose an extension to continuous and vector valued Z. The idea is to replace the observed cell entries in strata of the Mantel-Haenszel procedure by subject specific classification probabilities for the four possible values of (X,Y) predicted by a suitable statistical model. For situations where X and Y can be treated symmetrically we propose and explore the multinomial logistic model. Under the homogeneity hypothesis, which states that the odds ratio does not depend on Z, the logarithm of the odds ratio estimator can be expressed as a simple linear combination of three parameters of this model. Methods for testing the homogeneity hypothesis are proposed. The relationship between this method and binary logistic regression is explored. A numerical example using survey data is presented.     

Stoichiometry of Kir channels with phosphatidylinositol bisphosphate

Phosphatidylinositol bisphosphate (PIP2) is the most abundant phosphoinositide in the plasma membranes of cells and its interaction with many ion channel proteins has proven to be a critical factor enabling ion channel gating. All members of the inwardly rectifying potassium (Kir) channel family depend on PIP2 for their activity, displaying distinct affinities and stereospecificities of interaction with the phosphoinositide. Here, we explored the stoichiometry of Kir channels with PIP2. We first showed that PIP2 regulated the activity of Kir3.4 channels mainly by altering their bursting behavior. Detailed burst analysis indicates that the channels assumed up to four open states and a connectivity of four between open and closed states depending on the available PIP2 levels. Moreover, by controlling the number of PIP2-sensitive subunits in the stoichiometry of a tetrameric Kir2.1 channel, we showed that characteristic channel activity was obtained when at least two wild-type subunits were present. Our studies support a kinetic model for gating of Kir channels by PIP2, where each of the four open states corresponds to the channel activated by one to four PIP2 molecules.     

Stoichiometry of Kir channels with phosphatidylinositol bisphosphate

Phosphatidylinositol bisphosphate (PIP(2)) is the most abundant phosphoinositide in the plasma membrane of cells and its interaction with many ion channel proteins has proven to be a critical factor enabling ion channel gating. All members of the inwardly rectifying potassium (Kir) channel family depend on PIP(2) for their activity, displaying distinct affinities and stereospecificities of interaction with the phosphoinositide. Here, we explored the stoichiometry of Kir channels with PIP(2). We first showed that PIP(2) regulated the activity of Kir3.4 channels mainly by altering their bursting behavior. Detailed burst analysis indicates that the channels assumed up to four open states and a connectivity of four between open and closed states depending on the available PIP(2) levels. Moreover, by controlling the number of PIP(2)-sensitive subunits in the stoichiometry of a tetrameric Kir2.1 channel, we showed that characteristic channel activity was obtained when at least two wild-type subunits were present. Our studies support a kinetic model for gating of Kir channels by PIP(2), where each of the four open states corresponds to the channel activated by one to four PIP(2) molecules.     

Shaker potassium channel gating. III: Evaluation of kinetic models for activation

Predictions of different classes of gating models involving identical conformational changes in each of four subunits were compared to the gating behavior of Shaker potassium channels without N-type inactivation. Each model was tested to see if it could simulate the voltage dependence of the steady state open probability, and the kinetics of the single-channel currents, macroscopic ionic currents and macroscopic gating currents using a single set of parameters. Activation schemes based upon four identical single-step activation processes were found to be incompatible with the experimental results, as were those involving a concerted, opening transition. A model where the opening of the channel requires two conformational changes in each of the four subunits can adequately account for the steady state and kinetic behavior of the channel. In this model, the gating in each subunit is independent except for a stabilization of the open state when all four subunits are activated, and an unstable closed conformation that the channel enters after opening. A small amount of negative cooperativity between the subunits must be added to account quantitatively for the dependence of the activation time course on holding voltage.     

Blocking the gate to ligand entry in human hemoglobin

His(E7) to Trp replacements in HbA lead to markedly biphasic bimolecular CO rebinding after laser photolysis. For isolated mutant subunits, the fraction of fast phase increases with increasing [CO], suggesting a competition between binding to an open conformation with an empty E7 channel and relaxation to blocked or closed, slowly reacting states. The rate of conformational relaxation of the open state is ～18,000 s(-1) in α subunits and ～10-fold faster in β subunits, ～175,000 s(-1). Crystal structures were determined for tetrameric α(WT)β(Trp-63) HbCO, α(Trp-58)β(WT) deoxyHb, and Trp-64 deoxy- and CO-Mb as controls. In Trp-63(E7) βCO, the indole side chain is located in the solvent interface, blocking entry into the E7 channel. Similar blocked Trp-64(E7) conformations are observed in the mutant Mb crystal structures. In Trp-58(E7) deoxy-α subunits, the indole side chain fills both the channel and the distal pocket, forming a completely closed state. The bimolecular rate constant for CO binding, k'(CO), to the open conformations of both mutant Hb subunits is ～80-90 μm(-1) s(-1), whereas k'(CO) for the completely closed states is 1000-fold slower, ～0.08 μm(-1) s(-1). A transient intermediate with k'(CO) ≈ 0.7 μm(-1) s(-1) is observed after photolysis of Trp-63(E7) βCO subunits and indicates that the indole ring blocks the entrance to the E7 channel, as observed in the crystal structures of Trp(E7) deoxyMb and βCO subunits. Thus, either blocking or completely filling the E7 channel dramatically slows bimolecular binding, providing strong evidence that the E7 channel is the major pathway (≥90%) for ligand entry in human hemoglobin.     

Visible light induction of an electron paramagnetic resonance split signal in Photosystem II in the S(2) state reveals the importance of charges in the oxygen-evolving center during catalysis: a unifying model

Cryogenic illumination of Photosystem II (PSII) can lead to the trapping of the metastable radical Y(Z)(?), the radical form of the redox-active tyrosine residue D1-Tyr161 (known as Y(Z)). Magnetic interaction between this radical and the CaMn(4) cluster of PSII gives rise to so-called split electron paramagnetic resonance (EPR) signals with characteristics that are dependent on the S state. We report here the observation and characterization of a split EPR signal that can be directly induced from PSII centers in the S(2) state through visible light illumination at 10 K. We further show that the induction of this split signal takes place via a Mn-centered mechanism, in the same way as when using near-infrared light illumination [Koulougliotis, D., et al. (2003) Biochemistry 42, 3045-3053]. On the basis of interpretations of these results, and in combination with literature data for other split signals induced under a variety of conditions (temperature and light quality), we propose a unified model for the mechanisms of split signal induction across the four S states (S(0), S(1), S(2), and S(3)). At the heart of this model is the stability or instability of the Y(Z)(?)(D1-His190)(+) pair that would be formed during cryogenic oxidation of Y(Z). Furthermore, the model is closely related to the sequence of transfers of protons and electrons from the CaMn(4) cluster during the S cycle and further demonstrates the utility of the split signals in probing the immediate environment of the oxygen-evolving center in PSII.     

Molecular dynamics simulations of ethanol binding to the transmembrane domain of the glycine receptor: implications for the channel potentiation mechanism

The glycine receptor (GlyR) is potentiated by ethanol and other anesthetics. The potentiation mechanism at the molecular level is unknown and remains elusive, but mutagenic studies have shown that ethanol and other volatile anesthetics bind to a pocket between TM1, TM2, and TM3. The present study extends previous studies (Cheng et al., Proteins 2007;68:581-593) wherein we conducted homology modeling and molecular dynamics (MD) simulations to construct models of the homopentameric alpha1 subunits of the GlyR transmembrane domain in open and closed states. To understand the potentiation of GlyR by ethanol we compare the binding of ethanol molecules to the channel in these different states. We observe that ethanol stably resides inside solvent-accessible cavities found in the open state of GlyR that are formed by I229 (of TM1) in one subunit and S267 and A288 (of TM2 and TM3, respectively) in the adjacent subunit. The volume of these putative binding pockets is state-dependent. Selective binding to the open states of receptors has been proposed to explain the potentiating actions of this class of anesthetics. In accordance with this model, our MD simulations suggest that the potentiation of ethanol on GlyR may be effected through preferential binding of ethanol molecules to an inter-subunit binding pocket in the open state.     

An extracellular domain of the accessory β1 subunit is required for modulating BK channel voltage sensor and gate

A family of tissue-specific auxiliary β subunits modulates large conductance voltage- and calcium-activated potassium (BK) channel gating properties to suit their diverse functions. Paradoxically, β subunits both promote BK channel activation through a stabilization of voltage sensor activation and reduce BK channel openings through an increased energetic barrier of the closed-to-open transition. The molecular determinants underlying β subunit function, including the dual gating effects, remain unknown. In this study, we report the first identification of a β1 functional domain consisting of Y74, S104, Y105, and I106 residues located in the extracellular loop of β1. These amino acids reside within two regions of highest conservation among related β1, β2, and β4 subunits. Analysis in the context of the Horrigan-Aldrich gating model revealed that this domain functions to both promote voltage sensor activation and also reduce intrinsic gating. Free energy calculations suggest that the dual effects of the β1 Y74 and S104-I106 domains can be largely accounted for by a relative destabilization of channels in open states that have few voltage sensors activated. These results suggest a unique and novel mechanism for β subunit modulation of voltage-gated potassium channels wherein interactions between extracellular β subunit residues with the external portions of the gate and voltage sensor regulate channel opening.     

Naturally occurring mutations at the acetylcholine receptor binding site independently alter ACh binding and channel gating

By defining functional defects in a congenital myasthenic syndrome (CMS), we show that two mutant residues, located in a binding site region of the acetylcholine receptor (AChR) epsilon subunit, exert opposite effects on ACh binding and suppress channel gating. Single channel kinetic analysis reveals that the first mutation, epsilon N182Y, increases ACh affinity for receptors in the resting closed state, which promotes sequential occupancy of the binding sites and discloses rate constants for ACh occupancy of the nonmutant alphadelta site. Studies of the analogous mutation in the delta subunit, deltaN187Y, disclose rate constants for ACh occupancy of the nonmutant alpha epsilon site. The second CMS mutation, epsilon D175N, reduces ACh affinity for receptors in the resting closed state; occupancy of the mutant site still promotes gating because a large difference in affinity is maintained between closed and open states. epsilon D175N impairs overall gating, however, through an effect independent of ACh occupancy. When mapped on a structural model of the AChR binding site, epsilon N182Y localizes to the interface with the alpha subunit, and epsilon D175 to the entrance of the ACh binding cavity. Both epsilon N182Y and epsilon D175 show state specificity in affecting closed relative to desensitized state affinities, suggesting that the protein chain harboring epsilon N182 and epsilon D175 rearranges in the course of receptor desensitization. The overall results show that key residues at the ACh binding site differentially stabilize the agonist bound to closed, open and desensitized states, and provide a set point for gating of the channel.     

Mutations stabilizing an open conformation within the external region of the permeation pathway of the potassium channel KcsA

Four subunits of the bacterial Streptomyces lividans protein KcsA form a K+ channel which can be functionally reconstituted in vitro. Here we show that substitution of the tyrosine residue 82 by cysteine, valine or threonine, but not by glycine, led to functional channel types. Like the wild-type (WT) and an L81C channel, the mutant channels exhibit an internal pH-sensitive side and are cation selective. Based on the relative positions of the blocker tetraethylammonium within the electric field, the external entryways of the channels are concluded to have similar dimensions. For inward currents, the WT and the mutant channels vary in the occupancy of their subconductance states and concomitantly in their mean currents. Rectification properties are scarcely (L81C), little (Y82C) or considerably (Y82T and Y82V) altered. The data suggest that the amino acid type in position 82 stabilizes to varying degrees an open conformation within the external region of the permeation pathway.     

Modulation of gating currents of the Ca(v)3.1 calcium channel by alpha 2 delta 2 and gamma 5 subunits

Modulatory effects of auxiliary alpha(2)delta(2) and gamma(5) subunits on intramembrane charge movement originating from the expressed Ca(v)3.1 calcium channel were investigated. Inward current was blocked by 1mM La(3+). Voltage dependences of Q(on) and Q(off), kinetics of ON- and OFF-charge movement, and I(max)/Q(max) ratio were measured in the absence and the presence of an auxiliary subunit. The alpha(2)delta(2) subunit accelerated significantly both ON- and OFF-charge movement. I(max)/Q(max) ratio and Q(on)-V, Q(off)-V relations were not affected. Coexpression of the alpha(2)delta(2) subunit may accelerate channel transitions between individual closed states, but not the transition from the last closed channel state into an open state. Coexpression of the gamma(5) subunit accelerated the decay of the ON-charge transient and enhanced I(max)/Q(max) ratio. These effects suggest improvement of the coupling between the charge movement and the channel opening due to facilitation of transitions between individual closed states and the transition between the last closed state and an open state.     

Ligand-induced conformational change in the alpha7 nicotinic receptor ligand binding domain

Molecular dynamics simulations of a homology model of the ligand binding domain of the alpha7 nicotinic receptor are conducted with a range of bound ligands to induce different conformational states. Four simulations of 15 ns each are run with no ligand, antagonist d-tubocurarine (dTC), agonist acetylcholine (ACh), and agonist ACh with potentiator Ca(2+), to give insight into the conformations of the active and inactive states of the receptor and suggest the mechanism for conformational change. The main structural factor distinguishing the active and inactive states is that a more open, symmetric arrangement of the five subunits arises for the two agonist simulations, whereas a more closed and asymmetric arrangement results for the apo and dTC cases. Most of the difference arises in the lower portion of the ligand binding domain near its connection to the adjacent transmembrane domain. The transfer of the more open state to the transmembrane domain could then promote ion flow through the channel. Variation in how subunits pack together with no ligand bound appears to give rise to asymmetry in the apo case. The presence of dTC expands the receptor but induces rotations in alternate directions in adjacent subunits that lead to an asymmetric arrangement as in the apo case. Ca(2+) appears to promote a slightly greater expansion in the subunits than ACh alone by stabilizing the C-loop and ACh positions. Although the simulations are unlikely to be long enough to view the full conformational changes between open and closed states, a collection of different motions at a range of length scales are observed that are likely to participate in the conformational change.     

The role of subunit cooperativity on ryanodine receptor 2 calcium signaling

The ryanodine receptor type 2 (RyR2) is composed of four subunits that control calcium (Ca) release in cardiac cells. RyR2 serves primarily as a Ca sensor and can respond to rapid sub-millisecond pulses of Ca while remaining shut at resting concentrations. However, it is not known how the four subunits interact for the RyR2 to function as an effective Ca sensor. To address this question, and to understand the role of subunit cooperativity in Ca-mediated signal transduction, we have developed a computational model of the RyR2 composed of four interacting subunits. We first analyze the statistical properties of a single RyR2 tetramer, where each subunit can exist in a closed or open conformation. Our findings indicate that the number of subunits in the open state is a crucial parameter that dictates RyR2 kinetics. We find that three or four open subunits are required for the RyR2 to harness cooperative interactions to respond to sub-millisecond changes in Ca, while at the same time remaining shut at the resting Ca levels in the cardiac cell. If the required number of open subunits is lowered to one or two, the RyR2 cannot serve as a robust Ca sensor, as the large cooperativity required to stabilize the closed state prevents channel activation. Using this four-subunit model, we analyze the kinetics of Ca release from a RyR2 cluster. We show that the closure of a cluster of RyR2 channels is highly sensitive to the balance of cooperative interactions between closed and open subunits. Based on this result, we analyze how specific interactions between RyR2 subunits can induce persistent Ca leak from the sarcoplasmic reticulum (SR), which is believed to be arrhythmogenic. Thus, these results provide a framework to analyze how a pharmacologic or genetic modification of RyR2 subunit cooperativity can induce abnormal Ca cycling that can potentially lead to life-threatening arrhythmias.     

An NMDA Receptor Gating Mechanism Developed from MD Simulations Reveals Molecular Details Underlying Subunit-Specific Contributions

N-methyl-D-aspartate (NMDA) receptors are obligate heterotetrameric ligand-gated ion channels that play critical roles in learning and memory. Here, using targeted molecular dynamics simulations, we developed an atomistic model for the gating of the GluN1/GluN2A NMDA receptor. Upon agonist binding, lobe closure of the ligand-binding domain produced outward pulling of the M3-D2 linkers, leading to outward movements of the C-termini of the pore-lining M3 helices and opening of the channel. The GluN2A subunits, similar to the distal (B/D) subunits in the homotetrameric GluA2 α-amino-3-hydroxy-5-methyl-4-isoxazoleproprionate receptor, had greater M3 outward movements and thus contributed more to channel gating than the GluN1 subunits. Our gating model is validated by functional studies, including cysteine modification data indicating wider accessibility to the GluN1 M3 helices than to the GluN2A M3 helices from the lumen of the open channel, and reveals why the Lurcher mutation in GluN1 has a stronger ability in maintaining channel opening than the counterpart in GluN2A. The resulting structural model for the open state provides an explanation for the Ca²⁺ permeability of NMDA receptors, and the structural differences between the closed and open states form the basis for drug design.     

Two-dimensional probability density analysis of single channel currents from reconstituted acetylcholine receptors and sodium channels

Two-dimensional probability density analysis of single channel current recordings was applied to two purified channel proteins reconstituted in planar lipid bilayers: Torpedo acetylcholine receptors and voltage-sensitive sodium channels from rat brain. The information contained in the dynamic history of the gating process, i.e., the time sequence of opening and closing events was extracted from two-dimensional distributions of transitions between identifiable states. This approach allows one to identify kinetic models consistent with the observables. Gating of acetylcholine receptors expresses "memory" of the transition history: the receptor has two channel open (O) states; the residence time in each of them strongly depends on both the preceding open time and the intervening closed interval. Correspondingly, the residence time in the closed (C) states depends on both the preceding open time and the preceding closed time. This result confirms the scheme that considers, at least, two transition pathways between the open and closed states and extends the details of the model in that it defines that the short-lived open state is primarily entered from long-lived closed states while the long-lived open state is accessed mainly through short-lived closed states. Since ligand binding to the acetylcholine-binding sites is a reaction with channel closed states, we infer that the longest closed state (approximately 19 ms) is unliganded, the intermediate closed state (approximately 2 ms) is singly liganded and makes transitions to the short open state (approximately 0.5 ms) and the shortest closed state (approximately 0.4 ms) is doubly liganded and isomerizes to long open states (approximately 5 ms). This is the simplest interpretation consistent with available data. In contrast, sodium channels modified with batrachotoxin to eliminate inactivation show no correlation in the sequence of channel opening and closing events, i.e., have no memory of the transition history. This result is, therefore, consistent with any kinetic scheme that considers a single transition pathway between open and closed states, and confirms the C-C-O model previously inferred from one-dimensional distribution analysis. The strategy described should be of general validity in the analysis of single channel events from channel proteins in both natural and reconstituted membranes.     

EPR properties of a g=2 broad signal trapped in an S1 state in Ca2+-depleted Photosystem II

We investigated a new EPR signal that gives a broad line shape around g=2 in Ca(2+)-depleted Photosystem (PS) II. The signal was trapped by illumination at 243 K in parallel with the formation of Y(Z)*. The ratio of the intensities between the g=2 broad signal and the Y(Z)* signal was 1:3, assuming a Gaussian line shape for the former. The g=2 broad signal and the Y(Z)* signal decayed together in parallel with the appearance of the S(2) state multiline at 243 K. The g=2 broad signal was assigned to be an intermediate S(1)X* state in the transition from the S(1) to the S(2) state, where X* represents an amino acid radical nearby manganese cluster, such as D1-His337. The signal is in thermal equilibrium with Y(Z)*. Possible reactions in the S state transitions in Ca(2+)-depleted PS II were discussed.