Transfer of ion binding site from ether-à-go-go to Shaker: Mg2+ binds to resting state to modulate channel opening

In ether-à-go-go (eag) K⁺ channels, extracellular divalent cations bind to the resting voltage sensor and thereby slow activation. Two eag-specific acidic residues in S2 and S3b coordinate the bound ion. Residues located at analogous positions are ∼4 Å apart in the x-ray structure of a Kv1.2/Kv2.1 chimera crystallized in the absence of a membrane potential. It is unknown whether these residues remain in proximity in Kv1 channels at negative voltages when the voltage sensor domain is in its resting conformation. To address this issue, we mutated Shaker residues I287 and F324, which correspond to the binding site residues in eag, to aspartate and recorded ionic and gating currents in the presence and absence of extracellular Mg²⁺. In I287D+F324D, Mg²⁺ significantly increased the delay before ionic current activation and slowed channel opening with no readily detectable effect on closing. Because the delay before Shaker opening reflects the initial phase of voltage-dependent activation, the results indicate that Mg²⁺ binds to the voltage sensor in the resting conformation. Supporting this conclusion, Mg²⁺ shifted the voltage dependence and slowed the kinetics of gating charge movement. Both the I287D and F324D mutations were required to modulate channel function. In contrast, E283, a highly conserved residue in S2, was not required for Mg²⁺ binding. Ion binding affected activation by shielding the negatively charged side chains of I287D and F324D. These results show that the engineered divalent cation binding site in Shaker strongly resembles the naturally occurring site in eag. Our data provide a novel, short-range structural constraint for the resting conformation of the Shaker voltage sensor and are valuable for evaluating existing models for the resting state and voltage-dependent conformational changes that occur during activation. Comparing our data to the chimera x-ray structure, we conclude that residues in S2 and S3b remain in proximity throughout voltage-dependent activation.     

Activation and Inhibition of TMEM16A Calcium-Activated Chloride Channels

Calcium-activated chloride channels (CaCC) encoded by family members of transmembrane proteins of unknown function 16 (TMEM16) have recently been intensely studied for functional properties as well as their physiological roles as chloride channels in various tissues. One technical hurdle in studying these channels is the well-known channel rundown that frequently impairs the precision of electrophysiological measurements for the channels. Using experimental protocols that employ fast-solution exchange, we circumvented the problem of channel rundown by normalizing the Ca²⁺-induced current to the maximally-activated current obtained within a time period in which the channel rundown was negligible. We characterized the activation of the TMEM16A-encoded CaCC (also called ANO1) by Ca²⁺, Sr²⁺, and Ba²⁺, and discovered that Mg²⁺ competes with Ca²⁺ in binding to the divalent-cation binding site without activating the channel. We also studied the permeability of the ANO1 pore for various anions and found that the anion occupancy in the pore–as revealed by the permeability ratios of these anions–appeared to be inversely correlated with the apparent affinity of the ANO1 inhibition by niflumic acid (NFA). On the other hand, the NFA inhibition was neither affected by the degree of the channel activation nor influenced by the types of divalent cations used for the channel activation. These results suggest that the NFA inhibition of ANO1 is likely mediated by altering the pore function but not through changing the channel gating. Our study provides a precise characterization of ANO1 and documents factors that can affect divalent cation activation and NFA inhibition of ANO1.     

Kv Channel S1-S2 Linker Working as a Binding Site of Human β-Defensin 2 for Channel Activation Modulation

Among the three extracellular domains of the tetrameric voltage-gated K⁺ (Kv) channels consisting of six membrane-spanning helical segments named S1–S6, the functional role of the S1-S2 linker still remains unclear because of the lack of a peptide ligand. In this study, the Kv1.3 channel S1-S2 linker was reported as a novel receptor site for human β-defensin 2 (hBD2). hBD2 shifts the conductance-voltage relationship curve of the human Kv1.3 channel in a positive direction by nearly 10.5 mV and increases the activation time constant for the channel. Unlike classical gating modifiers of toxin peptides from animal venoms, which generally bind to the Kv channel S3-S4 linker, hBD2 only targets residues in both the N and C termini of the S1-S2 linker to influence channel gating and inhibit channel currents. The increment and decrement of the basic residue number in a positively charged S4 sensor of Kv1.3 channel yields conductance-voltage relationship curves in the positive direction by ∼31.2 mV and 2–4 mV, which suggests that positively charged hBD2 is anchored in the channel S1-S2 linker and is modulating channel activation through electrostatic repulsion with an adjacent S4 helix. Together, these findings reveal a novel peptide ligand that binds with the Kv channel S1-S2 linker to modulate channel activation. These findings also highlight the functional importance of the Kv channel S1-S2 linker in ligand recognition and modification of channel activation.     

Intracellular linkers are involved in Mg2+-dependent modulation of the Eag potassium channel

Modulation of activation kinetics by divalent ions is one of the characteristic features of Eag channels. Here, we report that Mg²⁺-dependent deceleration of Eag channel activation is significantly attenuated by a G297E mutation, which exhibits a gain-of-function phenotype in Drosophila by suppressing the effect of shaker mutation on behavior and neuronal excitability. The G297 residue is located in the intracellular linker of transmembrane segments S2 and S3, and is thus not involved in direct binding of Mg²⁺ ions. Moreover, mutation of the only positively charged residue in the other intracellular linker between S4 and S5 also results in a dramatic reduction of Mg²⁺-dependent modulation of Eag activation kinetics. Collectively, the two mutations in eag eliminate or even paradoxically reverse the effect of Mg²⁺ on channel activation and inactivation kinetics. Together, these results suggest an important role of the intracellular linker regions in gating processes of Eag channels.Key words: Drosophila potassium channel, activation, magnesium, gating, gain-of-function mutantThe Drosophila ether-à-go-go channel (Eag) is the founding member of an evolutionarily conserved subfamily of voltage-gated K⁺ channels,^1–5 which includes HERG (human eag-related gene), a channel that plays an important role in regulating cardiac excitability and maintaining normal cardiac rhythm.⁶ As demonstrated by previous studies from many groups, this family of channels is critical to the function of a variety of biological processes, including memory formation,⁷ signal transduction,^8,9 cell proliferation and tumor progression.^10–12In flies, mutation of eag increases neuronal excitability. When combined with a loss-of-function mutation in another voltage-gated K⁺ channel, shaker (sh), the eag phenotype is further enhanced, suggesting that both K⁺ channels contribute to the repolarization of presynaptic nerve terminals.^13–16 Recently, Gardnell et al., identified a gain-of-function mutation in the Eag channel that suppresses the effect of sh mutation on behavior and neuronal excitability in Drosophila.¹⁷ The mutation is caused by a single amino acid substitution (G297E) in the S2–S3 linker of the Eag channel protein. Interestingly, they have determined that extracellular Mg²⁺ is required for the gain-of-function eag mutant to suppress sh phenotypes. It thus raises the possibility that the G297E mutation may affect Eag channel properties regulated by the divalent ion.Eag channels are uniquely regulated by a variety of external divalent cations, including Mg²⁺, Mn²⁺, and Ni²⁺.^18–21 The major effect of these divalent ions is to slow activation kinetics, which presumably reflects a switch of Eag channels from a fast to a slow mode of activation gating.²² Based on previous studies, the divalent ions appear to modulate activation process of the Eag K⁺ channel through binding to acidic residues located in an extracellular- facing crevice between transmembrane segments S2 and S3 of the voltage sensor domain.^20,23,24 However, the molecular and physical mechanisms underlying divalent ion-dependent switch of gating modes in the Eag channel, particularly how does ion binding affects voltage sensor and its coupling to the activation gate, are yet to be determined.^24,25In this study, we carried out detailed analysis of the G297E mutant Eag channel in a heterologous expression system. Our data reveal that this mutation resulted in a significant decrease in Mg²⁺-induced deceleration of Eag channel activation kinetics, which provided a mechanistic basis for its gain-of-function phenotype in flies. In addition, we identified another mutation in the other intracellular linker that, in conjunction with G297E, eliminated Mg²⁺-dependent regulation of channel activation. Our findings set a stage for further determination of molecular detail of Eag channel activation processes.     

Probing Pore Constriction in a Ligand-gated Ion Channel by Trapping a Metal Ion in the Pore upon Agonist Dissociation

Eukaryotic pentameric ligand-gated ion channels (pLGICs) are receptors activated by neurotransmitters to rapidly transport ions across cell membranes, down their electrochemical gradients. Recent crystal structures of two prokaryotic pLGICs were interpreted to imply that the extracellular side of the transmembrane pore constricts to close the channel (Hilf, R. J., and Dutzler, R. (2009) Nature 457, 115–118; Bocquet, N., Nury, H., Baaden, M., Le Poupon, C., Changeux, J. P., Delarue, M., and Corringer, P. J. (2009) Nature 457, 111–114). Here, we utilized a eukaryotic acetylcholine (ACh)-serotonin chimeric pLGIC that was engineered with histidines to coordinate a metal ion within the channel pore, at its cytoplasmic side. In a previous study, the access of Zn²⁺ ions to the engineered histidines had been explored when the channel was either at rest (closed) or active (open) (Paas, Y., Gibor, G., Grailhe, R., Savatier-Duclert, N., Dufresne, V., Sunesen, M., de Carvalho, L. P., Changeux, J. P., and Attali, B. (2005) Proc. Natl. Acad. Sci. U.S.A. 102, 15877–15882). In this study, the interactions of Zn²⁺ with the pore were probed upon agonist (ACh) dissociation that triggers the transition of the receptor from the active conformation to the resting conformation (i.e. during deactivation). Application of Zn²⁺ onto ACh-bound open receptors obstructed their pore and prevented ionic flow. Removing ACh from its extracellular binding sites to trigger deactivation while Zn²⁺ is still bound led to tight trapping of Zn²⁺ within the pore. Together with single-channel recordings, made to explore single pore-blocking events, we show that dissociation of ACh causes the gate to shut on a Zn²⁺ ion that effectively acts as a “foot in the door.” We infer that, upon deactivation, the cytoplasmic side of the pore of the ACh-serotonin receptor chimera constricts to close the channel.     

The structure of MgtE in the absence of magnesium provides new insights into channel gating

MgtE is a Mg²⁺ channel conserved in organisms ranging from prokaryotes to eukaryotes, including humans, and plays an important role in Mg²⁺ homeostasis. The previously determined MgtE structures in the Mg²⁺-bound, closed-state, and structure-based functional analyses of MgtE revealed that the binding of Mg²⁺ ions to the MgtE cytoplasmic domain induces channel inactivation to maintain Mg²⁺ homeostasis. There are no structures of the transmembrane (TM) domain for MgtE in Mg²⁺-free conditions, and the pore-opening mechanism has thus remained unclear.Here, we determined the cryo-electron microscopy (cryo-EM) structure of the MgtE-Fab complex in the absence of Mg²⁺ ions. The Mg²⁺-free MgtE TM domain structure and its comparison with the Mg²⁺-bound, closed-state structure, together with functional analyses, showed the Mg²⁺-dependent pore opening of MgtE on the cytoplasmic side and revealed the kink motions of the TM2 and TM5 helices at the glycine residues, which are important for channel activity. Overall, our work provides structure-based mechanistic insights into the channel gating of MgtE.

MgtE is a magnesium-selective ion channel whose gating is regulated by cytoplasmic magnesium concentration; this cryo-EM study reveals how MgtE undergoes magnesium-dependent structural changes to open the pore on the cytoplasmic side.     

Conserved Gating Elements in TRPC4 and TRPC5 Channels

TRPC4 and TRPC5 proteins share 65% amino acid sequence identity and form Ca²⁺-permeable nonselective cation channels. They are activated by stimulation of receptors coupled to the phosphoinositide signaling cascade. Replacing a conserved glycine residue within the cytosolic S4–S5 linker of both proteins by a serine residue forces the channels into an open conformation. Expression of the TRPC4_G503S and TRPC5_G504S mutants causes cell death, which could be prevented by buffering the Ca²⁺ of the culture medium. Current-voltage relationships of the TRPC4_G503S and TRPC5_G504S mutant ion channels resemble that of fully activated TRPC4 and TRPC5 wild-type channels, respectively. Modeling the structure of the transmembrane domains and the pore region (S4-S6) of TRPC4 predicts a conserved serine residue within the C-terminal sequence of the predicted S6 helix as a potential interaction site. Introduction of a second mutation (S623A) into TRPC4_G503S suppressed the constitutive activation and partially rescued its function. These results indicate that the S4–S5 linker is a critical constituent of TRPC4/C5 channel gating and that disturbance of its sequence allows channel opening independent of any sensor domain.     

Structural Determinants of Skeletal Muscle Ryanodine Receptor Gating

Ryanodine receptor type 1 (RyR1) releases Ca²⁺ from intracellular stores upon nerve impulse to trigger skeletal muscle contraction. Effector binding at the cytoplasmic domain tightly controls gating of the pore domain of RyR1 to release Ca²⁺. However, the molecular mechanism that links effector binding to channel gating is unknown due to lack of structural data. Here, we used a combination of computational and electrophysiological methods and cryo-EM densities to generate structural models of the open and closed states of RyR1. Using our structural models, we identified an interface between the pore-lining helix (Tyr-4912–Glu-4948) and a linker helix (Val-4830–Val-4841) that lies parallel to the cytoplasmic membrane leaflet. To test the hypothesis that this interface controls RyR1 gating, we designed mutations in the linker helix to stabilize either the open (V4830W and T4840W) or closed (H4832W and G4834W) state and validated them using single channel experiments. To further confirm this interface, we designed mutations in the pore-lining helix to stabilize the closed state (Q4947N, Q4947T, and Q4947S), which we also validated using single channel experiments. The channel conductance and selectivity of the mutations that we designed in the linker and pore-lining helices were indistinguishable from those of WT RyR1, demonstrating our ability to modulate RyR1 gating without affecting ion permeation. Our integrated computational and experimental approach significantly advances the understanding of the structure and function of an unusually large ion channel.     

Divalent cations potentiate TRPV1 channel by lowering the heat activation threshold

Transient receptor potential vanilloid type 1 (TRPV1) channel responds to a wide spectrum of physical and chemical stimuli. In doing so, it serves as a polymodal cellular sensor for temperature change and pain. Many chemicals are known to strongly potentiate TRPV1 activation, though how this is achieved remains unclear. In this study we investigated the molecular mechanism underlying the gating effects of divalent cations Mg²⁺ and Ba²⁺. Using a combination of fluorescence imaging and patch-clamp analysis, we found that these cations potentiate TRPV1 gating by most likely promoting the heat activation process. Mg²⁺ substantially lowers the activation threshold temperature; as a result, a significant fraction of channels are heat-activated at room temperature. Although Mg²⁺ also potentiates capsaicin- and voltage-dependent activation, these processes were found either to be not required (in the case of capsaicin) or insufficient (in the case of voltage) to mediate the activating effect. In support of a selective effect on heat activation, Mg²⁺ and Ba²⁺ cause a Ca²⁺-independent desensitization that specifically prevents heat-induced channel activation but does not prevent capsaicin-induced activation. These results can be satisfactorily explained within an allosteric gating framework in which divalent cations strongly promote the heat-dependent conformational change or its coupling to channel activation, which is further coupled to the voltage- and capsaicin-dependent processes.     

Hydrophobic interaction between contiguous residues in the S6 transmembrane segment acts as a stimuli integration node in the BK channel

Large-conductance Ca²⁺- and voltage-activated K⁺ channel (BK) open probability is enhanced by depolarization, increasing Ca²⁺ concentration, or both. These stimuli activate modular voltage and Ca²⁺ sensors that are allosterically coupled to channel gating. Here, we report a point mutation of a phenylalanine (F380A) in the S6 transmembrane helix that, in the absence of internal Ca²⁺, profoundly hinders channel opening while showing only minor effects on the voltage sensor active–resting equilibrium. Interpretation of these results using an allosteric model suggests that the F380A mutation greatly increases the free energy difference between open and closed states and uncouples Ca²⁺ binding from voltage sensor activation and voltage sensor activation from channel opening. However, the presence of a bulky and more hydrophobic amino acid in the F380 position (F380W) increases the intrinsic open–closed equilibrium, weakening the coupling between both sensors with the pore domain. Based on these functional experiments and molecular dynamics simulations, we propose that F380 interacts with another S6 hydrophobic residue (L377) in contiguous subunits. This pair forms a hydrophobic ring important in determining the open–closed equilibrium and, like an integration node, participates in the communication between sensors and between the sensors and pore. Moreover, because of its effects on open probabilities, the F380A mutant can be used for detailed voltage sensor experiments in the presence of permeant cations.     

Positions of the cytoplasmic end of BK α S0 helix relative to S1–S6 and of β1 TM1 and TM2 relative to S0–S6

The large-conductance, voltage- and Ca²⁺-gated K⁺ (BK) channel consists of four α subunits, which form a voltage- and Ca²⁺-gated channel, and up to four modulatory β subunits. The β1 subunit is expressed in smooth muscle, where it slows BK channel kinetics and shifts the conductance–voltage (G-V) curve to the left at [Ca²⁺] > 2 µM. In addition to the six transmembrane (TM) helices, S1–S6, conserved in all voltage-dependent K⁺ channels, BK α has a unique seventh TM helix, S0, which may contribute to the unusual rightward shift in the G-V curve of BK α in the absence of β1 and to a leftward shift in its presence. Such a role is supported by the close proximity of S0 to S3 and S4 in the voltage-sensing domain. Furthermore, on the extracellular side of the membrane, one of the two TM helices of β1, TM2, is adjacent to S0. We have now analyzed induced disulfide bond formation between substituted Cys residues on the cytoplasmic side of the membrane. There, in contrast, S0 is closest to the S2–S3 loop, from which position it is displaced on the addition of β1. The cytoplasmic ends of β1 TM1 and TM2 are adjacent and are located between the S2–S3 loop of one α subunit and S1 of a neighboring α subunit and are not adjacent to S0; i.e., S0 and TM2 have different trajectories through the membrane. In the absence of β1, 70% of disulfide bonding of W43C (S0) and L175C (S2–S3) has no effect on V₅₀ for activation, implying that the cytoplasmic end of S0 and the S2–S3 loop move in concert, if at all, during activation. Otherwise, linking them together in one state would obstruct the transition to the other state, which would certainly change V₅₀.     

Bimodal regulation of an Elk subfamily K+ channel by phosphatidylinositol 4,5-bisphosphate

Phosphatidylinositol 4,5-bisphosphate (PIP₂) regulates Shaker K⁺ channels and voltage-gated Ca²⁺ channels in a bimodal fashion by inhibiting voltage activation while stabilizing open channels. Bimodal regulation is conserved in hyperpolarization-activated cyclic nucleotide–gated (HCN) channels, but voltage activation is enhanced while the open channel state is destabilized. The proposed sites of PIP₂ regulation in these channels include the voltage-sensor domain (VSD) and conserved regions of the proximal cytoplasmic C terminus. Relatively little is known about PIP₂ regulation of Ether-á-go-go (EAG) channels, a metazoan-specific family of K⁺ channels that includes three gene subfamilies, Eag (Kv10), Erg (Kv11), and Elk (Kv12). We examined PIP₂ regulation of the Elk subfamily potassium channel human Elk1 to determine whether bimodal regulation is conserved within the EAG K⁺ channel family. Open-state stabilization by PIP₂ has been observed in human Erg1, but the proposed site of regulation in the distal C terminus is not conserved among EAG family channels. We show that PIP₂ strongly inhibits voltage activation of Elk1 but also stabilizes the open state. This stabilization produces slow deactivation and a mode shift in voltage gating after activation. However, removal of PIP₂ has the net effect of enhancing Elk1 activation. R347 in the linker between the VSD and pore (S4–S5 linker) and R479 near the S6 activation gate are required for PIP₂ to inhibit voltage activation. The ability of PIP₂ to stabilize the open state also requires these residues, suggesting an overlap in sites central to the opposing effects of PIP₂ on channel gating. Open-state stabilization in Elk1 requires the N-terminal eag domain (PAS domain + Cap), and PIP₂-dependent stabilization is enhanced by a conserved basic residue (K5) in the Cap. Our data shows that PIP₂ can bimodally regulate voltage gating in EAG family channels, as has been proposed for Shaker and HCN channels. PIP₂ regulation appears fundamentally different for Elk and KCNQ channels, suggesting that, although both channel types can regulate action potential threshold in neurons, they are not functionally redundant.     

Susceptibility of the guard-cell K+-uptake channel KST1 to Zn2+ requires histidine residues in the S3-S4 linker and in the channel pore

 Potassium channels are inhibited by several mono- and divalent cations. To identify sites involved in the interaction between K⁺ channels and cationic effectors, we expressed the potato (Solanum tuberosum L.) guard-cell K⁺-uptake channel KST1 in Xenopus oocytes. This channel was reversibly blocked by extracellular Zn²⁺ in the micromolar range. In the presence of this heavy metal, steady-state currents were reduced in a pH-dependent but voltage-independent manner. Since Zn²⁺-inhibition was less effective at elevated external proton concentrations, we generated alanine mutants with respect to both extracellular histidines in KST1. Whereas substitution of the pore histidine H271 resulted in a reduced blockade by Zn²⁺, the channel mutant KST1-H160A in the S3-S4 linker lost most of its Zn²⁺ sensitivity. Since both histidines alter the susceptibility of KST1 to Zn²⁺, the block may predominantly result from these two sites. We thus conclude that the S3-S4 linker is involved in the formation of the outer pore. Received: 3 May 1999 / Accepted: 8 July 1999     

Clustered Distribution of Calcium Sensitivities: An Indication of Hetero-tetrameric Gating Components in Ca2+-activated K+ Channels Reconstituted from Avian Nasal Gland Cells

High-conductance, Ca²⁺-activated K⁺ channels from the basolateral membrane of rabbit distal colon epithelial cells were reconstituted into planar phospholipid bilayers to examine the effect of Mg²⁺ on the single-channel properties. Mg²⁺ decreases channel current and conductance in a concentration-dependent manner from both the cytoplasmic and the extracellular side of the channel. In contrast to other K⁺ channels, Mg²⁺ does not cause rectification of current through colonic Ca²⁺-activated K⁺ channels. In addition, cytoplasmic Mg²⁺ decreases the reversal potential of the channel. The Mg²⁺-induced decrease in channel conductance is relieved by high K⁺ concentrations, indicating competitive interaction between K⁺ and Mg²⁺. The monovalent organic cation choline also decreases channel conductance and reversal potential, suggesting that the effect is unspecific. The inhibition of channel current by Mg²⁺ and choline most likely is a result of electrostatic screening of negative charges located superficially in the channel entrance. But in addition to charge, other properties appear to be necessary for channel inhibition, as Na⁺ and Ba²⁺ are no (or only weak) inhibitors. Mg²⁺ and possibly other cations may play a role in the regulation of current through these channels. Received: 25 August 1995/Revised: 16 November 1995     

Aromatic–aromatic interactions between residues in KCa3.1 pore helix and S5 transmembrane segment control the channel gating process

The Ca²⁺-activated potassium channel KCa3.1 is emerging as a therapeutic target for a large variety of health disorders. One distinguishing feature of KCa3.1 is that the channel open probability at saturating Ca²⁺ concentrations (Pomax) is low, typically 0.1–0.2 for KCa3.1 wild type. This observation argues for the binding of Ca²⁺ to the calmodulin (CaM)–KCa3.1 complex, promoting the formation of a preopen closed-state configuration leading to channel opening. We have previously shown that the KCa3.1 active gate is most likely located at the level of the selectivity filter. As Ca²⁺-dependent gating of KCa3.1 originates from the binding of Ca²⁺ to CaM in the C terminus, the hypothesis of a gate located at the level of the selectivity filter requires that the conformational change initiated in the C terminus be transmitted to the S5 and S6 transmembrane helices, with a resulting effect on the channel pore helix directly connected to the selectivity filter. A study was thus undertaken to determine to what extent the interactions between the channel pore helix with the S5 and S6 transmembrane segments contribute to KCa3.1 gating. Molecular dynamics simulations first revealed that the largest contact area between the pore helix and the S5 plus S6 transmembrane helices involves residue F248 at the C-terminal end of the pore helix. Unitary current recordings next confirmed that modulating aromatic–aromatic interactions between F248 and W216 of the S5 transmembrane helical segment and/or perturbing the interactions between F248 and residues in S6 surrounding the glycine hinge G274 cause important changes in Pomax. This work thus provides the first evidence for a key contribution of the pore helix in setting Pomax by stabilizing the channel closed configuration through aromatic–aromatic interactions involving F248 of the pore helix. We propose that the interface pore helix/S5 constitutes a promising site for designing KCa3.1 potentiators.     

Modulation of P2X4 pore closure by magnesium,potassium, and ATP

The P2X4 receptor plays a prominent role in cellular responses to extracellular ATP. Through classical all-atom molecular dynamics (MD) simulations totaling 24 μs we have investigated how metal-complexed ATP stabilizes the channel's open state and prevents its closing. We have identified two metal-binding sites, Mg²⁺ and potassium K⁺, one at the intersection of the three subunits in the ectodomain (MBS1) and the second one near the ATP-binding site (MBS2), similar to those characterized in Gulf Coast P2X. Our data indicate that when Mg²⁺ and K⁺ ions are complexed with ATP, the channel is locked into an open state. Interestingly, irrespective of the number of bound ATP molecules, Mg²⁺ ions bound to the MBS2 impeded the collapse of the open-state protein to a closed state by stabilizing the ATP-protein interactions. However, when Mg²⁺ in the MBS2 was replaced with K⁺ ions, as might be expected when in equilibrium with an extracellular solution, the interactions between the subunits were weakened and the pore collapsed. This collapse was apparent when fewer than two ATPs were bound to MBS2 in the presence of K⁺. Therefore, the different capacities of common cations to stabilize the channel may underlie a mechanism governing P2X4 channel gating in physiological systems. This study therefore provides structural insights into the differential modulation of ATP activation of P2X4 by Mg²⁺ and K⁺.     

Four Ca2+ Ions Activate TRPM2 Channels by Binding in Deep Crevices near the Pore but Intracellularly of the Gate

TRPM2 is a tetrameric Ca²⁺-permeable channel involved in immunocyte respiratory burst and in postischaemic neuronal death. In whole cells, TRPM2 activity requires intracellular ADP ribose (ADPR) and intra- or extracellular Ca²⁺, but the mechanism and the binding sites for Ca²⁺ activation remain unknown. Here we study TRPM2 gating in inside-out patches while directly controlling intracellular ligand concentrations. Concentration jump experiments at various voltages and Ca²⁺ dependence of steady-state single-channel gating kinetics provide unprecedented insight into the molecular mechanism of Ca²⁺ activation. In patches excised from Xenopus laevis oocytes expressing human TRPM2, coapplication of intracellular ADPR and Ca²⁺ activated ∼50-pS nonselective cation channels; K_1/2 for ADPR was ∼1 µM at saturating Ca²⁺. Intracellular Ca²⁺ dependence of TRPM2 steady-state opening and closing rates (at saturating [ADPR] and low extracellular Ca²⁺) reveals that Ca²⁺ activation is a consequence of tighter binding of Ca²⁺ in the open rather than in the closed channel conformation. Four Ca²⁺ ions activate TRPM2 with a Monod-Wymann-Changeux mechanism: each binding event increases the open-closed equilibrium constant ∼33-fold, producing altogether 10⁶-fold activation. Experiments in the presence of 1 mM of free Ca²⁺ on the extracellular side clearly show that closed channels do not sense extracellular Ca²⁺, but once channels have opened Ca²⁺ entering passively through the pore slows channel closure by keeping the “activating sites” saturated, despite rapid continuous Ca²⁺-free wash of the intracellular channel surface. This effect of extracellular Ca²⁺ on gating is gradually lost at progressively depolarized membrane potentials, where the driving force for Ca²⁺ influx is diminished. Thus, the activating sites lie intracellularly from the gate, but in a shielded crevice near the pore entrance. Our results suggest that in intact cells that contain micromolar ADPR a single brief puff of Ca²⁺ likely triggers prolonged, self-sustained TRPM2 activity.     

Intracellular calcium strongly potentiates agonist-activated TRPC5 channels

TRPC5 is a calcium (Ca²⁺)-permeable nonselective cation channel expressed in several brain regions, including the hippocampus, cerebellum, and amygdala. Although TRPC5 is activated by receptors coupled to phospholipase C, the precise signaling pathway and modulatory signals remain poorly defined. We find that during continuous agonist activation, heterologously expressed TRPC5 currents are potentiated in a voltage-dependent manner (∼5-fold at positive potentials and ∼25-fold at negative potentials). The reversal potential, doubly rectifying current–voltage relation, and permeability to large cations such as N-methyl-d-glucamine remain unchanged during this potentiation. The TRPC5 current potentiation depends on extracellular Ca²⁺: replacement by Ba²⁺ or Mg²⁺ abolishes it, whereas the addition of 10 mM Ca²⁺ accelerates it. The site of action for Ca²⁺ is intracellular, as simultaneous fura-2 imaging and patch clamp recordings indicate that potentiation is triggered at ∼1 µM [Ca²⁺]. This potentiation is prevented when intracellular Ca²⁺ is tightly buffered, but it is promoted when recording with internal solutions containing elevated [Ca²⁺]. In cell-attached and excised inside-out single-channel recordings, increases in internal [Ca²⁺] led to an ∼10–20-fold increase in channel open probability, whereas single-channel conductance was unchanged. Ca²⁺-dependent potentiation should result in TRPC5 channel activation preferentially during periods of repetitive firing or coincident neurotransmitter receptor activation.     

State-dependent block of Orai3 TM1 and TM3 cysteine mutants: Insights into 2-APB activation

After endoplasmic reticulum (ER) Ca²⁺ store depletion, Orai channels in the plasma membrane (PM) are activated directly by ER-resident stromal interacting molecule (STIM) proteins to form the Ca²⁺-selective Ca²⁺ release-activated Ca²⁺ (CRAC) channel. Of the three human Orai channel homologues, only Orai3 can be activated by high concentrations (>50 µM) of 2-aminoethyl diphenylborinate (2-APB). 2-APB activation of Orai3 occurs without STIM1–Orai3 interaction or store depletion, and results in a cationic, nonselective current characterized by biphasic inward and outward rectification. Here we use cysteine scanning mutagenesis, thiol-reactive reagents, and patch-clamp analysis to define the residues that assist in formation of the 2-APB–activated Orai3 pore. Mutating transmembrane (TM) 1 residues Q83, V77, and L70 to cysteine results in potentiated block by cadmium ions (Cd²⁺). TM1 mutants E81C, G73A, G73C, and R66C form channels that are not sensitive to 2-APB activation. We also find that Orai3 mutant V77C is sensitive to block by 2-aminoethyl methanethiosulfonate (MTSEA), but not 2-(trimethylammonium)ethyl methanethiosulfonate (MTSET). Block induced by reaction with MTSEA is state dependent, as it occurs only when Orai3-V77C channels are opened by either 2-APB or by cotransfection with STIM1 and concurrent passive store depletion. We also analyzed TM3 residue E165. Mutation E165A in Orai3 results in diminished 2-APB–activated currents. However, it has little effect on store-operated current density. Furthermore, mutation E165C results in Cd²⁺-induced block that is state dependent: Cd²⁺ only blocks 2-APB–activated, not store-operated, mutant channels. Our data suggest that the dilated pore of 2-APB–activated Orai3 is lined by TM1 residues, but also allows for TM3 E165 to approach the central axis of the channel that forms the conducting pathway, or pore.     

A conserved gating element in TRPV6 channels

The Ca²⁺-selective tetrameric Transient Receptor Potential Vanilloid 6 (TRPV6) channel is an inwardly rectifying ion channel. The constitutive current endures Ca²⁺-induced inactivation as a result of the activation of phospholipase C followed depletion of phosphatidylinositol 4,5-bisphosphate, and calmodulin binding. Replacing a glycine residue within the cytosolic S4-S5 linker of the human TRPV6 protein, glycine 516, which is conserved in all TRP channel proteins, by a serine residue forces the channels into an open conformation thereby enhancing constitutive Ca²⁺ entry and preventing inactivation. Introduction of a second mutation (T621A) into TRPV6_G516S reduces constitutive activity and partially rescues the TRPV6 function. According to the recently revealed crystal structure of the rat TRPV6 the T621 is adjacent to the distal end of the transmembrane segment 6 (S6) within a short linker between S6 and the helix formed by the TRP domain. These results indicate that the S4-S5 linker and the S6-TRP-domain linker are critical constituents of TRPV6 channel gating and that disturbance of their sequences foster constitutive Ca²⁺ entry.