BK channel opening involves side-chain reorientation of multiple deep-pore residues

Three deep-pore locations, L312, A313, and A316, were identified in a scanning mutagenesis study of the BK (Ca²⁺-activated, large-conductance K⁺) channel S6 pore, where single aspartate substitutions led to constitutively open mutant channels (L312D, A313D, and A316D). To understand the mechanisms of the constitutive openness of these mutant channels, we individually mutated these three sites into the other 18 amino acids. We found that charged or polar side-chain substitutions at each of the sites resulted in constitutively open mutant BK channels, with high open probability at negative voltages, as well as a loss of voltage and Ca²⁺ dependence. Given the fact that multiple pore residues in BK displayed side-chain hydrophilicity-dependent constitutive openness, we propose that BK channel opening involves structural rearrangement of the deep-pore region, where multiple residues undergo conformational changes that may increase the exposure of their side chains to the polar environment of the pore.Large-conductance, Ca²⁺-activated K⁺ (BK) channels regulate physiological processes such as neurotransmitter release, smooth muscle contraction, and hair cell frequency tuning (1–12). BK channel proteins are homotetramers formed by BK α-subunits, which then associate with different β- or γ-subunits in a tissue-specific manner (13–22). The α-subunit of the BK (K_Ca1.1) channel is encoded by the KCNMA1 gene, first discovered in Drosophila as the slowpoke mutation (dSlo) (23, 24), and later identified in mouse (mSlo1) and human (hSlo1) (25, 26). Each α-subunit has seven transmembrane segments (S0–S6), with the S6 segments lining the pore. A similar structural arrangement is found in other members of the K⁺ channel protein family.From a functional point of view, the gating behavior of BK channels can be described as a central C⇔O (i.e., closed⇔open) transition, influenced by voltage sensor movement and/or Ca²⁺ binding (27–31). The structural basis for this C⇔O transition is believed to be conformational changes of the S6 segment and/or the selectivity filter (32, 33), controlling the passage of K⁺ ions across the membrane.Molecular details of gating-related conformational changes have been probed with mutagenesis-based methods for BK and related channels. Although it has been demonstrated that voltage-gated (Kv) K⁺ channels open by a cytoplasmic S6 “bundle crossing” gate (34–37), evidence has accumulated that the opening conformational change of BK, like cyclic nucleotide-gated (CNG) channels, occurs deeper in the pore, closer to the selectivity filter (38–46). With the goal of further understanding the opening conformational change in BK channels, we used a histidine substitution/protonation strategy and identified a residue in BK S6 (M314 in hSlo1) whose side chain turns more toward the pore when the channel is open. The open conformation can be stabilized by the presence of side-chain charges at this location, with the aspartate mutant being the most effective in keeping the channel open in neutral pH (47).To uncover more dynamic details at other pore residues during BK channel gating, we scanned the S6 segment cytoplasmic to the selectivity filter with single aspartate substitutions (I308D→N328D). Because the S6 residues of K⁺ channels reside on the interface between a polar (the water and ion filled pore) and a nonpolar (the rest of the protein in the membrane) environment, we expect the charged side chains of substituted aspartates to prefer the more aqueous environment of the pore, making the conformations with such side-chain orientation energetically favorable. If these conformations correspond to any functional states of the channel, such states may be stabilized, and will be functionally measurable. Our previous studies identified the M314D mutant channels as favoring the open state, consistent with greater exposure of this side chain to the pore upon opening.In addition, the S6-formed pore is believed to host a “gate” that, when closed, prevents the passage of ions. The nature of such a gate, in its closed conformation, is proposed to be an occlusion structure formed by the side chains of amino acid residues at the gate location. In the structural model of the closed KcsA channel, at three residue locations (T107, A111, V115) to the intracellular end of its TM2 (equivalent of S6 in mammalian K⁺ channels) (Fig. 1), one could see the side chains at equivalent locations from the four subunits come very close to each other in the pore, occluding passage of K⁺ (48, 49). In the structural model of an open KcsA channel, pore diameters at the same locations are significantly larger (50, 51).Open in a separate windowFig. 1.Sequence alignment and structural models. (A) Sequence alignment of KcsA (accession no. {"type":"entrez-protein","attrs":{"text":"NP_631700","term_id":"21225921","term_text":"NP_631700"}}NP_631700), Shaker (accession no. {"type":"entrez-protein","attrs":{"text":"CAA29917","term_id":"288442","term_text":"CAA29917"}}CAA29917), and hSlo1 (accession no. {"type":"entrez-protein","attrs":{"text":"AAB65837","term_id":"507922","term_text":"AAB65837"}}AAB65837), starting from the threonine in the “TVGYGD” signature sequence of the K⁺ channel selectivity filter. The numbers by the channel name indicate the sequence number for the threonine in the corresponding channel. Highlighted in yellow are T107, A111, and V115 of KcsA, and P475, V478, and F481 of Shaker. Three residues of hSlo1 are highlighted as follows: L312, blue; A313, red; A316, green. The brown bar below the sequences indicates the selectivity filter, and the gray bar indicates residues scanned by aspartate substitution. (B) Side, top, and bottom views of two KcsA structure models: 1R3J, the closed conformation; and 3F5W, the open conformation. Only the P and S6 helices are shown. In the side views, one of the four subunits was taken away to better view the pore. The brown vertical lines indicate ion permeation through the selectivity filter. The equivalent residues to L312, A313, and A316 are colored the same way as in A.One way to functionally locate such a tightly packed occlusion structure is to try to disrupt it with small, charged, or large side-chain amino acid substitutions. Application of this method on the Shaker K⁺ channels, together with prior evidence from cysteine accessibility experiments, identified V478 or F481 as candidate locations for the gate (52). With location P475, the aspartate mutant is constitutively open, the result of a shifted energetic balance between the closed and the open states (52, 53) (Fig. 1). If the BK channel gate is also formed by specific, intersubunit hydrophobic interactions between four equivalent residues in the tetramer, such as suggested by the KcsA structure, the charges on the substituted aspartate side chains may also prevent such interactions and reveal the location of the gate.     

Cyclic nucleotide-gated channel 18 is an essential Ca2+ channel in pollen tube tips for pollen tube guidance to ovules in Arabidopsis

In flowering plants, pollen tubes are guided into ovules by multiple attractants from female gametophytes to release paired sperm cells for double fertilization. It has been well-established that Ca²⁺ gradients in the pollen tube tips are essential for pollen tube guidance and that plasma membrane Ca²⁺ channels in pollen tube tips are core components that regulate Ca²⁺ gradients by mediating and regulating external Ca²⁺ influx. Therefore, Ca²⁺ channels are the core components for pollen tube guidance. However, there is still no genetic evidence for the identification of the putative Ca²⁺ channels essential for pollen tube guidance. Here, we report that the point mutations R491Q or R578K in cyclic nucleotide-gated channel 18 (CNGC18) resulted in abnormal Ca²⁺ gradients and strong pollen tube guidance defects by impairing the activation of CNGC18 in Arabidopsis. The pollen tube guidance defects of cngc18-17 (R491Q) and of the transfer DNA (T-DNA) insertion mutant cngc18-1 (+/−) were completely rescued by CNGC18. Furthermore, domain-swapping experiments showed that CNGC18’s transmembrane domains are indispensable for pollen tube guidance. Additionally, we found that, among eight Ca²⁺ channels (including six CNGCs and two glutamate receptor-like channels), CNGC18 was the only one essential for pollen tube guidance. Thus, CNGC18 is the long-sought essential Ca²⁺ channel for pollen tube guidance in Arabidopsis.Pollen tubes deliver paired sperm cells into ovules for double fertilization, and signaling communication between pollen tubes and female reproductive tissues is required to ensure the delivery of sperm cells into the ovules (1). Pollen tube guidance is governed by both female sporophytic and gametophytic tissues (2, 3) and can be separated into two categories: preovular guidance and ovular guidance (1). For preovular guidance, diverse signaling molecules from female sporophytic tissues have been identified, including the transmitting tissue-specific (TTS) glycoprotein in tobacco (4), γ-amino butyric acid (GABA) in Arabidopsis (5), and chemocyanin and the lipid transfer protein SCA in Lilium longiflorum (6, 7). For ovular pollen tube guidance, female gametophytes secrete small peptides as attractants, including LUREs in Torenia fournieri (8) and Arabidopsis (9) and ZmEA1 in maize (10, 11). Synergid cells, central cells, egg cells, and egg apparatus are all involved in pollen tube guidance, probably by secreting different attractants (9–15). Additionally, nitric oxide (NO) and phytosulfokine peptides have also been implicated in both preovular and ovular pollen tube guidance (16–18). Thus, pollen tubes could be guided by diverse attractants in a single plant species.Ca²⁺ gradients at pollen tube tips are essential for both tip growth and pollen tube guidance (19–27). Spatial modification of the Ca²⁺ gradients leads to the reorientation of pollen tube growth in vitro (28, 29). The Ca²⁺ gradients were significantly increased in pollen tubes attracted to the micropyles by synergid cells in vivo, compared with those not attracted by ovules (30). Therefore, the Ca²⁺ gradients in pollen tube tips are essential for pollen tube guidance. The Ca²⁺ gradients result from external Ca²⁺ influx, which is mainly mediated by plasma membrane Ca²⁺ channels in pollen tube tips. Thus, the Ca²⁺ channels are the key components for regulating the Ca²⁺ gradients and are consequently essential for pollen tube guidance. Using electrophysiological techniques, inward Ca²⁺ currents were observed in both pollen grain and pollen tube protoplasts (31–36), supporting the presence of plasma membrane Ca²⁺ channels in pollen tube tips. Recently, a number of candidate Ca²⁺ channels were identified in pollen tubes, including six cyclic nucleotide-gated channels (CNGCs) and two glutamate receptor-like channels (GLRs) in Arabidopsis (37–40). Three of these eight channels, namely CNGC18, GLR1.2, and GLR3.7, were characterized as Ca²⁺-permeable channels (40, 41) whereas the ion selectivity of the other five CNGCs has not been characterized. We hypothesized that the Ca²⁺ channel essential for pollen tube guidance could be among these eight channels.In this research, we first characterized the remaining five CNGCs as Ca²⁺ channels. We further found that CNGC18, out of the eight Ca²⁺ channels, was the only one essential for pollen tube guidance in Arabidopsis and that its transmembrane domains were indispensable for pollen tube guidance.     

Using protein backbone mutagenesis to dissect the link between ion occupancy and C-type inactivation in K+ channels

K⁺ channels distinguish K⁺ from Na⁺ in the selectivity filter, which consists of four ion-binding sites (S1–S4, extracellular to intracellular) that are built mainly using the carbonyl oxygens from the protein backbone. In addition to ionic discrimination, the selectivity filter regulates the flow of ions across the membrane in a gating process referred to as C-type inactivation. A characteristic of C-type inactivation is a dependence on the permeant ion, but the mechanism by which permeant ions modulate C-type inactivation is not known. To investigate, we used amide-to-ester substitutions in the protein backbone of the selectivity filter to alter ion binding at specific sites and determined the effects on inactivation. The amide-to-ester substitutions in the protein backbone were introduced using protein semisynthesis or in vivo nonsense suppression approaches. We show that an ester substitution at the S1 site in the KcsA channel does not affect inactivation whereas ester substitutions at the S2 and S3 sites dramatically reduce inactivation. We determined the structure of the KcsA S2 ester mutant and found that the ester substitution eliminates K⁺ binding at the S2 site. We also show that an ester substitution at the S2 site in the K_vAP channel has a similar effect of slowing inactivation. Our results link C-type inactivation to ion occupancy at the S2 site. Furthermore, they suggest that the differences in inactivation of K⁺ channels in K⁺ compared with Rb⁺ are due to different ion occupancies at the S2 site.Potassium channels are a ubiquitous family of integral membrane proteins that facilitate the selective conduction of K⁺ ions across cellular membranes (1). K⁺ selectivity is achieved by a structural element in the K⁺ channel pore called the selectivity filter (2). The selectivity filter consists of four sequential ion-binding sites (labeled S1–S4, from the outside to inside) that are built using protein backbone carbonyl oxygen atoms and the threonine side chain from the protein sequence T-V-G-Y-G (Fig. 1A) (4, 5).Open in a separate windowFig. 1.Ester substitutions in the selectivity filter of the KcsA channel. (A) Close-up view of the selectivity filter of the wild-type KcsA channel [Protein Data Bank (PDB): 1K4C]. Two opposite subunits are shown in stick representation, and the K⁺ ions bound are shown as purple spheres. The amide bonds (1′–4′) and the ion-binding sites in the selectivity filter (S1–S4) are labeled. (B) Macroscopic currents for the KcsA channels were elicited at +100 mV by a rapid change in pH from 7.5 to 3.0. (C) Single-channel currents recorded at steady-state conditions at pH 3.0. The pH 3.0 solution is 10 mM succinate, 200 mM KCl, and the pH 7.5 solution is 10 mM Hepes–KOH, 200 mM KCl. The data for KcsA^WT are from ref. 3.In addition to selective conduction of K⁺, the selectivity filter acts as a gate to regulate the flow of ions through the pore (6–8). During this gating process, conformational changes at the selectivity filter convert it from a conductive to a nonconductive state. In voltage-gated K⁺ (K_v) channels, this gating process is referred to as C-type inactivation (9). C-type inactivation is a physiologically important process as it plays a direct role in regulating neuronal firing and in pacing cardiac action potentials (8). The KcsA K⁺ channel from Streptomyces lividans undergoes an inactivation process that is functionally similar to C-type inactivation in a eukaryotic K_v channel (10–13). As the KcsA channel is easily amenable to structural studies, it has become an important model system for understanding the structure of the selectivity filter in the C-type–inactivated state and the forces that drive inactivation (14, 15).One of the hallmarks of C-type inactivation is a dependence on the permeant ion (6, 7). The rate of C-type inactivation decreases when the K⁺ concentration is increased or when the permeant ion is changed from K⁺ to Rb⁺ (16, 17). Crystallographic studies on K⁺ channels have shown that a change in the permeant ion or its concentration results in changes in the ion occupancy at the binding sites in the selectivity filter (18, 19). For example, K⁺ and Rb⁺ at similar concentrations show different occupancies at the ion-binding sites, and the channel exhibits different rates of inactivation in K⁺ compared with Rb⁺ (3, 16, 20), which suggests a link between ion occupancy at the selectivity filter and inactivation (21, 22). The influence of permeant ions on inactivation has been proposed to arise from a “foot in the door”-like effect in which ion binding at a specific site prevents inactivation, similar to the presence of a foot in the doorway that prevents a door from closing (16, 23). The binding site responsible for the foot in the door effect is suspected to be at the extracellular side of the channel, but the exact location of the binding site, whether in the selectivity filter or at the extracellular mouth of the filter, is not known (6).In this study, we investigate this link between ion binding at the selectivity filter and inactivation. The approach that we use is to alter ion binding at the selectivity filter sites and to determine the effect on inactivation. The S1–S3 ion-binding sites in the selectivity filter are constructed by backbone carbonyl oxygens. Therefore, conventional site-directed mutagenesis does not allow us to alter these sites. Instead, we use chemical synthesis and nonsense suppression approaches to introduce amide-to-ester substitutions in the protein backbone to perturb ion binding to specific sites in the selectivity filter (24, 25).Amide-to-ester substitutions have previously been used to engineer the protein backbone for studies on protein stability and folding (26). Ester bonds are isosteric to amide bonds but have altered hydrogen-bonding properties and reduced electronegativity at the carbonyl oxygen (27). This reduction in the electronegativity of the carbonyl oxygen, by roughly one-half compared with an amide bond, perturbs ion binding to the selectivity filter. Amide-to-ester substitutions have previously been reported in the selectivity filters of the Kir2.1 and the KcsA K⁺ channels (28, 29). In the Kir2.1 channel, an ester substitution for the 3′ amide bond (see Fig. 1A for nomenclature) was found to reduce channel conductance and to produce distinct subconductance levels. In the KcsA channel, an ester substitution for the 1′ amide bond was found to reduce channel conductance, and a crystal structure of the ester mutant showed that the ester substitution decreased ion occupancy at the S1 site. Neither of these studies examined the effect of the ester substitutions on inactivation.Here we substitute the 1′, 2′, and 3′ amide bonds in the selectivity filter of the KcsA K⁺ channel with esters and investigate the effect on inactivation. We determine the crystal structure of the 2′ ester mutant of the KcsA channel to examine the effect of the ester substitution on the structure and ion occupancy of the selectivity filter. We also investigate the effect of an ester substitution at the 2′ amide bond in the selectivity filter on inactivation in the voltage-gated K⁺ channel, K_vAP. Our results show that the S1 and S2 sites in the selectivity filter do not act as the foot in the door sites to prevent inactivation. Unexpectedly, we find that a lack of ion binding at the S2 site reduces inactivation.     

Transmembrane allosteric coupling of the gates in a potassium channel

It has been hypothesized that transmembrane allostery is the basis for inactivation of the potassium channel KcsA: opening the intracellular gate is spontaneously followed by ion expulsion at the extracellular selectivity filter. This suggests a corollary: following ion expulsion at neutral pH, a spontaneous global conformation change of the transmembrane helices, similar to the motion involved in opening, is expected. Consequently, both the low potassium state and the low pH state of the system could provide useful models for the inactivated state. Unique NMR studies of full-length KcsA in hydrated bilayers provide strong evidence for such a mutual coupling across the bilayer: namely, upon removing ambient potassium ions, changes are seen in the NMR shifts of carboxylates E118 and E120 in the pH gate in the hinges of the inner transmembrane helix (98–103), and in the selectivity filter, all of which resemble changes seen upon acid-induced opening and inhibition and suggest that ion release can trigger channel helix opening.Potassium channel activation and inactivation is fundamental to many physiological functions including muscle contraction and the generation of synaptic action potentials (1). KcsA is a 160-residue pH-activated homotetrameric K⁺ channel isolated from the soil bacterium Streptomyces lividans (2, 3) with high sequence homology and functional similarity to mammalian potassium channels (4). It has provided an excellent model for studies of ion-conduction by X-ray crystallography (3, 5, 6), electrophysiology (7, 8), and NMR (9–21). Like many potassium channels, it exhibits (4, 6, 22, 23) slow, spontaneous inactivation involving the residues near the extracellular selectivity filter subsequent to channel activation. Recent results from X-ray crystallography and molecular dynamics suggest that the gates are coupled and that inactivation is prompted by channel opening, mediated via a series of intrasubunit steric contacts involving F103 with T74, T75, and M96 and an intersubunit contact with the neighboring I100 side chain (4–6, 24, 25). In separate experiments, the extracellular gate has been observed to respond directly to ambient [K⁺]: at high [K⁺] it exists in a conductive form, and at low K⁺ it collapses into a nonconductive state (3). Our NMR studies suggest that the low [K⁺] state and the low pH inactivated state may be similar; this conclusion is supported by the effect of the mutation E71A and the pattern of chemical shift perturbations in the selectivity filter when the ion is depleted (9, 19). Meanwhile, X-ray crystallography studies suggest that mutants (E71A) unable to undergo inactivation are also unable to expel ions (26).An established similarity of the low pH and the low [K⁺] states would clarify the importance of allosteric coupling and have the practical consequence that the well-behaved low K⁺ state could serve as a useful structural proxy for the otherwise fleeting inactivated state. For these reasons we tested this correspondence using NMR experiments. If the low K⁺ state is similar to the inactivated state of KcsA achieved by lowering the pH, it is expected that structural changes indicative of channel opening observed at low [K⁺] would occur not only in the selectivity filter but also in the pH gate and the hinge region. However, some studies imply that these two gates might be uncoupled or weakly coupled. For example, X-ray crystallographic studies of KcsA, where K⁺ sensitivity was largely isolated to the selectivity filter (3). In this work, we asked whether, by contrast, full-length wild-type KcsA (160 aa) reconstituted into hydrated lipid bilayers exhibits global structural changes upon ion expulsion suggestive of channel opening. To accomplish this, nearly complete ¹³C and ¹⁵N chemical shift assignments were obtained for the transmembrane and loop regions from four-dimensional (4D) solid-state nuclear magnetic resonance (SSNMR) (27), providing numerous reporters for conformational change during ion binding. In low [K⁺] ion conditions at neutral pH, not only does KcsA expel the K⁺ ions from the inner selectivity filter sites, but the channel also exhibits chemical shift perturbations at the pH gate and the hinge of the inner transmembrane helix, suggesting features akin to the inhibited state that is present at low pH and high [K⁺]. That these two distinct conditions result in a nearly identical state of the channel offers strong evidence for transmembrane allostery in the inactivation process.     

Dynamics transitions at the outer vestibule of the KcsA potassium channel during gating

In K⁺ channels, the selectivity filter, pore helix, and outer vestibule play a crucial role in gating mechanisms. The outer vestibule is an important structurally extended region of KcsA in which toxins, blockers, and metal ions bind and modulate the gating behavior of K⁺ channels. Despite its functional significance, the gating-related structural dynamics at the outer vestibule are not well understood. Under steady-state conditions, inactivating WT and noninactivating E71A KcsA stabilize the nonconductive and conductive filter conformations upon opening the activation gate. Site-directed fluorescence polarization of 7-nitrobenz-2-oxa-1,3-diazol-4-yl (NBD)-labeled outer vestibule residues shows that the outer vestibule of open/conductive conformation is highly dynamic compared with the motional restriction experienced by the outer vestibule during inactivation gating. A wavelength-selective fluorescence approach shows a change in hydration dynamics in inactivated and noninactivated conformations, and supports a possible role of restricted/bound water molecules in C-type inactivation gating. Using a unique restrained ensemble simulation method, along with distance measurements by EPR, we show that, on average, the outer vestibule undergoes a modest backbone conformational change during its transition to various functional states, although the structural dynamics of the outer vestibule are significantly altered during activation and inactivation gating. Taken together, our results support the role of a hydrogen bond network behind the selectivity filter, side-chain conformational dynamics, and water molecules in the gating mechanisms of K⁺ channels.The functional behavior of K⁺ channels is defined by a series of structural rearrangements associated with the processes of activation and inactivation gating (1–6). In response to a prolonged stimulus and in the absence of an N-terminal inactivating particle, most K⁺ channels become nonconductive through a process known as C-type inactivation (7). This C-type inactivation is crucial in controlling the firing patterns in excitable cells and is fundamental in determining the length and frequency of the cardiac action potential (8). C-type inactivation is inhibited by high extracellular K⁺ (9, 10), and the blocker tetraethylammonium (TEA) (11) can also be slowed down in the presence of permeant ions with a long residence time in the selectivity filter (Rb⁺, Cs⁺, and NH⁴⁺) (10).The prokaryotic pH-gated K⁺ channel KcsA shares most of the mechanistic properties of C-type inactivation in voltage-dependent K⁺ channels (5, 6, 12–16). Recent crystal structures of open/inactivated KcsA reveal that there is a remarkable correlation between the degree of opening at the activation gate and the conformation and ion occupancy of the selectivity filter (5). In KcsA, the selectivity filter is stabilized by a hydrogen bond network, with key interactions between residues Glu71, Asp80, and Trp67 and a bound water molecule (17). Disrupting this hydrogen bond network favors the conductive conformation of the selectivity filter (12, 13, 15).Early electrophysiological experiments have suggested that the outer vestibule (around T449 residue in Shaker and Y82 residue in KcsA) undergoes significant conformational rearrangement during C-type inactivation gating (16, 18, 19). However, comparison of the WT KcsA crystal structure, where the filter is in its conductive conformation, with either the structure obtained with low K⁺ (collapsed filter) (17) or the crystal structure of open-inactivated KcsA with maximum opening (inactivated filter) (5) does not show major conformational changes in the outer vestibule that would explain these results (Fig. 1A). We have suggested that this apparent discrepancy can be understood if we take into consideration the potential differences in the dynamic behavior of the outer vestibule changes as the K⁺ channel undergoes its gating cycle (16).Open in a separate windowFig. 1.Comparison of outer vestibule conformation in KcsA structures with conductive and collapsed/inactivated filters. (A) High-K⁺ KcsA structure [Protein Data Bank (PDB) ID code 1K4C; yellow] is compared with a low-K⁺ KcsA structure (PDB ID code 1K4D; blue) in the closed state (Left) and open/inactivated conformation (PDB ID code 3F5W; green) (Right). The outer vestibule residues are depicted as red spheres, and relevant residues are labeled. (B) Schematic representation of typical macroscopic currents elicited by pH-jump experiments in WT (inactivating) and E71A (noninactivating) KcsA channels at a depolarizing membrane potential is shown. Conditions that stabilize the closed, open/inactivated, and open/conductive conformations at the steady state are indicated with a black circle. (C) Effect of opening the lower gate on the mobility of spin-labeled outer vestibule residues in palmitoyloleoylphosphatidyl choline/palmitoyloleoylphosphatidyl glycerol (POPC/POPG) (3:1, moles/moles) reconstituted WT (Left) and noninactivating mutant E71A (Right) backgrounds for the closed (pH 7, red) and open (pH 4, black) states of KcsA, as determined by continuous wave (CW) EPR. The spectra shown are amplitude-normalized. Details are provided in SI Materials and Methods.We have probed the gating-induced structural dynamics at the outer vestibule of KcsA using site-directed fluorescence and site-directed spin labeling and pulsed EPR approaches in combination with a recently developed computational method, restrained ensemble (RE) simulations. RE simulation was used to constrain the outer vestibule using experimentally derived distance histograms in different functional states (closed, open/inactivated, and open/conductive) and to monitor the extent of backbone conformational changes during gating. To this end, we took advantage of our ability to stabilize both the open/conductive (E71A mutant) and the open/inactivated (WT) conformations of KcsA upon opening the activation gate under steady-state conditions (Fig. 1B).Our data show that the outer vestibule in the open/conductive conformation is highly dynamic. In addition, the red edge excitation shift (REES) points to a change in hydration dynamics between conductive and nonconductive outer vestibule conformations, suggesting a role of restricted water molecules in C-type inactivation gating. We suggest that, on average, the backbone conformation of the outer vestibule does not change significantly between different functional states but that local dynamics change significantly, underlining the importance of the hydrogen bond network behind the selectivity filter and the microscopic observables (e.g., dynamics of hydration) in K⁺ channel gating and C-type inactivation.     

Rab3-interacting molecules 2α and 2β promote the abundance of voltage-gated CaV1.3 Ca2+ channels at hair cell active zones

Ca²⁺ influx triggers the fusion of synaptic vesicles at the presynaptic active zone (AZ). Here we demonstrate a role of Ras-related in brain 3 (Rab3)–interacting molecules 2α and β (RIM2α and RIM2β) in clustering voltage-gated Ca_V1.3 Ca²⁺ channels at the AZs of sensory inner hair cells (IHCs). We show that IHCs of hearing mice express mainly RIM2α, but also RIM2β and RIM3γ, which all localize to the AZs, as shown by immunofluorescence microscopy. Immunohistochemistry, patch-clamp, fluctuation analysis, and confocal Ca²⁺ imaging demonstrate that AZs of RIM2α-deficient IHCs cluster fewer synaptic Ca_V1.3 Ca²⁺ channels, resulting in reduced synaptic Ca²⁺ influx. Using superresolution microscopy, we found that Ca²⁺ channels remained clustered in stripes underneath anchored ribbons. Electron tomography of high-pressure frozen synapses revealed a reduced fraction of membrane-tethered vesicles, whereas the total number of membrane-proximal vesicles was unaltered. Membrane capacitance measurements revealed a reduction of exocytosis largely in proportion with the Ca²⁺ current, whereas the apparent Ca²⁺ dependence of exocytosis was unchanged. Hair cell-specific deletion of all RIM2 isoforms caused a stronger reduction of Ca²⁺ influx and exocytosis and significantly impaired the encoding of sound onset in the postsynaptic spiral ganglion neurons. Auditory brainstem responses indicated a mild hearing impairment on hair cell-specific deletion of all RIM2 isoforms or global inactivation of RIM2α. We conclude that RIM2α and RIM2β promote a large complement of synaptic Ca²⁺ channels at IHC AZs and are required for normal hearing.Tens of Ca_V1.3 Ca²⁺ channels are thought to cluster within the active zone (AZ) membrane underneath the presynaptic density of inner hair cells (IHCs) (1–4). They make up the key signaling element, coupling the sound-driven receptor potential to vesicular glutamate release (5–7). The mechanisms governing the number of Ca²⁺ channels at the AZ as well as their spatial organization relative to membrane-tethered vesicles are not well understood. Disrupting the presynaptic scaffold protein Bassoon diminishes the numbers of Ca²⁺ channels and membrane-tethered vesicles at the AZ (2, 8). However, the loss of Bassoon is accompanied by the loss of the entire synaptic ribbon, which makes it challenging to distinguish the direct effects of gene disruption from secondary effects (9).Among the constituents of the cytomatrix of the AZ, RIM1 and RIM2 proteins are prime candidates for the regulation of Ca²⁺ channel clustering and function (10, 11). The family of RIM proteins has seven identified members (RIM1α, RIM1β, RIM2α, RIM2β, RIM2γ, RIM3γ, and RIM4γ) encoded by four genes (RIM1–RIM4). All isoforms contain a C-terminal C₂ domain but differ in the presence of additional domains. RIM1 and RIM2 interact with Ca²⁺ channels, most other proteins of the cytomatrix of the AZ, and synaptic vesicle proteins. They interact directly with the auxiliary β (Ca_Vβ) subunits (12, 13) and pore-forming Ca_Vα subunits (14, 15). In addition, RIMs are indirectly linked to Ca²⁺ channels via RIM-binding protein (14, 16, 17). A regulation of biophysical channel properties has been demonstrated in heterologous expression systems for RIM1 (12) and RIM2 (13).A role of RIM1 and RIM2 in clustering Ca²⁺ channels at the AZ was demonstrated by analysis of RIM1/2-deficient presynaptic terminals of cultured hippocampal neurons (14), auditory neurons in slices (18), and Drosophila neuromuscular junction (19). Because α-RIMs also bind the vesicle-associated protein Ras-related in brain 3 (Rab3) via the N-terminal zinc finger domain (20), they are also good candidates for molecular coupling of Ca²⁺ channels and vesicles (18, 21, 22). Finally, a role of RIMs in priming of vesicles for fusion is the subject of intense research (18, 21–27). RIMs likely contribute to priming via disinhibiting Munc13 (26) and regulating vesicle tethering (27). Here, we studied the expression and function of RIM in IHCs. We combined molecular, morphologic, and physiologic approaches for the analysis of RIM2α knockout mice [RIM2α SKO (28); see Methods] and of hair cell-specific RIM1/2 knockout mice (RIM1/2 cDKO). We demonstrate that RIM2α and RIM2β are present at IHC AZs of hearing mice, positively regulate the number of synaptic Ca_V1.3 Ca²⁺ channels, and are required for normal hearing.     

Major diversification of voltage-gated K+ channels occurred in ancestral parahoxozoans

We examined the origins and functional evolution of the Shaker and KCNQ families of voltage-gated K⁺ channels to better understand how neuronal excitability evolved. In bilaterians, the Shaker family consists of four functionally distinct gene families (Shaker, Shab, Shal, and Shaw) that share a subunit structure consisting of a voltage-gated K⁺ channel motif coupled to a cytoplasmic domain that mediates subfamily-exclusive assembly (T1). We traced the origin of this unique Shaker subunit structure to a common ancestor of ctenophores and parahoxozoans (cnidarians, bilaterians, and placozoans). Thus, the Shaker family is metazoan specific but is likely to have evolved in a basal metazoan. Phylogenetic analysis suggested that the Shaker subfamily could predate the divergence of ctenophores and parahoxozoans, but that the Shab, Shal, and Shaw subfamilies are parahoxozoan specific. In support of this, putative ctenophore Shaker subfamily channel subunits coassembled with cnidarian and mouse Shaker subunits, but not with cnidarian Shab, Shal, or Shaw subunits. The KCNQ family, which has a distinct subunit structure, also appears solely within the parahoxozoan lineage. Functional analysis indicated that the characteristic properties of Shaker, Shab, Shal, Shaw, and KCNQ currents evolved before the divergence of cnidarians and bilaterians. These results show that a major diversification of voltage-gated K⁺ channels occurred in ancestral parahoxozoans and imply that many fundamental mechanisms for the regulation of action potential propagation evolved at this time. Our results further suggest that there are likely to be substantial differences in the regulation of neuronal excitability between ctenophores and parahoxozoans.Voltage-gated K⁺ channels are highly conserved among bilaterian metazoans and play a central role in the regulation of excitation in neurons and muscle. Understanding the functional evolution of these channels may therefore provide important insights into how neuromuscular excitation evolved within the Metazoa. Three major gene families, Shaker, KCNQ, and Ether-a-go-go (EAG) encode all voltage-gated K⁺ channels in bilaterians (1, 2). In this study, we examine the functional evolution and origins of the Shaker and KCNQ gene families. Shaker family channels can be definitively identified by a unique subunit structure that includes both a voltage-gated K⁺ channel core and a family-specific cytoplasmic domain within the N terminus known as the T1 domain. T1 mediates assembly of Shaker family subunits into functional tetrameric channels (3, 4). KCNQ channels are also tetrameric but lack a T1 domain and use a distinct coiled-coil assembly domain in the C terminus (5, 6). KCNQ channels can be identified by the presence of this family-specific assembly motif and high amino acid conservation within the K⁺ channel core. Both channel families are found in cnidarians (1, 7) and thus predate the divergence of cnidarians and bilaterians, but their ultimate evolutionary origins have not yet been defined.Shaker family K⁺ channels serve diverse roles in the regulation of neuronal firing and can be divided into four gene subfamilies based on function and sequence homology: Shaker, Shab, Shal, and Shaw (8, 9). The T1 assembly domain is only compatible between subunits from the same gene subfamily (4, 10) and thus serves to keep the subfamilies functionally segregated. Shaker subfamily channels activate rapidly near action potential threshold and range from rapidly inactivating to noninactivating. Multiple roles for Shaker channels in neurons and muscles have been described, but their most unique and fundamental role may be that of axonal action potential repolarization. Shaker channels are clustered to the axon initial segment and nodes of Ranvier in vertebrate neurons (11–13) and underlie the delayed rectifier in squid giant axons (14). The Shaker subfamily is diverse in cnidarians (15, 16), and the starlet sea anemone Nematostella vectensis has functional orthologs of most identified Shaker current types observed in bilaterians (16).The Shab and Shal gene subfamilies encode somatodendritic delayed rectifiers and A currents, respectively (17–20). Shab channels are important for maintaining sustained firing (21, 22), whereas the Kv4-based A current modulates spike threshold and frequency (17). Shab and Shal channels are present in cnidarians, but cnidarian Shab channels have not been functionally characterized, and the only cnidarian Shal channels expressed to date display atypical voltage dependence and kinetics compared with bilaterian channels (23). Shaw channels are rapid, high-threshold channels specialized for sustaining fast firing in vertebrates (24, 25) but have a low activation threshold and may contribute to resting potential in Drosophila (19, 26, 27). A Caenorhabditis elegans Shaw has slow kinetics but a high activation threshold (28), and a single expressed cnidarian Shaw channel has the opposite: a low activation threshold but relatively fast kinetics (29). Thus, the ancestral properties and function of Shaw channels is not yet understood. Further functional characterization of cnidarian Shab, Shal, and Shaw channels would provide a better understanding of the evolutionary status of the Shaker family in early parahoxozoans.KCNQ family channels underlie the M current in vertebrate neurons (30) that regulates subthreshold excitability (31). The M current provides a fundamental mechanism for regulation of firing threshold through the Gq G-protein pathway because KCNQ channels require phosphatidylinositol 4,5-bisphosphate (PIP₂) for activation (32, 33). PIP₂ hydrolysis and subsequent KCNQ channel closure initiated by Gq-coupled receptors produces slow excitatory postsynaptic potentials, during which the probability of firing is greatly increased (32, 33). The key functional adaptations of KCNQ channels for this physiological role that can be observed in vitro are (i) a requirement for PIP₂ to couple voltage-sensor activation to pore opening (34, 35), and (ii) a hyperpolarized voltage–activation curve that allows channels to open below typical action potential thresholds. Both key features are found in vertebrate (30, 34, 36–38), Drosophila (39), and C. elegans (40) KCNQ channels, suggesting they may have been present in KCNQ channels in a bilaterian ancestor. Evolution of the M current likely represented a major advance in the ability to modulate the activity of neuronal circuits, but it is not yet clear when PIP₂-dependent KCNQ channels first evolved.Here, we examine the origins and functional evolution of the Shaker and KCNQ gene families. If we assume the evolution of neuronal signaling provided a major selective pressure for the functional diversification of voltage-gated K⁺ channels, then we can hypothesize that the appearance of these gene families might accompany the emergence of the first nervous systems or a major event in nervous system evolution. Recent phylogenies that place the divergence of ctenophores near the root of the metazoan tree suggest that the first nervous systems, or at least the capacity to make neurons, may have been present in a basal metazoan ancestor (41–43) (Fig. S1). One hypothesis then is that much of the diversity of metazoan voltage-gated channels should be shared between ctenophores and parahoxozoans [cnidarians, bilaterians, and placozoans (44)]. However, genome analysis indicates that many “typical” neuronal genes are missing in ctenophores and the sponges lack a nervous system, leading to the suggestion that extant nervous systems may have evolved independently in ctenophores and parahoxozoans (42, 45). Thus, a second hypothesis is that important steps in voltage-gated K⁺ channel evolution might have occurred separately in ctenophores and parahoxozoans. We tested these hypotheses by carefully examining the phylogenetic distribution and functional evolution of Shaker and KCNQ family K⁺ channels. Our results support a model in which major innovations in neuromuscular excitability occurred specifically within the parahoxozoan lineage.     

The Two-pore channel (TPC) interactome unmasks isoform-specific roles for TPCs in endolysosomal morphology and cell pigmentation

The two-pore channels (TPC1 and TPC2) belong to an ancient family of intracellular ion channels expressed in the endolysosomal system. Little is known about how regulatory inputs converge to modulate TPC activity, and proposed activation mechanisms are controversial. Here, we compiled a proteomic characterization of the human TPC interactome, which revealed that TPCs complex with many proteins involved in Ca²⁺ homeostasis, trafficking, and membrane organization. Among these interactors, TPCs were resolved to scaffold Rab GTPases and regulate endomembrane dynamics in an isoform-specific manner. TPC2, but not TPC1, caused a proliferation of endolysosomal structures, dysregulating intracellular trafficking, and cellular pigmentation. These outcomes required both TPC2 and Rab activity, as well as their interactivity, because TPC2 mutants that were inactive, or rerouted away from their endogenous expression locale, or deficient in Rab binding, failed to replicate these outcomes. Nicotinic acid adenine dinucleotide phosphate (NAADP)-evoked Ca²⁺ release was also impaired using either a Rab binding-defective TPC2 mutant or a Rab inhibitor. These data suggest a fundamental role for the ancient TPC complex in trafficking that holds relevance for lysosomal proliferative scenarios observed in disease.Two-pore channels (TPCs) are an ancient family of intracellular ion channels and a likely ancestral stepping stone in the evolution of voltage-gated Ca²⁺ and Na⁺ channels (1). Architecturally, TPCs resemble a halved voltage-gated Ca²⁺/Na⁺ channel with cytosolic NH₂ and COOH termini, comprising two repeats of six transmembrane spanning helices with a putative pore-forming domain between the fifth and sixth membrane-spanning regions. Since their discovery in vertebrate systems, many studies have investigated the properties of these channels (2–7) that may support such a lengthy evolutionary pedigree.In this context, demonstration that (i) the two human TPC isoforms (TPC1 and TPC2) are uniquely distributed within the endolysosomal system (2, 3) and that (ii) TPC channel activity is activated by the Ca²⁺ mobilizing molecule nicotinic acid adenine dinucleotide phosphate (NAADP) (4–6) generated considerable excitement that TPCs function as effectors of this mercurial second messenger long known to trigger Ca²⁺ release from “acidic stores.” The spectrum of physiological activities that have been linked to NAADP signaling over the last 25 years (8, 9) may therefore be realized through regulation of TPC activity. However, recent studies have questioned the idea that TPCs are NAADP targets (10, 11), demonstrating instead that TPCs act as Na⁺ channels regulated by the endolysosomal phosphoinositide PI(3,5)P₂. Such controversy (12, 13) underscores how little we know about TPC regulatory inputs and the dynamic composition of TPC complexes within cells.Here, to generate unbiased insight into the cell biology of the TPC complex, we report a proteomic analysis of human TPCs. The TPC interactome establishes a useful community resource as a “rosetta stone” for interrogating the cell biology of TPCs and their regulation. The dataset reveals a predomination of links between TPCs and effectors controlling membrane organization and trafficking, relevant for disease states involving lysosomal proliferation where TPC functionality may be altered (14).     

Role of protein dynamics in ion selectivity and allosteric coupling in the NaK channel

Flux-dependent inactivation that arises from functional coupling between the inner gate and the selectivity filter is widespread in ion channels. The structural basis of this coupling has only been well characterized in KcsA. Here we present NMR data demonstrating structural and dynamic coupling between the selectivity filter and intracellular constriction point in the bacterial nonselective cation channel, NaK. This transmembrane allosteric communication must be structurally different from KcsA because the NaK selectivity filter does not collapse under low-cation conditions. Comparison of NMR spectra of the nonselective NaK and potassium-selective NaK2K indicates that the number of ion binding sites in the selectivity filter shifts the equilibrium distribution of structural states throughout the channel. This finding was unexpected given the nearly identical crystal structure of NaK and NaK2K outside the immediate vicinity of the selectivity filter. Our results highlight the tight structural and dynamic coupling between the selectivity filter and the channel scaffold, which has significant implications for channel function. NaK offers a distinct model to study the physiologically essential connection between ion conduction and channel gating.Ion conduction through the pore domain of cation channels is regulated by two gates: an inner gate at the bundle crossing of the pore-lining transmembrane helices and an outer gate located at the selectivity filter (Fig. 1 B and C). These two gates are functionally coupled as demonstrated by C-type inactivation, in which channel opening triggers loss of conduction at the selectivity filter (1–4). A structural model for C-type inactivation has been developed for KcsA, with selectivity filter collapse occurring upon channel opening (4–10). In the reverse pathway, inactivation of the selectivity filter has been linked to changes at the inner gate (5–14). However, flux-dependent inactivation occurs in Na⁺ and Ca²⁺ channels as well and would likely require a structurally different mechanism to explain coupling between the selectivity filter and inner gate (7, 13–18).Open in a separate windowFig. 1.Crystal structures of the nonselective cation channel NaK and the potassium-selective NaK2K mutant show structural changes restricted to the area of the selectivity filter. Alignment of the WT NaK (gray; PDB 3E8H) and NaK2K (light blue; PDB 3OUF) selectivity filters shows a KcsA-like four-ion-binding-site selectivity filter is created by the NaK2K mutations (D66Y and N68D) (A), but no structural changes occur outside the vicinity of the selectivity filter (B). (C) Full-length NaK (green; PDB 2AHZ) represents a closed conformation. Alignment of this structure with NaK (gray) highlights the changes in the M2 hinge (arrow), hydrophobic cluster (residues F24, F28, and F94 shown as sticks), and constriction point (arrow; residue Q103 shown as sticks) upon channel opening. Two (A) or three monomers (B and C) from the tetramer are shown for clarity.This study provides experimental evidence of structural and dynamic coupling between the inner gate and selectivity filter in the NaK channel, a nonselective cation channel from Bacillus cereus (19). These results were entirely unexpected given the available high-resolution crystal structures (20, 21). The NaK channel has the same basic pore architecture as K⁺ channels (Fig. 1 B and C) and has become a second model system for investigating ion selectivity and gating due to its distinct selectivity filter sequence (₆₃TVGDGN₆₈) and structure (19–23). Most strikingly, there are only two ion binding sites in the selectivity filter of the nonselective NaK channel (Fig. 1A) (21, 24). However, mutation of two residues in the selectivity filter sequence converts the NaK selectivity filter to the canonical KcsA sequence (₆₃TVGYGD₆₈; Fig. 1 A and B), leading to K⁺ selectivity and a KcsA-like selectivity filter structure with four ion binding sites (21, 23). This K⁺-selective mutant of NaK is called NaK2K. Outside of the immediate vicinity of the two mutations in the selectivity filter, high-resolution crystal structures of NaK and NaK2K are essentially identical (Fig. 1B) with an all-atom rmsd of only 0.24 Å.NaK offers a distinct model to study the physiologically essential connection between ion conduction and channel gating because there is no evidence for any collapse or structural change in the selectivity filter. The NaK selectivity filter structure is identical in Na⁺ or K⁺ (22) and even in low-ion conditions (25), consistent with its nonselective behavior. Even the selective NaK2K filter appears structurally stable in all available crystal structures (25). Here we use NMR spectroscopy to study bicelle-solubilized NaK. Surprisingly, we find significant differences in the NMR spectra of NaK and NaK2K that extend throughout the protein and are not localized to the selectivity filter region. This, combined with NMR dynamics studies of NaK, suggests a dynamic pathway for transmembrane coupling between the inner gate and selectivity filter of NaK.     

Conformational cycle and ion-coupling mechanism of the Na+/hydantoin transporter Mhp1

Ion-dependent transporters of the LeuT-fold couple the uptake of physiologically essential molecules to transmembrane ion gradients. Defined by a conserved 5-helix inverted repeat that encodes common principles of ion and substrate binding, the LeuT-fold has been captured in outward-facing, occluded, and inward-facing conformations. However, fundamental questions relating to the structural basis of alternating access and coupling to ion gradients remain unanswered. Here, we used distance measurements between pairs of spin labels to define the conformational cycle of the Na⁺-coupled hydantoin symporter Mhp1 from Microbacterium liquefaciens. Our results reveal that the inward-facing and outward-facing Mhp1 crystal structures represent sampled intermediate states in solution. Here, we provide a mechanistic context for these structures, mapping them into a model of transport based on ion- and substrate-dependent conformational equilibria. In contrast to the Na⁺/leucine transporter LeuT, our results suggest that Na⁺ binding at the conserved second Na⁺ binding site does not change the energetics of the inward- and outward-facing conformations of Mhp1. Comparative analysis of ligand-dependent alternating access in LeuT and Mhp1 lead us to propose that different coupling schemes to ion gradients may define distinct conformational mechanisms within the LeuT-fold class.Secondary active transporters harness the energy of ion gradients to power the uphill movement of solutes across membranes. Mitchell (1) and others (2, 3) proposed and elaborated “alternating access” mechanisms wherein the transporter transitions between two conformational states that alternately expose the substrate binding site to the two sides of the membrane. The LeuT class of ion-coupled symporters consists of functionally distinct transporters that share a conserved scaffold of two sets of five transmembrane helices related by twofold symmetry around an axis nearly parallel to the membrane (4). Ions and substrates are bound near the middle of the membrane stabilized by electrostatic interactions with unwound regions of transmembrane helix (TM) 1 and often TM6 (4). The recurrence of this fold in transporters that play critical roles in fundamental physiological processes (5, 6) has spurred intense interest in defining the principles of alternating access.Despite rapid progress in structure determination of ion-coupled LeuT-fold transporters (7–11), extrapolation of these static snapshots to a set of conformational steps underlying alternating access (4, 7, 9–12) remains incomplete, often hindered by uncertainties in the mechanistic identities of crystal structures. Typically, transporter crystal structures are classified as inward-facing, outward-facing, or occluded on the basis of the accessibility of the substrate binding site (7–11). In a recent spectroscopic analysis of LeuT, we demonstrated that detergent selection and mutations of conserved residues appeared to stabilize conformations that were not detected in the wild-type (WT) LeuT and concurrently inhibited movement of structural elements involved in ligand-dependent alternating access (13). Therefore, although crystal structures define the structural context and identify plausible pathways of substrate binding and release, development of transport models requires confirming or assigning the mechanistic identity of these structures and framing them into ligand-dependent equilibria (14).Mhp1, an Na⁺-coupled symporter of benzyl-hydantoin (BH) from Microbacterium liquefaciens, was the first LeuT-fold member to be characterized by crystal structures purported to represent outward-facing, inward-facing, and outward-facing/occluded conformations of an alternating access cycle (8, 15). In these structures, solvent access to ligand-binding sites is defined by the relative orientation between a 4-helix bundle motif and a 4-helix scaffold motif (8). In Mhp1, alternating access between inward- and outward-facing conformations, was predicted from a computational analysis based on the inverted repeat symmetry of the LeuT fold and is referred to as the rocking-bundle model (16). The conservation of the inverted symmetry prompted proposal of the rocking-bundle mechanism as a general model for LeuT-fold transporters (16). Subsequent crystal structures of other LeuT-fold transporters (7, 9, 10) tempered this prediction because the diversity of the structural rearrangements implicit in these structures is seemingly inconsistent with a conserved conformational cycle.Another outstanding question pertains to the ion-coupling mechanism and the driving force of conformational changes. The implied ion-to-substrate stoichiometry varies across LeuT-fold ion-coupled transporters. For instance, LeuT (17) and BetP (18) require two Na⁺ ions that bind at two distinct sites referred to as Na1 and Na2 whereas Mhp1 (15) and vSGLT (19) appear to possess only the conserved Na2 site. Molecular dynamics (MD) simulations (20, 21) and electron paramagnetic resonance (EPR) analysis (13, 22) of LeuT demonstrated that Na⁺ binding favors an outward-facing conformation although it is unclear which Na⁺ site (or both) is responsible for triggering this conformational transition. Similarly, a role for Na⁺ in conformational switching has been uncovered in putative human LeuT-fold transporters, including hSGLT (23). In Mhp1, the sole Na2 site has been shown to modulate substrate affinity (15); however, its proposed involvement in gating of the intracellular side (12, 21) lacks experimental validation.Here, we used site-directed spin labeling (SDSL) (24) and double electron-electron resonance (DEER) spectroscopy (25) to elucidate the conformational changes underlying alternating access in Mhp1 and define the role of ion and substrate binding in driving transition between conformations. This methodology has been successfully applied to define coupled conformational cycles for a number of transporter classes (13, 26–32). We find that patterns of distance distributions between pairs of spin labels monitoring the intra- and extracellular sides of Mhp1 are consistent with isomerization between the crystallographic inward- and outward-facing conformations. A major finding is that this transition is driven by substrate but not Na⁺ binding. Although the amplitudes of the observed distance changes are in overall agreement with the rocking-bundle model deduced from the crystal structures of Mhp1 (8, 15) and predicted computationally (16), we present evidence that relative movement of bundle and scaffold deviate from strict rigid body. Comparative analysis of LeuT and Mhp1 alternating access reveal how the conserved LeuT fold harnesses the energy of the Na⁺ gradient through two distinct coupling mechanisms and supports divergent conformational cycles to effect substrate binding and release.     

Structural interactions of a voltage sensor toxin with lipid membranes

Protein toxins from tarantula venom alter the activity of diverse ion channel proteins, including voltage, stretch, and ligand-activated cation channels. Although tarantula toxins have been shown to partition into membranes, and the membrane is thought to play an important role in their activity, the structural interactions between these toxins and lipid membranes are poorly understood. Here, we use solid-state NMR and neutron diffraction to investigate the interactions between a voltage sensor toxin (VSTx1) and lipid membranes, with the goal of localizing the toxin in the membrane and determining its influence on membrane structure. Our results demonstrate that VSTx1 localizes to the headgroup region of lipid membranes and produces a thinning of the bilayer. The toxin orients such that many basic residues are in the aqueous phase, all three Trp residues adopt interfacial positions, and several hydrophobic residues are within the membrane interior. One remarkable feature of this preferred orientation is that the surface of the toxin that mediates binding to voltage sensors is ideally positioned within the lipid bilayer to favor complex formation between the toxin and the voltage sensor.Protein toxins from venomous organisms have been invaluable tools for studying the ion channel proteins they target. For example, in the case of voltage-activated potassium (Kv) channels, pore-blocking scorpion toxins were used to identify the pore-forming region of the channel (1, 2), and gating modifier tarantula toxins that bind to S1–S4 voltage-sensing domains have helped to identify structural motifs that move at the protein–lipid interface (3–5). In many instances, these toxin–channel interactions are highly specific, allowing them to be used in target validation and drug development (6–8).Tarantula toxins are a particularly interesting class of protein toxins that have been found to target all three families of voltage-activated cation channels (3, 9–12), stretch-activated cation channels (13–15), as well as ligand-gated ion channels as diverse as acid-sensing ion channels (ASIC) (16–21) and transient receptor potential (TRP) channels (22, 23). The tarantula toxins targeting these ion channels belong to the inhibitor cystine knot (ICK) family of venom toxins that are stabilized by three disulfide bonds at the core of the molecule (16, 17, 24–31). Although conventional tarantula toxins vary in length from 30 to 40 aa and contain one ICK motif, the recently discovered double-knot toxin (DkTx) that specifically targets TRPV1 channels contains two separable lobes, each containing its own ICK motif (22, 23).One unifying feature of all tarantula toxins studied thus far is that they act on ion channels by modifying the gating properties of the channel. The best studied of these are the tarantula toxins targeting voltage-activated cation channels, where the toxins bind to the S3b–S4 voltage sensor paddle motif (5, 32–36), a helix-turn-helix motif within S1–S4 voltage-sensing domains that moves in response to changes in membrane voltage (37–41). Toxins binding to S3b–S4 motifs can influence voltage sensor activation, opening and closing of the pore, or the process of inactivation (4, 5, 36, 42–46). The tarantula toxin PcTx1 can promote opening of ASIC channels at neutral pH (16, 18), and DkTx opens TRPV1 in the absence of other stimuli (22, 23), suggesting that these toxin stabilize open states of their target channels.For many of these tarantula toxins, the lipid membrane plays a key role in the mechanism of inhibition. Strong membrane partitioning has been demonstrated for a range of toxins targeting S1–S4 domains in voltage-activated channels (27, 44, 47–50), and for GsMTx4 (14, 50), a tarantula toxin that inhibits opening of stretch-activated cation channels in astrocytes, as well as the cloned stretch-activated Piezo1 channel (13, 15). In experiments on stretch-activated channels, both the d- and l-enantiomers of GsMTx4 are active (14, 50), implying that the toxin may not bind directly to the channel. In addition, both forms of the toxin alter the conductance and lifetimes of gramicidin channels (14), suggesting that the toxin inhibits stretch-activated channels by perturbing the interface between the membrane and the channel. In the case of Kv channels, the S1–S4 domains are embedded in the lipid bilayer and interact intimately with lipids (48, 51, 52) and modification in the lipid composition can dramatically alter gating of the channel (48, 53–56). In one study on the gating of the Kv2.1/Kv1.2 paddle chimera (53), the tarantula toxin VSTx1 was proposed to inhibit Kv channels by modifying the forces acting between the channel and the membrane. Although these studies implicate a key role for the membrane in the activity of Kv and stretch-activated channels, and for the action of tarantula toxins, the influence of the toxin on membrane structure and dynamics have not been directly examined. The goal of the present study was to localize a tarantula toxin in membranes using structural approaches and to investigate the influence of the toxin on the structure of the lipid bilayer.     

Cadmium–cysteine coordination in the BK inner pore region and its structural and functional implications

To probe structure and gating-associated conformational changes in BK-type potassium (BK) channels, we examined consequences of Cd²⁺ coordination with cysteines introduced at two positions in the BK inner pore. At V319C, the equivalent of valine in the conserved Kv proline-valine-proline (PVP) motif, Cd²⁺ forms intrasubunit coordination with a native glutamate E321, which would place the side chains of V319C and E321 much closer together than observed in voltage-dependent K⁺ (Kv) channel structures, requiring that the proline between V319C and E321 introduces a kink in the BK S6 inner helix sharper than that observed in Kv channel structures. At inner pore position A316C, Cd²⁺ binds with modest state dependence, suggesting the absence of an ion permeation gate at the cytosolic side of BK channel. These results highlight fundamental structural differences between BK and Kv channels in their inner pore region, which likely underlie differences in voltage-dependent gating between these channels.How transmembrane potential influences the opening and closing of ion channels, a process known as gating, is central to understanding how cellular excitability is regulated (1). For voltage-dependent K⁺ (Kv) channels, functional (2–4) and crystallographic (5, 6) studies have led to a compelling model of gating. In this model there are two key elements, both of which arise from properties of the cytosolic end of Kv channels: a cytosolic ion permeation gate formed by an interlaced arrangement of S6 inner helices termed the bundle crossing (2, 3), and a kink produced by the conserved proline-valine-proline (PVP) motif (Fig. 1A, boxed residues) to allow the C terminus of Kv S6 to form extensive contact with the S4–S5 linker (5, 6). Thus, the outward movement of the voltage sensors (VSDs) induced by transmembrane depolarization is thought to be transmitted to S6 through the S4–S5 linker to open the cytosolic ion permeation gate (6–8), and this enables access of cytosolic K⁺ ions to the Kv inner pore region.Open in a separate windowFig. 1.Cd²⁺ coordinates with cysteines separately introduced at two BK inner pore sites. (A) Sequence alignment of the S6 segment in the inner pore regions of Slo family channels, Kv channels, and the KcsA channel. The conserved glycine hinge and prolines in the PVP motif (boxed residues) are colored in red. The sites evaluated with cysteine substitution in BK S6 are underlined. A316 and V319 are indicated by arrowheads. (B) Radius profiles of Kv1.2 (blue) and KcsA (red) channels calculated from their crystal structures. The approximate locations of A316C and V319C are marked by horizontal dotted lines. SF, selectivity filter; IP, inner pore; BC, bundle crossing. (C–E) Currents of WT, V319C, and A316C recorded in 300 μM Ca²⁺ without (Left) and with (Right) 100 μM intracellular Cd²⁺. Voltage protocol was shown in E. (F) G–V relationships of WT (black circles), V319C (red triangles), and A316C (blue squares) in 300 μM Ca²⁺ without (open symbols) or with (filled symbols) 100 μM Cd²⁺. Boltzmann fit results (lines) are z = 1e, V_h = −3 mV (WT, control); z = 0.75e, V_h = −57 mV (WT, Cd²⁺); z = 0.94e, V_h = +69 mV (V319C, control); z = 0.6e, V_h = +136 mV (A316C, control); z = 0.32e, V_h = −66 mV (A316C, Cd²⁺).Although this model is generally accepted for Kv channel gating, it is not clear to what extent it applies to other K⁺ channels. For example, the large conductance, Ca²⁺-activated K⁺ (BK or Slo1) channel shares with Kv channels a similar set of four VSDs attached to a central pore and gate domain (PGD), such that both channels are voltage dependent (9). However, unlike that of Kv channels, the inner pore of a closed BK channels is accessible to large molecules such as quaternary ammonium (QA) blockers (10, 11) and methanethiosulfonate ethyltrimethylammonium (MTSET) (12), indicating that the cytosolic end of a closed BK channel cannot completely occlude K⁺ flow; this indicates that a gate extracellular to that proposed for Kv channels is required to securely prevent K⁺ flow in a closed BK channel. As a corollary, the underlying structural and conformational details required to couple VSD activation to channel opening may differ between BK and Kv channels.To provide new insight into differences between BK and Kv in the inner pore region, we have probed the cadmium (Cd²⁺) sensitivity of cysteine residues introduced in the BK S6 inner helix. Compared with cysteine modification by MTSET (12), Cd²⁺–Cys coordination provides two potential advantages for investigation of BK channel gating. First, Cd²⁺ coordination can involve multiple residues with strict distance and geometric requirements. This potentially provides information about the relative position of coordinating residues in the BK inner pore region, thereby placing important structural constraints on BK S6. Second, the size of Cd²⁺ is closer to that of a K⁺ ion compared with large probes such as QA blockers or MTSET; this allows a more accurate evaluation of whether there may be restriction to K⁺ flow at the cytosolic end of BK channel. Despite the evidence that large molecules can access the BK inner cavity in closed states, gated access of intracellularly applied Shaker ball peptide indicates that conformational changes do occur at the cytosolic end of BK channels (13). Furthermore, our previous study showed that the open-state modification of BK inner pore cysteines by MTSET is approximately two orders of magnitude faster than that in closed states (12), although the state dependence is weaker than that observed in Shaker channels (3). It is therefore important to determine the extent to which the BK cytosolic side may restrict inner pore access of K⁺ by using a probe similar in size to K⁺.Here we focus on two BK S6 residues, V319 and A316, which confer interesting sensitivity to Cd²⁺ on BK channels when replaced by cysteine. V319 is in register with Kv residues that contribute to the bundle crossing in Kv channels. The results reveal that Cd²⁺ coordination occurs between V319C and E321 in the same BK α-subunit, requiring that BK S6 adopts a novel kink at this level that is distinct from that in Kv channel structures. Furthermore, for A316C, which lines the BK inner pore, the Cd²⁺ coordination rate differs only modestly between open and closed states, suggesting there is no significant restriction of K⁺ access to the BK inner cavity in both states.     

Functional heterogeneity of the four voltage sensors of a human L-type calcium channel

Excitation-evoked Ca²⁺ influx is the fastest and most ubiquitous chemical trigger for cellular processes, including neurotransmitter release, muscle contraction, and gene expression. The voltage dependence and timing of Ca²⁺ entry are thought to be functions of voltage-gated calcium (Ca_V) channels composed of a central pore regulated by four nonidentical voltage-sensing domains (VSDs I–IV). Currently, the individual voltage dependence and the contribution to pore opening of each VSD remain largely unknown. Using an optical approach (voltage-clamp fluorometry) to track the movement of the individual voltage sensors, we discovered that the four VSDs of Ca_V1.2 channels undergo voltage-evoked conformational rearrangements, each exhibiting distinct voltage- and time-dependent properties over a wide range of potentials and kinetics. The voltage dependence and fast kinetic components in the activation of VSDs II and III were compatible with the ionic current properties, suggesting that these voltage sensors are involved in Ca_V1.2 activation. This view is supported by an obligatory model, in which activation of VSDs II and III is necessary to open the pore. When these data were interpreted in view of an allosteric model, where pore opening is intrinsically independent but biased by VSD activation, VSDs II and III were each found to supply ∼50 meV (∼2 kT), amounting to ∼85% of the total energy, toward stabilizing the open state, with a smaller contribution from VSD I (∼16 meV). VSD IV did not appear to participate in channel opening.Voltage-gated Ca²⁺ (Ca_V) channels respond to membrane depolarization by catalyzing Ca²⁺ influx. Ca_V-mediated elevation of intracellular [Ca²⁺] regulates such critical physiological functions as neurotransmitter and hormone release, axonal outgrowth, muscle contraction, and gene expression (1). Their relevance to human physiology is evident from the broad phenotypic consequences of Ca_V channelopathies (2). The voltage dependence of Ca_V-driven Ca²⁺ entry relies on the modular organization of the channel-forming α₁ subunit (Fig. 1), which consists of four repeated motifs (I–IV), each comprising six membrane-spanning helical segments (S1–S6) (Fig. 1A). Segments S1–S4 form a voltage-sensing domain (VSD), whereas segments S5 and S6 contribute to the Ca²⁺-conductive pore (1). The VSDs surround the central pore (Fig. 1B). VSDs are structurally and functionally conserved modules (3–5) capable of transducing a change in the cell membrane electrical potential into a change of ion-specific permeability or enzyme activity. VSDs sense depolarization by virtue of a signature motif of positively charged Arg or Lys at every third position of helix S4 (Fig. 1D), which rearranges in response to depolarization (4, 6–10). In contrast to voltage-gated K⁺ (K_V) channels but similar to pseudotetrameric voltage-gated Na⁺ (Na_V) channels, the amino acid sequences encoding each VSD have evolved independently (Fig. 1D). In addition to their distinct primary structure, the four Ca_V VSDs may also gain distinct functional properties from the asymmetrical association of auxiliary subunits, such as β, α₂δ, and calmodulin (1, 11–16) (Fig. 1C). The structural divergence among VSD-driven channels was foreseen by the classical Hodgkin–Huxley model (17), in which four independent “gating particles” control the opening of homotetrameric K_V channels and only three seem sufficient to open Na_V channels. An early study by Kostyuk et al. (18) suggested that only two gating particles are coupled to Ca_V channel opening. We recognize today that gating particles correspond to VSDs, and in Na_V channels, VSDs I–III control Na⁺ influx, whereas VSD IV is associated with fast inactivation (19–21).Open in a separate windowFig. 1.Ca_V membrane topology, putative structure, and S4 helix homology. (A) Ca_V channel-forming α₁ subunits consist of four concatenated repeats, each encompassing one voltage sensor domain (VSD) and a quarter of the central pore domain (PD) (1). Stars indicate the positions of fluorophore labeling. (B) The atomic structure of an Na_V channel (Protein Data Bank ID code 4EKW; top view) (56) shown as a structural representation for the Ca_V α₁ subunit. (C) The α₁ subunit asymmetrically associates with auxiliary β, α₂δ, and calmodulin (CaM) subunits (11–16). (D) Sequence alignment of VSD helix S4 from each of four Ca_V1.2 repeats and the archetypal homotetrameric Shaker K⁺ channel. Conserved, positively charged Arg or Lys is in blue. Residues substituted by Cys for fluorescent labeling are marked: F231 (VSD I), L614 (VSD II), V994 (VSD II), and S1324 (VSD IV).In this study, we used fluorometry to probe the properties of four individual VSDs in a human L-type calcium channel Ca_V1.2, which is a widely expressed regulator of physiological processes, such as cardiac and smooth muscle contractility (22). Although the collective transition of the Ca_V VSDs and the pore has been investigated in studies measuring total charge displacement (gating currents) (23, 24), the activation properties and functional roles of each VSD are unknown. Evidence for the role of each VSD in L-type Ca_V channel operation has been presented from charge neutralization studies, but a clear picture has yet to emerge. Work on a chimeric L-type channel suggests that VSDs I and III drive channel opening (25), whereas other studies on Ca_V1.2 favored the involvement of VSD II over VSD I, with the roles of VSDs III and IV remaining unclear (26, 27).The individual optical reports of four Ca_V1.2 VSDs revealed that each operates with distinct biophysical parameters. We found that VSDs II and III exhibit voltage- and time-dependent characteristics compatible with channel opening and that they can be considered rate-limiting for activation. We compared the voltage and time dependence of the fluorescent signals and ionic currents with the predictions of thermodynamic models relevant to Ca_V domain organization. We found that Ca_V1.2 activation is compatible with a model of allosteric VSD–pore coupling, where VSDs II and III are the primary drivers of channel opening with a smaller contribution by VSD I. We discuss the mechanism of Ca_V1.2 voltage sensitivity, which exhibits similarities to but also clear differences from the related pseudotetrameric Na_V1.4 channels.     

Hyperpolarization-activated,cyclic nucleotide-gated cation channels in Aplysia: Contribution to classical conditioning

Hyperpolarization-activated, cyclic nucleotide-gated cation (HCN) channels are critical regulators of neuronal excitability, but less is known about their possible roles in synaptic plasticity and memory circuits. Here, we characterized the HCN gene organization, channel properties, distribution, and involvement in associative and nonassociative forms of learning in Aplysia californica. Aplysia has only one HCN gene, which codes for a channel that has many similarities to the mammalian HCN channel. The cloned acHCN gene was expressed in Xenopus oocytes, which displayed a hyperpolarization-induced inward current that was enhanced by cGMP as well as cAMP. Similarly to its homologs in other animals, acHCN is permeable to K⁺ and Na⁺ ions, and is selectively blocked by Cs⁺ and ZD7288. We found that acHCN is predominantly expressed in inter- and motor neurons, including LFS siphon motor neurons, and therefore tested whether HCN channels are involved in simple forms of learning of the siphon-withdrawal reflex in a semiintact preparation. ZD7288 (100 μM) significantly reduced an associative form of learning (classical conditioning) but had no effect on two nonassociative forms of learning (intermediate-term sensitization and unpaired training) or baseline responses. The HCN current is enhanced by nitric oxide (NO), which may explain the postsynaptic role of NO during conditioning. HCN current in turn enhances the NMDA-like current in the motor neurons, suggesting that HCN channels contribute to conditioning through this pathway.Hyperpolarization-activated, cyclic nucleotide-gated (HCN), cation nonselective ion channels generate hyperpolarization-activated inward currents (I_h) and thus tend to stabilize membrane potential (1–3). In addition, binding of cyclic nucleotides (cAMP and cGMP) to the C-terminal cyclic nucleotide binding domain (CNBD) enhances I_h and thus couples membrane excitability with intracellular signaling pathways (2, 4). HCN channels are widely important for numerous systemic functions such as hormonal regulation, heart contractility, epilepsy, pain, central pattern generation, sensory perception (4–15), and learning and memory (16–24).However, in previous studies it has been difficult to relate the cellular effects of HCN channels directly to their behavioral effects, because of the immense complexity of the mammalian brain. We have therefore investigated the role of HCN channels in Aplysia, which has a numerically simpler nervous system (25). We first identified and characterized an HCN gene in Aplysia, and showed that it codes for a channel that has many similarities to the mammalian HCN channel. We found that the Aplysia HCN channel is predominantly expressed in motor neurons including LFS neurons in the siphon withdrawal reflex circuit (26, 27). We therefore investigated simple forms of learning of that reflex in a semiintact preparation (28–30) and found that HCN current is involved in classical conditioning and enhances the NMDA-like current in the motor neurons. These results provide a direct connection between HCN channels and behavioral learning and suggest a postsynaptic mechanism of that effect. HCN current in turn is enhanced by nitric oxide (NO), a transmitter of facilitatory interneurons, and thus may contribute to the postsynaptic role of NO during conditioning.     

Loss of Miro1-directed mitochondrial movement results in a novel murine model for neuron disease

Defective mitochondrial distribution in neurons is proposed to cause ATP depletion and calcium-buffering deficiencies that compromise cell function. However, it is unclear whether aberrant mitochondrial motility and distribution alone are sufficient to cause neurological disease. Calcium-binding mitochondrial Rho (Miro) GTPases attach mitochondria to motor proteins for anterograde and retrograde transport in neurons. Using two new KO mouse models, we demonstrate that Miro1 is essential for development of cranial motor nuclei required for respiratory control and maintenance of upper motor neurons required for ambulation. Neuron-specific loss of Miro1 causes depletion of mitochondria from corticospinal tract axons and progressive neurological deficits mirroring human upper motor neuron disease. Although Miro1-deficient neurons exhibit defects in retrograde axonal mitochondrial transport, mitochondrial respiratory function continues. Moreover, Miro1 is not essential for calcium-mediated inhibition of mitochondrial movement or mitochondrial calcium buffering. Our findings indicate that defects in mitochondrial motility and distribution are sufficient to cause neurological disease.Motor neuron diseases (MNDs), including ALS and spastic paraplegia (SP), are characterized by the progressive, length-dependent degeneration of motor neurons, leading to muscle atrophy, paralysis, and, in some cases, premature death. There are both inherited and sporadic forms of MNDs, which can affect upper motor neurons, lower motor neurons, or both. Although the molecular and cellular causes of most MNDs are unknown, many are associated with defects in axonal transport of cellular components required for neuron function and maintenance (1–6).A subset of MNDs is associated with impaired mitochondrial respiration and mitochondrial distribution. This observation has led to the hypothesis that neurodegeneration results from defects in mitochondrial motility and distribution, which, in turn, cause subcellular ATP depletion and interfere with mitochondrial calcium ([Ca²⁺]_m) buffering at sites of high synaptic activity (reviewed in ref. 7). It is not known, however, whether mitochondrial motility defects are a primary cause or a secondary consequence of MND progression. In addition, it has been difficult to isolate the primary effect of mitochondrial motility defects in MNDs because most mutations that impair mitochondrial motility in neurons also affect transport of other organelles and vesicles (1, 8–11).In mammals, the movement of neuronal mitochondria between the cell body and the synapse is controlled by adaptors called trafficking kinesin proteins (Trak1 and Trak2) and molecular motors (kinesin heavy chain and dynein), which transport the organelle in the anterograde or retrograde direction along axonal microtubule tracks (7, 12–24). Mitochondrial Rho (Miro) GTPase proteins are critical for transport because they are the only known surface receptors that attach mitochondria to these adaptors and motors (12–15, 18, 25, 26). Miro proteins are tail-anchored in the outer mitochondrial membrane with two GTPase domains and two predicted calcium-binding embryonic fibroblast (EF) hand motifs facing the cytoplasm (12, 13, 25, 27, 28). A recent Miro structure revealed two additional EF hands that were not predicted from the primary sequence (29). Studies in cultured cells suggest that Miro proteins also function as calcium sensors (via their EF hands) to regulate kinesin-mediated mitochondrial “stopping” in axons (15, 16, 26). Miro-mediated movement appears to be inhibited when cytoplasmic calcium is elevated in active synapses, effectively recruiting mitochondria to regions where calcium buffering and energy are needed. Despite this progress, the physiological relevance of these findings has not yet been tested in a mammalian animal model. In addition, mammals ubiquitously express two Miro orthologs, Miro1 and Miro2, which are 60% identical (12, 13). However, the individual roles of Miro1 and Miro2 in neuronal development, maintenance, and survival have no been evaluated.We describe two new mouse models that establish the importance of Miro1-mediated mitochondrial motility and distribution in mammalian neuronal function and maintenance. We show that Miro1 is essential for development/maintenance of specific cranial neurons, function of postmitotic motor neurons, and retrograde mitochondrial motility in axons. Loss of Miro1-directed retrograde mitochondrial transport is sufficient to cause MND phenotypes in mice without abrogating mitochondrial respiratory function. Furthermore, Miro1 is not essential for calcium-mediated inhibition of mitochondrial movement or [Ca²⁺]_m buffering. These findings have an impact on current models for Miro1 function and introduce a specific and rapidly progressing mouse model for MND.     

Medial septal GABAergic projection neurons promote object exploration behavior and type 2 theta rhythm

Exploratory drive is one of the most fundamental emotions, of all organisms, that are evoked by novelty stimulation. Exploratory behavior plays a fundamental role in motivation, learning, and well-being of organisms. Diverse exploratory behaviors have been described, although their heterogeneity is not certain because of the lack of solid experimental evidence for their distinction. Here we present results demonstrating that different neural mechanisms underlie different exploratory behaviors. Localized Ca_v3.1 knockdown in the medial septum (MS) selectively enhanced object exploration, whereas the null mutant (KO) mice showed enhanced-object exploration as well as open-field exploration. In MS knockdown mice, only type 2 hippocampal theta rhythm was enhanced, whereas both type 1 and type 2 theta rhythm were enhanced in KO mice. This selective effect was accompanied by markedly increased excitability of septo-hippocampal GABAergic projection neurons in the MS lacking T-type Ca²⁺ channels. Furthermore, optogenetic activation of the septo-hippocampal GABAergic pathway in WT mice also selectively enhanced object exploration behavior and type 2 theta rhythm, whereas inhibition of the same pathway decreased the behavior and the rhythm. These findings define object exploration distinguished from open-field exploration and reveal a critical role of T-type Ca²⁺ channels in the medial septal GABAergic projection neurons in this behavior.When confronted with an unfamiliar environment, or physical or social objects, animals often exhibit behavior patterns that can broadly be termed exploration, such as moving around the environment, touching or sniffing novel objects, and interacting with social stimuli (1). Social exploration involves complex processes that differ from those involved in the nonsocial exploration (2). Several distinctions were proposed to categorize the different forms of nonsocial exploratory behaviors from a motivational perspective (3). Behaviorally, two types of nonsocial exploration are observed in rodents and humans (3–5): object exploration and spatial or environmental exploration in the absence of objects. Object exploration is the behavior to explore discrete novel objects. This activity is elicited and sustained by the physical presence of an object. Several types of preference or “novelty” tests have been developed to investigate object exploration in rodents (3, 5–7). Environmental or spatial exploration in the absence of objects refers to the inquisitive activity of an animal in a new space, where the eliciting and sustaining stimulus is the “place” itself. Various forms of open-field tests have been used to investigate environmental or spatial exploration in rodents (3, 5, 8). Experimentally, however, the distinction can be less obvious because both can occur together (4, 7–9). Spatial exploration is suggested to be hippocampal-dependent (10)—although that is controversial (11)—whereas object exploration is suggested to be hippocampal-independent (12). Thus, it is still a matter of debate whether animal exploration belongs to a unitary category or not (9). To resolve this issue, neural definitions of these two previously proposed exploratory behaviors are needed.Interestingly, the medial septum (MS), where Ca_v3.1 T-type Ca²⁺ channels are highly expressed (13), is suggested to be critical for exploratory behaviors (5, 14–16). Moreover, the MS is also the nodal point for ascending afferent systems involved in the generation of hippocampal theta rhythms, the largest synchronous oscillatory signals in the mammalian brain, which are implicated in diverse brain functions (17, 18). Although the heterogeneity of hippocampal theta rhythms has long been under debate (19), recent studies based on genetic mutations in mice and optogenetics provide strong support for theta rhythm heterogeneity (20–22). However, their exact behavioral correlates are still debated. Ca_v3.1 Ca²⁺ channels play an important role in diverse behaviors, as well as the generation of physiologic and pathophysiologic brain rhythms (23). Notably, T-type, low-threshold Ca²⁺ currents are assumed to be a candidate ionic mechanism of theta rhythm genesis (24), analogous to the role of T-type channels in the generation of oscillations in the reticular nucleus of the thalamus (25). Nevertheless the involvement of T-type Ca²⁺ channels in hippocampal theta rhythms or exploratory behavior has not been examined. Here, we analyzed global KO mice and mice with MS-specific inactivation of the Ca_v3.1 gene encoding T-type Ca²⁺ channels, focusing on finding the neural mechanism that control the exploratory behaviors. Using a combination of tools, we provide evidence that object and open field exploratory behaviors are processed differently in the brain. Furthermore, Ca_v3.1 T-type Ca²⁺ channels in the septo-hippocampal GABAergic projection neurons are critically involved in controlling object exploration through modulating hippocampal type 2 theta rhythm.     

From the Cover: Ryanodine receptor fragmentation and sarcoplasmic reticulum Ca2+ leak after one session of high-intensity interval exercise

High-intensity interval training (HIIT) is a time-efficient way of improving physical performance in healthy subjects and in patients with common chronic diseases, but less so in elite endurance athletes. The mechanisms underlying the effectiveness of HIIT are uncertain. Here, recreationally active human subjects performed highly demanding HIIT consisting of 30-s bouts of all-out cycling with 4-min rest in between bouts (≤3 min total exercise time). Skeletal muscle biopsies taken 24 h after the HIIT exercise showed an extensive fragmentation of the sarcoplasmic reticulum (SR) Ca²⁺ release channel, the ryanodine receptor type 1 (RyR1). The HIIT exercise also caused a prolonged force depression and triggered major changes in the expression of genes related to endurance exercise. Subsequent experiments on elite endurance athletes performing the same HIIT exercise showed no RyR1 fragmentation or prolonged changes in the expression of endurance-related genes. Finally, mechanistic experiments performed on isolated mouse muscles exposed to HIIT-mimicking stimulation showed reactive oxygen/nitrogen species (ROS)-dependent RyR1 fragmentation, calpain activation, increased SR Ca²⁺ leak at rest, and depressed force production due to impaired SR Ca²⁺ release upon stimulation. In conclusion, HIIT exercise induces a ROS-dependent RyR1 fragmentation in muscles of recreationally active subjects, and the resulting changes in muscle fiber Ca²⁺-handling trigger muscular adaptations. However, the same HIIT exercise does not cause RyR1 fragmentation in muscles of elite endurance athletes, which may explain why HIIT is less effective in this group.It is increasingly clear that regular physical exercise plays a key role in the general well-being, disease prevention, and longevity of humans. Impaired muscle function manifesting as muscle weakness and premature fatigue development are major health problems associated with the normal aging process as well as with numerous common diseases (1). Physical exercise has a fundamental role in preventing and/or reversing these muscle problems, and training also improves the general health status in numerous diseases (2–4). On the other side of the spectrum, excessive muscle use can induce prolonged force depressions, which may set the limit on training tolerance and performance of top athletes (5, 6).Recent studies imply a key role of the sarcoplasmic reticulum (SR) Ca²⁺ release channel, the ryanodine receptor 1 (RyR1), in the reduced muscle strength observed in numerous physiological conditions, such as after strenuous endurance training (6), in situations with prolonged stress (7), and in normal aging (8, 9). Defective RyR1 function is also implied in several pathological states, including generalized inflammatory disorders (10), heart failure (11), and inherited conditions such as malignant hyperthermia (12) and Duchenne muscular dystrophy (13). In many of the above conditions, there is a link between the impaired RyR1 function and modifications induced by reactive oxygen/nitrogen species (ROS) (6, 8, 10, 12, 13). Conversely, altered RyR1 function may also be beneficial by increasing the cytosolic free [Ca²⁺] ([Ca²⁺]_i) at rest, which can stimulate mitochondrial biogenesis and thereby increase fatigue resistance (14–16). Intriguingly, effective antioxidant treatment hampers beneficial adaptations triggered by endurance training (17–19), and this effect might be due to antioxidants preventing ROS-induced modifications of RyR1 (20).A high-intensity interval training (HIIT) session typically consists of a series of brief bursts of vigorous physical exercise separated by periods of rest or low-intensity exercise. A major asset of HIIT is that beneficial adaptations can be obtained with much shorter exercise duration than with traditional endurance training (21–25). HIIT has been shown to effectively stimulate mitochondrial biogenesis in skeletal muscle and increase endurance in untrained and recreationally active healthy subjects (22, 26), whereas positive effects in elite endurance athletes are less clear (21, 27, 28). Moreover, HIIT improves health and physical performance in various pathological conditions, including cardiovascular disease, obesity, and type 2 diabetes (29, 30). Thus, short bouts of vigorous physical exercise trigger intracellular signaling of large enough magnitude and duration to induce extensive beneficial adaptations in skeletal muscle. The initial signaling that triggers these adaptations is not known.In this study, we tested the hypothesis that a single session of HIIT induces ROS-dependent RyR1 modifications. These modifications might cause prolonged force depression due to impaired SR Ca²⁺ release during contractions. Conversely, they may also initiate beneficial muscular adaptations due to increased SR Ca²⁺ leak at rest.