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.     

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.     

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.     

Voltage sensor movements of CaV1.1 during an action potential in skeletal muscle fibers

The skeletal muscle L-type Ca²⁺ channel (Ca_V1.1) works primarily as a voltage sensor for skeletal muscle action potential (AP)-evoked Ca²⁺ release. Ca_V1.1 contains four distinct voltage-sensing domains (VSDs), yet the contribution of each VSD to AP-evoked Ca²⁺ release remains unknown. To investigate the role of VSDs in excitation–contraction coupling (ECC), we encoded cysteine substitutions on each S4 voltage-sensing segment of Ca_V1.1, expressed each construct via in vivo gene transfer electroporation, and used in cellulo AP fluorometry to track the movement of each Ca_V1.1 VSD in skeletal muscle fibers. We first provide electrical measurements of Ca_V1.1 voltage sensor charge movement in response to an AP waveform. Then we characterize the fluorescently labeled channels’ VSD fluorescence signal responses to an AP and compare them with the waveforms of the electrically measured charge movement, the optically measured free myoplasmic Ca²⁺, and the calculated rate of Ca²⁺ release from the sarcoplasmic reticulum for an AP, the physiological signal for skeletal muscle fiber activation. A considerable fraction of the fluorescence signal for each VSD occurred after the time of peak Ca²⁺ release, and even more occurred after the earlier peak of electrically measured charge movement during an AP, and thus could not directly reflect activation of Ca²⁺ release or charge movement, respectively. However, a sizable fraction of the fluorometric signals for VSDs I, II, and IV, but not VSDIII, overlap the rising phase of charge moved, and even more for Ca²⁺ release, and thus could be involved in voltage sensor rearrangements or Ca²⁺ release activation.

During membrane depolarization of a skeletal muscle fiber, L-type Ca²⁺ channels (Ca_V1.1) in the transverse-tubule (TT) membrane serve as voltage-sensors for activation of Ca²⁺ release (1, 2) from the adjacent but structurally distinct sarcoplasmic reticulum (SR) via ryanodine receptor type-1 (RyR1) Ca²⁺ release channels in the SR terminal cisternae (3, 4). This series of events is known as excitation–contraction coupling (ECC) of skeletal muscle (5). Translocation of the voltage-sensing domains (VSDs) is also coupled to opening of Ca_V1.1 channel permeation pore in Ca_V1.1, which mediate inward Ca²⁺ current across the TT membrane (6). However, the L-type current is relatively small and slowly activating, and numerous reports indicate that Ca_V1.1 Ca²⁺ current may play little or no role in the events that initiate ECC (7–9). Although the role of Ca_V1.1 functioning as the voltage sensor for SR Ca²⁺ release in ECC has been clearly established, many aspects of how the Ca_V1.1 works as a voltage sensor during muscle fiber activation remain unknown. Surprisingly, the voltage-sensor movements during an action potential (AP), which is the physiological stimulus for the muscle fiber activation (10), have not been previously measured in muscle or in any other cell type.The skeletal muscle voltage-gated L-type Ca²⁺ channel is a heteromultimeric protein composed of a voltage-sensor and pore-forming α_1S subunit (Ca_V1.1), and auxiliary subunits β1a, α2δ1, and γ (11, 12). Ca_V1.1 contains four homologous domains (I to IV) (Fig. 1A). Each repeat is comprised of six (S1 to S6) transmembranal helical segments; the VSD is formed by the S1 to S4 segments, and the pore domain is formed by spanning helices S5 and S6 (Fig. 1A) (12, 13). The S4 segment in each VSD contains positively charged Arg or Lys (Fig. 1 A and B), a conserved motif across the family of voltage-gated channels, that undergoes conformational changes in response to membrane depolarization and it is thought to be the primary voltage-sensing region (14–17). Each Ca_V1.1 is composed of a central pore and four highly similar but distinct heterogenous VSD (VSD I–IV) in the three-dimensional space (Fig. 1C) (12). Because in other voltage-gated channels individual VSDs appear to be differentially involved in specific aspects of channel gating (18–21), here we hypothesized that not all the VSDs in Ca_V1.1 contribute equally to ECC. Intramembrane movements of Ca_V1.1 VSDs I–IV within the TTs during fiber depolarization generate a small electrical current (2), but neither the charge movement during the AP nor the contribution of the individual VSDs to voltage-gated Ca²⁺ release has been previously monitored.Open in a separate windowFig. 1.Ca_V1.1 membrane topology, VSDs S4 helix comparisons and structure. (A) Membrane topology of the Ca_V1.1 α1 subunit. The α1 subunit is composed by four homologous (but not identical) domains, consisting of six transmembrane domains, S1 to S6. S1 to S4 from each domain form a VSD, whereas S5 and S6 from all four domains form the pore domain. Intracellular loops connect the domains, the loops II-III and I-II are important for ECC. (B) Sequence alignment of VSDI to -IV of Ca_V1.1. Arg and Lys within S4 helices proposed critical for voltage sensing are highlighted in blue. Residues substituted by Cys for fluorescent labeling (VSDI L159C; VSDII L522C; VSDIII V893C; VSDIV S1231C) are indicated (star). (C) Extracellular view of cryoelectron microscopy reconstruction of Ca_V1.1 α1 subunits (domain I, green; domain II, cyan; domain III, red; and domain IV, blue) with relative location of mutation points marked (star) and calcium ions in the pore (central red dot). S4 helix delimitation is indicated with light blue highlight based on the cryoelectron microscopy structure and the rabbit Ca_V1.1 sequence (PDB ID code 5GJW and Uniprot ID {"type":"entrez-protein","attrs":{"text":"P07293","term_id":"116408"}}P07293) (12). Auxiliary subunits (i.e., β1a, α2δ1, γ, and STAC3), important for Ca_V1.1 function, are not illustrated.Here, we introduce a two-pronged approach to monitoring movement of VSD charges during an AP in adult muscle fibers. First, we electrically monitored the total VSD charge moved during a muscle AP using the AP voltage-clamp (APVC) technique, applied here to skeletal muscle fibers. Next, we introduce AP-fluorometry, a variant of the voltage-clamp fluorometry (VCF) (22), to optically track the movement of each VSD via a cysteine (Cys) substitution on the extracellular flank of the S4 of each VSD and its labeling with a cysteine-reacting fluorescent probe, which serves as an optical reporter of local rearrangements in response to an AP. Parallel optical recordings of AP and Ca²⁺ transients, and the calculated Ca²⁺ release rate from SR, were also obtained to establish the temporal correlation between AP, AP-elicited charge movement, AP-evoked Ca²⁺ transient, SR Ca²⁺ release flux, and VSD conformational changes.Our electrical measurements show that a peak of about 65% of the total available voltage-sensor charge is moved during a single muscle fiber AP. Our optical measurements show that the peak of the detected fluorescence change signals from fluorophores introduced into each of the VSDs occurs about 3 to 7 ms later than the peak of the electrically measured charge movement, indicating that the conformational changes of the fluorophores at the labeled sites is slower than the movement of the VSD charged residues. However, a sizable fraction of the fluorescence signal for VSDs I, II, and IV, but not VSDIII overlap the rising phase of charge moved, and even more for Ca²⁺ release, and thus could be involved in voltage sensor rearrangements or Ca²⁺ release activation.     

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).     

Ion-binding properties of a K+ channel selectivity filter in different conformations

K⁺ channels are membrane proteins that selectively conduct K⁺ ions across lipid bilayers. Many voltage-gated K⁺ (K_V) channels contain two gates, one at the bundle crossing on the intracellular side of the membrane and another in the selectivity filter. The gate at the bundle crossing is responsible for channel opening in response to a voltage stimulus, whereas the gate at the selectivity filter is responsible for C-type inactivation. Together, these regions determine when the channel conducts ions. The K⁺ channel from Streptomyces lividians (KcsA) undergoes an inactivation process that is functionally similar to K_V channels, which has led to its use as a practical system to study inactivation. Crystal structures of KcsA channels with an open intracellular gate revealed a selectivity filter in a constricted conformation similar to the structure observed in closed KcsA containing only Na⁺ or low [K⁺]. However, recent work using a semisynthetic channel that is unable to adopt a constricted filter but inactivates like WT channels challenges this idea. In this study, we measured the equilibrium ion-binding properties of channels with conductive, inactivated, and constricted filters using isothermal titration calorimetry (ITC). EPR spectroscopy was used to determine the state of the intracellular gate of the channel, which we found can depend on the presence or absence of a lipid bilayer. Overall, we discovered that K⁺ ion binding to channels with an inactivated or conductive selectivity filter is different from K⁺ ion binding to channels with a constricted filter, suggesting that the structures of these channels are different.K⁺ channels are found in all three domains of life, where they selectively conduct K⁺ ions across cell membranes. Specific stimuli trigger the activation of K⁺ channels, which results in a hinged movement of the inner helix bundle (1–7). This opening on the intracellular side of the membrane initiates ion conduction across the membrane by allowing ions to enter into the channel. After a period, many channels spontaneously inactivate to attenuate the response (8–17). The inactivation process is a timer that terminates the flow of ions in the presence of an activator to help shape the response of the system. Two dominant types of inactivation have been characterized in voltage-dependent channels: N-type and C-type (18). N-type inactivation is fast and involves an N-terminal positively charged “ball” physically plugging the pore of the channel when the membrane is depolarized. C-type inactivation, on the other hand, is a slower process involving a conformational change in the selectivity filter that is initiated by a functional link between the intracellular gate and the selectivity filter (10, 19).Several experimental observations indicate a role for the selectivity filter in C-type inactivation. First, mutations in and around the selectivity filter can alter the kinetics of inactivation (20–23). Second, increasing concentrations of extracellular K⁺ ions decrease the rate of inactivation, as if the ions are stabilizing the conductive conformation of the channel to prevent a conformational change in the selectivity filter (14, 16, 17, 22). Finally, a loss of selectivity of K⁺ over Na⁺ has been observed during the inactivation process in Shaker channels, suggesting a role for the selectivity filter (24, 25). Together, these data indicate that channels in their inactivated and conductive conformations interact with K⁺ ions differently, and suggest that C-type inactivation involves a conformational change in the selectivity filter. Although several structures of K⁺ channels in their conductive state have been solved using X-ray crystallography, there is at present no universally accepted model for the C-type inactivated channel (1, 3–5, 9, 19, 26–28) (Fig. 1B).Open in a separate windowFig. 1.Macroscopic recordings and structural models of KcsA K⁺ channel. (A) Macroscopic currents of WT KcsA obtained by a pH jump from pH 8 to pH 4 reveal channel inactivation. Two models representing the conformation of the channel are shown below. (B) Conductive [Left, Protein Data Bank (PDB) ID code 1K4C] and constricted (Right, PDB ID code 1K4D) conformations of the selectivity filter are shown as sticks, and the ion-binding sites are indicated with green spheres. The thermodynamic properties of the conductive, constricted, and inactivated (Middle) conformations are the subject of this study.Inactivation in the K⁺ channel from Streptomyces lividians (KcsA) has many of the same functional properties of C-type inactivation, which has made it a model to understand its structural features (20). KcsA channels transition from their closed to open gate upon changing the intracellular pH from high to low (Fig. 1A). The rapid flux of ions through the channel is then attenuated by channel inactivation, where most open WT channels are not conducting, suggesting that crystal structures of open KcsA channels would reveal the inactivated channel. In some crystal structures of truncated WT KcsA solved with an open gate, the selectivity filter appears in the constricted conformation, similar to the conformation observed in structures of the KcsA channel determined in the presence of only Na⁺ ions or low concentrations of K⁺ ions (3, 10, 29, 30) (Fig. 1B). Solid-state and solution NMR also indicate that the selectivity filter of the KcsA channel is in the constricted conformation when the cytoplasmic gate is open (31–33).However, a recently published study shows that even when the constricted conformation of KcsA’s selectivity filter is prevented by a nonnatural amino acid substitution, the channel inactivates like WT channels, suggesting the constricted filter does not correspond to the functionally observed inactivation in KcsA (28). In this study, we use isothermal titration calorimetry (ITC) to quantify the ion-binding properties of WT and mutant KcsA K⁺ channels with their selectivity filters in different conformations and EPR spectroscopy to determine the conformation of the channels’ intracellular gates. A comparison of these ion-binding properties leads us to conclude that the conductive and inactivated filters are energetically more similar to each other than the constricted and inactivated filters.     

Changing hydration level in an internal cavity modulates the proton affinity of a key glutamate in cytochrome c oxidase

Cytochrome c oxidase contributes to the transmembrane proton gradient by removing two protons from the high-pH side of the membrane each time the binuclear center active site is reduced. One proton goes to the binuclear center, whereas the other is pumped to the low-pH periplasmic space. Glutamate 286 (Glu286) has been proposed to serve as a transiently deprotonated proton donor. Using unrestrained atomistic molecular dynamics simulations, we show that the size of and water distribution in the hydrophobic cavity that holds Glu286 is controlled by the protonation state of the propionic acid of heme a₃, a group on the proton outlet pathway. Protonation of the propionate disrupts hydrogen bonding to two side chains, allowing a loop to swing open. Continuum electrostatics and atomistic free-energy perturbation calculations show that the resultant changes in hydration and electrostatic interactions lower the Glu proton affinity by at least 5 kcal/mol. These changes in the internal hydration level occur in the absence of major conformational transitions and serve to stabilize needed transient intermediates in proton transport. The trigger is not the protonation of the Glu of interest, but rather the protonation of a residue ∼10 Å away. Thus, unlike local water penetration to stabilize a new charge, this finding represents a specific role for water molecules in the protein interior, mediating proton transfers and facilitating ion transport.Water is essential to the structure, dynamics, and function of biomolecules (1, 2), and its role in protein folding, association (3), and dynamics (4, 5) has been well documented. The highly polar and polarizable water molecules play diverse roles in protein interiors. Water can aid catalysis in enzyme active sites (6–8). Water or water chains are often observed in proteins that are (9, 10) proton or ion transporters or pumps (11–14). Internal cavities holding functional water molecules are believed to have a fairly constant level of hydration throughout the protein reaction cycle, unless significant conformational changes occur (15). Water penetration in response to the ionization or reduction of internal groups has been extensively discussed (16, 17), although it is usually described as part of protein''s local dielectric response.Cytochrome c oxidase (CcO) adds to the transmembrane proton gradient through proton transport coupled to electron transfer reactions (12, 18, 19). In the overall reaction, electrons from four cytochromes c are transferred to oxygen to make two water molecules at the binuclear center (BNC). The four protons needed for chemistry are bound only from the high-pH, N side of the membrane. Coupled to the process, four more protons are transferred across the membrane from the high- to low-pH (P) side of the membrane. Thus, eight charges are transferred across the membrane as each O₂ is reduced.Glu286 is a required, conserved residue that is expected to transfer protons from the D channel either to the BNC or the proton-loading site (PLS) each time CcO is reduced (Fig. 1). Experiments assign a functional pK_a to Glu286 near 9.4 (20). Thus, at higher pH, proton binding to the Glu becomes rate-limiting for steady-state turnover. The current understanding of the reaction cycle shows that protons are pumped in each of the four distinct BNC redox states (12, 18, 19). The reaction mechanism needs Glu286 to be deprotonated twice to pass a proton to the PLS and to the BNC in each CcO reduction step. Previous continuum electrostatics (21–24) and semimacroscopic (25, 26) calculations obtained pK_a values for Glu286 near 9–10. However, recent microscopic calculations have found significantly higher pK_a values of more than 12 (17, 27), making it unclear how a proton could be lost from this site, whereas others do not address the proton affinity of the essential Glu (28, 29). The discrepancy between experiment and simulations may result from technical issues such as the use of static protein structures and limited sampling of protonation states of titratable groups, or it may arise from changes in the protein that have been missed. Thus, a key question remaining is how the proton affinity of this essential Glu is modulated so it can donate a proton to the PLS and the BNC through the reaction cycle.Open in a separate windowFig. 1.Illustration of key residues near the hydrophobic cavity in CcO and general proton pathways to and from Glu286.In this work, computational studies show the hydration level of an internal cavity near Glu286 changes substantially without needing global conformational changes. Rather, the structure of an internal loop is controlled or anchored by the protonation state of the D-propionic acid of heme a₃. This potentially important motion has not been noted in previous computational studies in which part of the protein structure was constrained (21, 27, 28). Both continuum electrostatics and quantum mechanical/classical mechanical (QM/MM) free-energy simulations show that the resultant changes in Glu286 hydration level and electrostatic interactions significantly affect its pK_a (proton affinity). These findings point to a molecular mechanism to modulate the timing of proton transfers in the CcO proton pumping cycle by modifying the proton affinity of this key acid. More generally, the results show that changes in protein internal hydration may occur with only small, distal conformational changes, and these can serve as an important regulatory mechanism in ion transport, thus going beyond being part of generic dielectric response of proteins.     

Rapid stimulus-evoked astrocyte Ca2+ elevations and hemodynamic responses in mouse somatosensory cortex in vivo

Increased neuron and astrocyte activity triggers increased brain blood flow, but controversy exists over whether stimulation-induced changes in astrocyte activity are rapid and widespread enough to contribute to brain blood flow control. Here, we provide evidence for stimulus-evoked Ca²⁺ elevations with rapid onset and short duration in a large proportion of cortical astrocytes in the adult mouse somatosensory cortex. Our improved detection of the fast Ca²⁺ signals is due to a signal-enhancing analysis of the Ca²⁺ activity. The rapid stimulation-evoked Ca²⁺ increases identified in astrocyte somas, processes, and end-feet preceded local vasodilatation. Fast Ca²⁺ responses in both neurons and astrocytes correlated with synaptic activity, but only the astrocytic responses correlated with the hemodynamic shifts. These data establish that a large proportion of cortical astrocytes have brief Ca²⁺ responses with a rapid onset in vivo, fast enough to initiate hemodynamic responses or influence synaptic activity.Brain function emerges from signaling in and between neurons and associated astrocytes, which causes fluctuations in cerebral blood flow (CBF) (1–5). Astrocytes are ideally situated for controlling activity-dependent increases in CBF because they closely associate with synapses and contact blood vessels with their end-feet (1, 6). Whether or not astrocytic Ca²⁺ responses develop often or rapidly enough to account for vascular signals in vivo is still controversial (7–10). Ca²⁺ responses are of interest because intracellular Ca²⁺ is a key messenger in astrocytic communication and because enzymes that synthesize the vasoactive substances responsible for neurovascular coupling are Ca²⁺-dependent (1, 4). Neuronal activity releases glutamate at synapses and activates metabotropic glutamate receptors on astrocytes, and this activation can be monitored by imaging cytosolic Ca²⁺ changes (11). Astrocytic Ca²⁺ responses are often reported to evolve on a slow (seconds) time scale, which is too slow to account for activity-dependent increases in CBF (8, 10, 12, 13). Furthermore, uncaging of Ca²⁺ in astrocytes triggers vascular responses in brain slices through specific Ca²⁺-dependent pathways with a protracted time course (14, 15). More recently, stimulation of single presynaptic neurons in hippocampal slices was shown to evoke fast, brief, local Ca²⁺ elevations in astrocytic processes that were essential for local synaptic functioning in the adult brain (16, 17). This work prompted us to reexamine the characteristics of fast, brief astrocytic Ca²⁺ signals in vivo with special regard to neurovascular coupling, i.e., the association between local increases in neural activity and the concomitant rise in local blood flow, which constitutes the physiological basis for functional neuroimaging.Here, we describe how a previously undescribed method of analysis enabled us to provide evidence for fast Ca²⁺ responses in a main fraction of astrocytes in mouse whisker barrel cortical layers II/III in response to somatosensory stimulation. The astrocytic Ca²⁺ responses were brief enough to be a direct consequence of synaptic excitation and correlated with stimulation-induced hemodynamic responses. Fast Ca²⁺ responses in astrocyte end-feet preceded the onset of dilatation in adjacent vessels by hundreds of milliseconds. This finding might suggest that communication at the gliovascular interface contributes considerably to neurovascular coupling.     

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.     

Anaerobic biosynthesis of the lower ligand of vitamin B12

Vitamin B₁₂ (cobalamin) is required by humans and other organisms for diverse metabolic processes, although only a subset of prokaryotes is capable of synthesizing B₁₂ and other cobamide cofactors. The complete aerobic and anaerobic pathways for the de novo biosynthesis of B₁₂ are known, with the exception of the steps leading to the anaerobic biosynthesis of the lower ligand, 5,6-dimethylbenzimidazole (DMB). Here, we report the identification and characterization of the complete pathway for anaerobic DMB biosynthesis. This pathway, identified in the obligate anaerobic bacterium Eubacterium limosum, is composed of five previously uncharacterized genes, bzaABCDE, that together direct DMB production when expressed in anaerobically cultured Escherichia coli. Expression of different combinations of the bza genes revealed that 5-hydroxybenzimidazole, 5-methoxybenzimidazole, and 5-methoxy-6-methylbenzimidazole, all of which are lower ligands of cobamides produced by other organisms, are intermediates in the pathway. The bza gene content of several bacterial and archaeal genomes is consistent with experimentally determined structures of the benzimidazoles produced by these organisms, indicating that these genes can be used to predict cobamide structure. The identification of the bza genes thus represents the last remaining unknown component of the biosynthetic pathway for not only B₁₂ itself, but also for three other cobamide lower ligands whose biosynthesis was previously unknown. Given the importance of cobamides in environmental, industrial, and human-associated microbial metabolism, the ability to predict cobamide structure may lead to an improved ability to understand and manipulate microbial metabolism.Vitamin B₁₂ (cobalamin) is required by the majority of animals, protists, and prokaryotes, although B₁₂ and other cobamide cofactors are synthesized exclusively by a subset of prokaryotes (1). Cobalamin is a member of the cobamide family of cofactors that are composed of a central cobalt ion coordinated by a tetrapyrrolic corrin ring, an upper ligand, and a lower ligand covalently tethered to the corrin ring by a nucleotide loop (Fig. 1A) (1). The lower ligand of cobalamin is 5,6-dimethylbenzimidazole (DMB). Purines, phenolic compounds, and other substituted benzimidazoles have also been found as cobamide lower ligands (2).Open in a separate windowFig. 1.Vitamin B₁₂ structure and the labeling pattern of DMB. (A) Structure of cyanocobalamin (vitamin B₁₂). Cbi and the lower ligand DMB are indicated. (B) AIR is a branch point in the biosynthesis of thiamin (8), purines (27), and benzimidazoles (as shown in this study). The symbols represent the origins of atoms in each product: ●, formyltetrahydrofolate; #, glutamine; *, glycine; shaded circle, erythrose 4-phosphate or threose; ▪, methionine.Distinct oxygen-requiring (aerobic) and oxygen-sensitive (anaerobic) cobamide biosynthesis pathways have been characterized, and each contains ∼30 genes (1). The biosynthetic pathway for DMB remained elusive until the discovery of the bluB gene, the product of which fragments reduced flavin mononucleotide (FMNH₂) to produce DMB in an oxygen-dependent reaction (3–6). Currently, the only unidentified genes in the cobamide biosynthesis pathway are those required for the anaerobic biosynthesis of DMB and other benzimidazoles (7). Based on previous isotope-labeling studies, performed mainly in the anaerobic bacterium Eubacterium limosum, the biosynthesis of DMB was proposed to branch from the purine biosynthetic pathway, similar to the pyrimidine ring of the cofactor thiamin pyrophosphate (Fig. 1B) (2, 8–15). Here, we sought to identify the genes in Eubacterium limosum required for the biosynthesis of DMB.     

Aberrant calcium signaling by transglutaminase-mediated posttranslational modification of inositol 1,4,5-trisphosphate receptors

The inositol 1,4,5-trisphosphate receptor (IP₃R) in the endoplasmic reticulum mediates calcium signaling that impinges on intracellular processes. IP₃Rs are allosteric proteins comprising four subunits that form an ion channel activated by binding of IP₃ at a distance. Defective allostery in IP₃R is considered crucial to cellular dysfunction, but the specific mechanism remains unknown. Here we demonstrate that a pleiotropic enzyme transglutaminase type 2 targets the allosteric coupling domain of IP₃R type 1 (IP₃R1) and negatively regulates IP₃R1-mediated calcium signaling and autophagy by locking the subunit configurations. The control point of this regulation is the covalent posttranslational modification of the Gln2746 residue that transglutaminase type 2 tethers to the adjacent subunit. Modification of Gln2746 and IP₃R1 function was observed in Huntington disease models, suggesting a pathological role of this modification in the neurodegenerative disease. Our study reveals that cellular signaling is regulated by a new mode of posttranslational modification that chronically and enzymatically blocks allosteric changes in the ligand-gated channels that relate to disease states.Ligand-gated ion channels function by allostery that is the regulation at a distance; the allosteric coupling of ligand binding with channel gating requires reversible changes in subunit configurations and conformations (1). Inositol 1,4,5-trisphosphate receptors (IP₃Rs) are ligand-gated ion channels that release calcium ions (Ca²⁺) from the endoplasmic reticulum (ER) (2, 3). IP₃Rs are allosteric proteins comprising four subunits that assemble a calcium channel with fourfold symmetry about an axis perpendicular to the ER membrane. The subunits of three IP₃R isoforms (IP₃R1, IP₃R2, and IP₃R3) are structurally divided into three domains: the IP₃-binding domain (IBD), the regulatory domain, and the channel domain (3–6). Fitting of the IBD X-ray structures (7, 8) to a cryo-EM map (9) indicates that the IBD activates a remote Ca²⁺ channel by allostery (8); however, the current X-ray structure only spans 5% of each tetramer, such that the mechanism underlying allosteric coupling of the IBD to channel gating remains unknown.The IP₃R in the ER mediates intracellular calcium signaling that impinges on homeostatic control in various subsequent intracellular processes. Deletion of the genes encoding the type 1 IP₃R (IP₃R1) leads to perturbations in long-term potentiation/depression (3, 10, 11) and spinogenesis (12), and the human genetic disease spinocerebellar ataxia 15 is caused by haploinsufficiency of the IP₃R1 gene (13–15). Dysregulation of IP₃R1 is also implicated in neurodegenerative diseases including Huntington disease (HD) (16–18) and Alzheimer’s disease (AD) (19–22). IP₃Rs also control fundamental cellular processes—for example, mitochondrial energy production (23, 24), autophagy regulation (24–27), ER stress (28), hepatic gluconeogenesis (29), pancreatic exocytosis (30), and macrophage inflammasomes (31). On the other hand, excessive IP₃R function promotes cell death processes including apoptosis by activating mitochondrial or calpain pathways (2, 17). Considering these versatile roles of IP₃Rs, appropriate IP₃R structure and function are essential for living systems, and aberrant regulation of IP₃R closely relates to various diseases.Several factors such as cytosolic molecules, interacting proteins, and posttranslational modifications control the IP₃-induced Ca²⁺ release (IICR) through allosteric sites in IP₃Rs. Cytosolic Ca²⁺ concentrations strictly control IICR in a biphasic manner with activation at low concentrations and inhibition at higher concentrations. The critical Ca²⁺ sensor for activation is conserved among the three isoforms of IP₃ and ryanodine receptors, and this sensor is located in the regulatory domain outside the IBD and the channel domain (32). A putative ATP regulatory region is deleted in opisthotonos mice, and IICR is also regulated by this mutation in the regulatory domain (33). Various interacting proteins, such as cytochrome c, Bcl-2-family proteins, ataxin-3, huntingtin (Htt) protein, Htt-associated protein 1A (HAP1A), and G-protein–coupled receptor kinase-interacting protein 1 (GIT1), target allosteric sites in the carboxyl-terminal tail (3–5). The regulatory domain and the carboxyl-terminal tail also undergo phosphorylation by the protein kinases A/G and B/Akt and contain the apoptotic cleavage sites for the protease caspase-3 (4, 5). These factors allosterically regulate IP₃R structure and function to control cellular fates; therefore, understanding the allosteric coupling of the IBD to channel gating will elucidate the regulatory mechanism of these factors.Transglutaminase (TG) catalyses protein cross-linking between a glutamine (Gln) residue and a lysine (Lys) residue via an N^ε-(γ-glutamyl)lysine isopeptide bond (34, 35). TG type 2 (TG2) is a Ca²⁺-dependent enzyme with widespread distribution and is highly inducible by various stimulations such as oxidative stress, cytokines, growth factors, and retinoic acid (RA) (34, 35). TG2 is considered a significant disease-modifying factor in neurodegenerative diseases including HD, AD, and Parkinson’s diseases (PD) (34, 36–45) because TG2 might enzymatically stabilize aberrant aggregates of proteins implicated in these diseases—that is, mutant Htt, β-amyloid, and α-synuclein; however, the causal role of TG2 in Ca²⁺ signaling in brain pathogenesis has been unclear. Ablation of TG2 in HD mouse models is associated with increased lifespan and improved motor function (46, 47). However, TG2 knockout mice do not show impaired Htt aggregation, suggesting that TG2 may play a causal role in these disorders rather than TG2-dependent cross-links in aberrant protein aggregates (47, 48).In this study, we discovered a new mode of chronic and irreversible allosteric regulation in IP₃R1 in which covalent modification of the receptor at Gln2746 is catalyzed by TG2. We demonstrate that up-regulation of TG2 modifies IP₃R1 structure and function in HD models and propose an etiologic role of this modification in the reduction of neuronal signaling and subsequent processes during the prodromal state of the neurodegenerative disease.     

De novo production of the plant-derived alkaloid strictosidine in yeast

The monoterpene indole alkaloids are a large group of plant-derived specialized metabolites, many of which have valuable pharmaceutical or biological activity. There are ∼3,000 monoterpene indole alkaloids produced by thousands of plant species in numerous families. The diverse chemical structures found in this metabolite class originate from strictosidine, which is the last common biosynthetic intermediate for all monoterpene indole alkaloid enzymatic pathways. Reconstitution of biosynthetic pathways in a heterologous host is a promising strategy for rapid and inexpensive production of complex molecules that are found in plants. Here, we demonstrate how strictosidine can be produced de novo in a Saccharomyces cerevisiae host from 14 known monoterpene indole alkaloid pathway genes, along with an additional seven genes and three gene deletions that enhance secondary metabolism. This system provides an important resource for developing the production of more complex plant-derived alkaloids, engineering of nonnatural derivatives, identification of bottlenecks in monoterpene indole alkaloid biosynthesis, and discovery of new pathway genes in a convenient yeast host.Monoterpene indole alkaloids (MIAs) are a diverse family of complex nitrogen-containing plant-derived metabolites (1, 2). This metabolite class is found in thousands of plant species from the Apocynaceae, Loganiaceae, Rubiaceae, Icacinaceae, Nyssaceae, and Alangiaceae plant families (2, 3). Many MIAs and MIA derivatives have medicinal properties; for example, vinblastine, vincristine, and vinflunine are approved anticancer therapeutics (4, 5). These structurally complex compounds can be difficult to chemically synthesize (6, 7). Consequently, industrial production relies on extraction from the plant, but these compounds are often produced in small quantities as complex mixtures, making isolation challenging, laborious, and expensive (8–10). Reconstitution of plant pathways in microbial hosts is proving to be a promising approach to access plant-derived compounds as evidenced by the successful production of terpenes, flavonoids, and benzylisoquinoline alkaloids in microorganisms (11–19). Microbial hosts can also be used to construct hybrid biosynthetic pathways to generate modified natural products with potentially enhanced bioactivities (8, 20, 21). Across numerous plant species, strictosidine is believed to be the core scaffold from which all 3,000 known MIAs are derived (1, 2). Strictosidine undergoes a variety of redox reactions and rearrangements to form the thousands of compounds that comprise the MIA natural product family (Fig. 1) (1, 2). Due to the importance of strictosidine, the last common biosynthetic intermediate for all known MIAs, we chose to focus on heterologous production of this complex molecule (1). Therefore, strictosidine reconstitution represents the necessary first step for heterologous production of high-value MIAs.Open in a separate windowFig. 1.Strictosidine, the central intermediate in monoterpene indole alkaloid (MIA) biosynthesis, undergoes a series of reactions to produce over 3,000 known MIAs such as vincristine, quinine, and strychnine.     

Network compensation of cyclic GMP-dependent protein kinase II knockout in the hippocampus by Ca2+-permeable AMPA receptors

Gene knockout (KO) does not always result in phenotypic changes, possibly due to mechanisms of functional compensation. We have studied mice lacking cGMP-dependent kinase II (cGKII), which phosphorylates GluA1, a subunit of AMPA receptors (AMPARs), and promotes hippocampal long-term potentiation (LTP) through AMPAR trafficking. Acute cGKII inhibition significantly reduces LTP, whereas cGKII KO mice show no LTP impairment. Significantly, the closely related kinase, cGKI, does not compensate for cGKII KO. Here, we describe a previously unidentified pathway in the KO hippocampus that provides functional compensation for the LTP impairment observed when cGKII is acutely inhibited. We found that in cultured cGKII KO hippocampal neurons, cGKII-dependent phosphorylation of inositol 1,4,5-trisphosphate receptors was decreased, reducing cytoplasmic Ca²⁺ signals. This led to a reduction of calcineurin activity, thereby stabilizing GluA1 phosphorylation and promoting synaptic expression of Ca²⁺-permeable AMPARs, which in turn induced a previously unidentified form of LTP as a compensatory response in the KO hippocampus. Calcineurin-dependent Ca²⁺-permeable AMPAR expression observed here is also used during activity-dependent homeostatic synaptic plasticity. Thus, a homeostatic mechanism used during activity reduction provides functional compensation for gene KO in the cGKII KO hippocampus.Some gene deletions yield no phenotypic changes because of functional compensation by closely related or duplicate genes (1). However, such duplicate gene activity may not be the main compensatory mechanism in mouse (2), although this possibility is still controversial (3). A second mechanism of compensation is provided by alternative metabolic pathways or regulatory networks (4). Although such compensatory mechanisms have been extensively studied, especially in yeast and nematode (1), the roles of metabolic and network compensatory pathways are not well understood in mouse.Long-term potentiation (LTP) and long-term depression (LTD) are long-lasting forms of synaptic plasticity that are thought to be the cellular basis for learning and memory and proper formation of neural circuits during development (5). NMDA receptor (NMDAR)-mediated synaptic plasticity is a generally agreed postsynaptic mechanism in the hippocampus (5). In particular, synaptic Ca²⁺ influx through NMDARs is critical for LTP and LTD through control of various protein kinases and phosphatases (6). LTP is in part dependent upon the activation of protein kinases, which phosphorylate target proteins (6). Several kinases are activated during the induction of LTP, including cAMP-dependent protein kinase (PKA) and cGMP-dependent protein kinases (cGKs) (6). In contrast, LTD results from activation of phosphatases that dephosphorylate target proteins (6), and calcineurin, a Ca²⁺/calmodulin-dependent protein phosphatase, is important for LTD expression (7). AMPA receptors (AMPARs) are postsynaptic glutamate receptors that mediate rapid excitatory transmission in the central nervous system (8). During LTP, activated kinases phosphorylate AMPARs, leading to synaptic trafficking of the receptors to increase synapse activity (5). For LTD, activation of postsynaptic phosphatases induces internalization of AMPARs from the synaptic membrane, thereby reducing synaptic strength (5). Therefore, both protein kinases and phosphatases control synaptic trafficking of AMPARs, underlying LTP and LTD.AMPARs are tetrameric ligand-gated ion channels that consist of a combinatorial assembly of four subunits (GluA1–4) (9). Studies of GluA1 knockout (KO) mice show that GluA1 is critical for LTP in the CA1 region of the hippocampus (10). GluA1 homomers, like all GluA2-lacking/GluA1-containing receptors, are sensitive to polyamine block and are Ca²⁺-permeable, whereas GluA2-containing AMPARs are Ca²⁺-impermeable (9). Moreover, GluA1 is the major subunit that is trafficked from recycling endosomes to the synaptic membrane in response to neuronal activity (11). Phosphorylation of GluA1 within its intracellular carboxyl-terminal domain (CTD) can regulate AMPAR membrane trafficking (12). Several CTD phosphorylations regulate trafficking (6). In particular, PKA and cGKII both phosphorylate serine 845 of GluA1, increasing the level of extrasynaptic receptors (13, 14). Therefore, activation of PKA and cGKII during LTP induction increases GluA1 phosphorylation, which enhances AMPAR activity at synapses. On the other hand, calcineurin dephosphorylates serine 845 of GluA1, which enables GluA1-containing AMPARs to be endocytosed from the plasma membrane during LTD (15, 16). This removes synaptic AMPARs, leading to reduction of receptor function during LTD. Taken together, the activity-dependent trafficking of synaptic GluA1 is regulated by the status of phosphorylation in the CTD, which provides a critical mechanism underlying LTP and LTD.Several studies have shown that acute inhibition of cGKII impairs hippocampal LTP (13, 17, 18). However, cGKII KO animals show apparently normal LTP in the hippocampus (19), suggesting that a form of functional compensation takes place in the KO hippocampus. Here, we show that cGKII KO reduces Ca²⁺ signals by decreasing cGKII-dependent phosphorylation of inositol 1,4,5-trisphosphate receptors (IP3Rs), which in turn lowers calcineurin activity in hippocampal neurons, which stabilizes phosphorylation of GluA1 in homomeric, Ca²⁺-permeable AMPARs (CPARs). This elevates CPARs at the synapse as a previously unidentified compensatory mechanism for hippocampal LTP in cGKII-deficient animals that is alternative to the form of LTP expressed in WT.     

G protein-gated IKACh channels as therapeutic targets for treatment of sick sinus syndrome and heart block

Dysfunction of pacemaker activity in the sinoatrial node (SAN) underlies “sick sinus” syndrome (SSS), a common clinical condition characterized by abnormally low heart rate (bradycardia). If untreated, SSS carries potentially life-threatening symptoms, such as syncope and end-stage organ hypoperfusion. The only currently available therapy for SSS consists of electronic pacemaker implantation. Mice lacking L-type Ca_v1.3 Ca²⁺ channels (Ca_v1.3^−/−) recapitulate several symptoms of SSS in humans, including bradycardia and atrioventricular (AV) dysfunction (heart block). Here, we tested whether genetic ablation or pharmacological inhibition of the muscarinic-gated K⁺ channel (I_KACh) could rescue SSS and heart block in Ca_v1.3^−/− mice. We found that genetic inactivation of I_KACh abolished SSS symptoms in Ca_v1.3^−/− mice without reducing the relative degree of heart rate regulation. Rescuing of SAN and AV dysfunction could be obtained also by pharmacological inhibition of I_KACh either in Ca_v1.3^−/− mice or following selective inhibition of Ca_v1.3-mediated L-type Ca²⁺ (I_Ca,L) current in vivo. Ablation of I_KACh prevented dysfunction of SAN pacemaker activity by allowing net inward current to flow during the diastolic depolarization phase under cholinergic activation. Our data suggest that patients affected by SSS and heart block may benefit from I_KACh suppression achieved by gene therapy or selective pharmacological inhibition.Pacemaker activity of the sinoatrial node (SAN) controls heart rate under physiological conditions. Abnormal generation of SAN automaticity underlies “sick sinus” syndrome (SSS), a pathological condition manifested when heart rate is not sufficient to meet the physiological requirements of the organism (1). Typical hallmarks of SSS include SAN bradycardia, chronotropic incompetence, SAN arrest, and/or exit block (1–3). SSS carries incapacitating symptoms, such as fatigue and syncope (1–3). A significant percentage of patients with SSS present also with tachycardia-bradycardia syndrome (3). SSS can also be associated with atrioventricular (AV) conduction block (heart block) (1–3). Although aging is a known intrinsic cause of SSS (4), this disease appears also in the absence of any associated cardiac pathology and displays a genetic legacy (1, 2). Heart disease or drug intake can induce acquired SSS (2). Symptomatic SSS requires the implantation of an electronic pacemaker. SSS accounts for about half of all pacemaker implantations in the United States (5, 6). The incidence of SSS has been forecasted to increase during the next 50 y, particularly in the elder population (7). Furthermore, it has been estimated that at least half of SSS patients will need to be electronically paced (7). Although pacemakers are continuously ameliorated, they remain costly and require lifelong follow-up. Moreover, the implantation of an electronic pacemaker remains difficult in pediatric patients (8). Development of alternative and complementary pharmacological or molecular therapies for SSS management could improve quality of life and limit the need for implantation of electronic pacemakers.Recently, the genetic bases of some inherited forms of SSS have been elucidated (recently reviewed in 1, 9) with the discovery of mutations in genes encoding for ion channels involved in cardiac automaticity (4, 9, 10). Notably, loss of function of L-type Ca_v1.3 Ca²⁺ channels is central in some inherited forms of SSS. For instance, loss of function in Ca_v1.3-mediated L-type Ca²⁺ (I_Ca,L) current causes the sinoatrial node dysfunction and deafness syndrome (SANDD) (10). Affected individuals with SANDD present with profound deafness, bradycardia, and dysfunction of AV conduction (10). Mutation in ankyrin-B causes SSS by reduced membrane targeting of Ca_v1.3 channels (11). The relevance of Ca_v1.3 channels to SSS is demonstrated also by work on the pathophysiology of congenital heart block, where down-regulation of Ca_v1.3 channels by maternal Abs causes heart block in infants (12). Additionally, recent data show that chronic iron overload induces acquired SSS via a reduction in Ca_v1.3-mediated I_Ca,L (13).In mice and humans, Ca_v1.3 channels are expressed in the SAN, atria, and the AV node but are absent in adult ventricular tissue (14, 15). Ca_v1.3-mediated I_Ca,L plays a major role in the generation of the diastolic depolarization in SAN and AV myocytes, thereby constituting important determinants of heart rate and AV conduction velocity (14, 16). The heart rate of mice lacking Ca_v1.3 channels (Ca_v1.3^−/− mice) fairly recapitulates the hallmarks of SSS and associated symptoms, including bradycardia and tachycardia-bradycardia syndrome (17, 18). In addition, severe AV dysfunction is recorded in Ca_v1.3^−/− mice to variable degrees. Typically, these mice show first- and second-degree AV block (16, 17, 19). Complete AV block with dissociated atrial and ventricular rhythms can also be observed in these animals. The phenotype of Ca_v1.3^−/− mice thus constitutes a unique model for developing new therapeutic strategies against SSS (10).The muscarinic-gated K⁺ channel (I_KACh) is involved in the negative chronotropic effect of the parasympathetic nervous system on heart rate (20, 21). Two subunits of the G-protein activated inwardly rectifying K⁺ channels (GIRK1 and GIRK4) of the GIRK/Kir3 subfamily assemble as heterotetramers to form cardiac I_KACh channels (22). Indeed, both Girk1^−/− and Girk4^−/− mice lack cardiac I_KACh (20, 21, 23). We recently showed that silencing of the hyperpolarization-activated current “funny” (I_f) channel in mice induces a complex arrhythmic profile that can be rescued by concurrent genetic ablation of Girk4 (24). In this study, we tested the effects of genetic ablation and pharmacological inhibition of I_KACh on the Ca_v1.3^−/− mouse model of SSS. We found that Girk4 ablation or pharmacological inhibition of I_KACh rescues SSS and AV dysfunction in Ca_v1.3^−/−. Thus, our study shows that I_KACh targeting may be pursued as a therapeutic strategy for treatment of SSS and heart block.     

Selective molecular transport through the protein shell of a bacterial microcompartment organelle

Bacterial microcompartments are widespread prokaryotic organelles that have important and diverse roles ranging from carbon fixation to enteric pathogenesis. Current models for microcompartment function propose that their outer protein shell is selectively permeable to small molecules, but whether a protein shell can mediate selective permeability and how this occurs are unresolved questions. Here, biochemical and physiological studies of structure-guided mutants are used to show that the hexameric PduA shell protein of the 1,2-propanediol utilization (Pdu) microcompartment forms a selectively permeable pore tailored for the influx of 1,2-propanediol (the substrate of the Pdu microcompartment) while restricting the efflux of propionaldehyde, a toxic intermediate of 1,2-propanediol catabolism. Crystal structures of various PduA mutants provide a foundation for interpreting the observed biochemical and phenotypic data in terms of molecular diffusion across the shell. Overall, these studies provide a basis for understanding a class of selectively permeable channels formed by nonmembrane proteins.The complex behavior of biological systems depends fundamentally on the controlled movement of molecules between cellular compartments. Such processes occur in a wide range of biological contexts through the movement of ions and small molecules across lipid bilayers via proteins—channels and pumps—embedded in the bilayer. Achievements in understanding molecular transport in transmembrane systems have contributed to scientific disciplines from cell biology and physiology to membrane biophysics (1, 2). Interestingly, there exists a second type of system for molecular transport through proteins that is fundamentally different and much less understood. Hundreds of species of bacteria produce large subcellular organelles known as microcompartments (MCPs), which consist of metabolic enzymes encapsulated within proteinaceous shells reminiscent of viral capsids (reviewed in ref. 3). For MCPs to function, substrates and products must move across their outer protein shell, which lacks any lipid-based membrane. In the last several years, 3D structures of the proteins that comprise MCP shells have revealed narrow pores through their centers that have been hypothesized to be the routes by which substrates enter (and products escape from) MCPs (4; reviewed in ref. 5). However, experimental evidence to support this key hypothesis and the molecular principles involved is lacking.The overarching function of MCPs is to optimize metabolic pathways that have toxic or volatile intermediates. MCPs are present across at least 11 different bacterial phyla, where they carry out diverse metabolic processes (6–12). The carboxysome MCP is used to enhance CO₂ fixation in nearly all bacteria that use the Calvin cycle, and it has been estimated that 25% of the carbon fixation on Earth occurs within this proteinaceous bacterial organelle (9). The 1,2-propanediol utilization (Pdu) and ethanolamine utilization (Eut) MCPs are used to optimize 1,2-propanediol (1,2-PD) and ethanolamine catabolism, respectively (13–15), and the degradation of these compounds is thought to promote enteric pathogenesis (12, 16, 17). Although the Pdu and Eut MCPs, the carboxysome, and other metabolically diverse MCPs of unknown function encapsulate distinct sets of enzymes, all have shells built from homologous proteins suggesting they operate by conserved functional principles. Most models of MCP function propose that the protein shell acts as a diffusion barrier that allows passage of substrates (and products) while limiting the escape of a toxic or volatile metabolic intermediate such as CO₂ or toxic aldehyde (9, 18), but selective permeability by MCP shells has not been established experimentally.The shells of MCPs are assembled primarily from a family of small proteins that have so-called bacterial microcompartment (BMC) domains (5). Many BMC domain proteins form flat, hexagonally shaped oligomers that tile into extended sheets that form the basis of the MCP shell (4, 19, 20) (Fig. 1). In most cases, MCP shells are composed of four to eight different types of functionally diversified BMC domain proteins, some of which have pores proposed to mediate the selective movement of metabolites across the shell (4, 7, 8, 18). For example, the PduA shell protein from the Pdu MCP has a small central pore (∼6 Å) that is lined with numerous hydrogen-bond donors and acceptors, leading to a suggested role in the preferential movement of 1,2-PD over the less polar propionaldehyde (a toxic intermediate) (21). In addition, a subgroup of BMC proteins have been crystallized in two distinct conformations where the central pore is either fully closed or opened widely (12–15 Å), suggesting that a gating mechanism might control the movement of larger molecules (such as enzymatic cofactors) across the MCP shell (22, 23). However, no physiological or biochemical studies demonstrating transport or selective movement specifically through any MCP pore have been reported. As a result, the idea that the MCP protein shell is capable of mediating selective diffusion has lacked a clear experimental basis. Furthermore, a recent alternative model for MCP function proposes that enzymes embedded in or tightly associated with the shell could move metabolites into MCPs by vectorial catalysis, in which case functional pores might not be required for metabolite movement (24).Open in a separate windowFig. 1.Structure and function of the propanediol utilization (Pdu) bacterial microcompartment. A few thousand shell proteins (mostly of the BMC family) encapsulate a series of enzymes for metabolizing 1,2-PD. The protein shell of the Pdu MCP has been hypothesized to be selectively permeable allowing substrates such as 1,2-PD to enter through small pores in the center of hexameric shell proteins while restricting the efflux of propionaldehyde, which is toxic to the cell. For clarity, the reaction scheme has been simplified by omitting some of the steps involved in coenzyme B₁₂ recycling.Here, we use the known structure of PduA—a canonical, hexameric BMC-type shell protein in the Pdu MCP—to design a series of mutant shell proteins having central pores with altered sizes and physicochemical properties. A combination of physiological studies on mutant bacteria, biochemical studies on isolated mutant MCPs, and crystal structure studies on the mutant shell proteins, show that the PduA pore serves as a key route for entry of the metabolic substrate (1,2-PD), and that the chemical properties of the PduA pore are tuned to limit the escape of the toxic propionaldehyde intermediate.     

Overexpression of junctophilin-2 does not enhance baseline function but attenuates heart failure development after cardiac stress

Heart failure is accompanied by a loss of the orderly disposition of transverse (T)-tubules and a decrease of their associations with the junctional sarcoplasmic reticulum (jSR). Junctophilin-2 (JP2) is a structural protein responsible for jSR/T-tubule docking. Animal models of cardiac stresses demonstrate that down-regulation of JP2 contributes to T-tubule disorganization, loss of excitation-contraction coupling, and heart failure development. Our objective was to determine whether JP2 overexpression attenuates stress-induced T-tubule disorganization and protects against heart failure progression. We therefore generated transgenic mice with cardiac-specific JP2 overexpression (JP2-OE). Baseline cardiac function and Ca²⁺ handling properties were similar between JP2-OE and control mice. However, JP2-OE mice displayed a significant increase in the junctional coupling area between T-tubules and the SR and an elevated expression of the Na⁺/Ca²⁺ exchanger, although other excitation-contraction coupling protein levels were not significantly changed. Despite similar cardiac function at baseline, overexpression of JP2 provided significantly protective benefits after pressure overload. This was accompanied by a decreased percentage of surviving mice that developed heart failure, as well as preservation of T-tubule network integrity in both the left and right ventricles. Taken together, these data suggest that strategies to maintain JP2 levels can prevent the progression from hypertrophy to heart failure.In working ventricular myocytes, normal excitation-contraction (E-C) coupling requires precise communication between voltage-gated L-type Ca²⁺ channels (Cav1.2) located in clusters within transverse (T)-tubules and, less frequently, on the plasmalemma, and Ca²⁺ release channels/ryanodine receptor channels (RyRs) that are also clustered on the junctional sarcoplasmic reticulum (jSR) membrane (1–4). In normal hearts, flat jSR cisternae containing a continuous row of polymerized calsequestrin (CsQ2) either wrap around a T-tubule segment or abut against the plasmalemma (5, 6) and are coupled to the surface membranes via apposed clusters of RyR2 and Cav1.2 (7). These junctional sites are called dyads. However, although the jSR cisternae constitute a single continuous compartment, the clusters of RyR2 do not occupy the whole jSR surface but are in smaller groups (8, 9). Hence, each dyad is composed of several smaller RyR2/Cav1.2 complexes, also called couplons. Functional interaction between Cav1.2 and RyR2 at these sites ensure synchronous SR Ca²⁺ release and coordinated contraction (1, 10, 11). There is evidence that impaired cardiac E-C coupling/Ca²⁺ handling is a key mediator of heart failure (12, 13). One underlying mechanism for the defective Ca²⁺ release is the progressive loss of T-tubule network organization and of the relationship between RyR2 and Cav1.2 (14–16). Therefore, preventing loss of jSR/T-tubule junctions and of T-tubule organization may represent a new strategy for therapeutic intervention in heart failure.In normal cardiomyocytes, the formation of dyads requires junctophilin 2 (JP2), a structural protein that provides a physical connection between the T-tubule and SR membranes (17). JP2’s eight N-terminal “membrane occupation and recognition nexus” domains bind to the plasmalemma (T-tubules), and its C-terminal transmembrane domain tethers the opposite end to the SR membrane (17). Decreased JP2 levels have been observed in human heart failure patients and in failing hearts from animal models of cardiac disease (16, 18–22). Knockdown of JP2 results in acute heart failure that is associated with the loss of junctional membrane complex, disrupted T-tubule organization, and Ca²⁺ handling dysfunction (23). In addition, embryonic myocytes with JP2 deficiency have defective cardiac dyads, including more SR segments with no T-tubule couplings as well as reduced intracellular Ca²⁺ transients (17). These data collectively suggest that loss of JP2 contributes to the functional defects in heart failure. Therefore, interesting questions are: Is the JP2 deficiency effect linked to the resultant disruption of jSR/T-tubule junctions and of T-tubule network integrity, as suggested by previous findings (16–18, 23)? Conversely, could exogenous overexpression of JP2 in cardiomyocytes improve Ca²⁺ handling and protect against the development of heart failure?To answer this question, we generated transgenic mice with cardiac-specific overexpression of JP2. Moderate overexpression of JP2 led to a significant increase in the junctional coupling area between T-tubule and SR membrane, but surprisingly, it did not enhance cardiac function or increase SR Ca²⁺ release at baseline. However, interestingly, JP2-overexpressing mice were resistant to left ventricular pressure overload-induced heart failure, demonstrating that JP2 overexpression is protective. These data suggest that preventing the loss of JP2 could be a potential therapeutic strategy for heart failure treatment.     

Structural basis for gating charge movement in the voltage sensor of a sodium channel

Voltage-dependent gating of ion channels is essential for electrical signaling in excitable cells, but the structural basis for voltage sensor function is unknown. We constructed high-resolution structural models of resting, intermediate, and activated states of the voltage-sensing domain of the bacterial sodium channel NaChBac using the Rosetta modeling method, crystal structures of related channels, and experimental data showing state-dependent interactions between the gating charge-carrying arginines in the S4 segment and negatively charged residues in neighboring transmembrane segments. The resulting structural models illustrate a network of ionic and hydrogen-bonding interactions that are made sequentially by the gating charges as they move out under the influence of the electric field. The S4 segment slides 6–8 Å outward through a narrow groove formed by the S1, S2, and S3 segments, rotates ∼30°, and tilts sideways at a pivot point formed by a highly conserved hydrophobic region near the middle of the voltage sensor. The S4 segment has a 3₁₀-helical conformation in the narrow inner gating pore, which allows linear movement of the gating charges across the inner one-half of the membrane. Conformational changes of the intracellular one-half of S4 during activation are rigidly coupled to lateral movement of the S4–S5 linker, which could induce movement of the S5 and S6 segments and open the intracellular gate of the pore. We confirmed the validity of these structural models by comparing with a high-resolution structure of a NaChBac homolog and showing predicted molecular interactions of hydrophobic residues in the S4 segment in disulfide-locking studies.Voltage-gated sodium (Na_V) channels are responsible for initiation and propagation of action potentials in nerve, muscle, and endocrine cells (1, 2). They are members of the structurally homologous superfamily of voltage-gated ion channel proteins that also includes voltage-gated potassium (K_V), voltage-gated calcium (Ca_V), and cyclic nucleotide-gated (CNG) channels (3). Mammalian Na_V and Ca_V channels consist of four homologous domains (I through IV), each containing six transmembrane segments (S1 through S6) and a membrane-reentrant pore loop between the S5 and S6 segments (1, 3). Segments S1–S4 of the channel form the voltage-sensing domain (VSD), and segments S5 and S6 and the membrane-reentrant pore loop form the pore. The bacterial Na_V channel NaChBac and its relatives consist of tetramers of four identical subunits, which closely resemble one domain of vertebrate Na_V and Ca_V channels, but provide much simpler structures for studying the mechanism of voltage sensing (4, 5). The hallmark feature of the voltage-gated ion channels is the steep voltage dependence of activation, which derives from the voltage-driven outward movement of gating charges in response to the membrane depolarization (6, 7). The S4 transmembrane segment in the VSD has four to seven arginine residues spaced at 3-aa intervals, which serve as gating charges in the voltage-sensing mechanism (8–15). The intracellular S4–S5 linker that connects the VSD to the pore plays a key role in coupling voltage-dependent conformational changes in the VSD to opening and closing of the pore (16). The gating charges are pulled in by the internally negative transmembrane electric field and released to move out on depolarization. Their outward movement must be catalyzed by the voltage sensor to reduce the large thermodynamic barrier to movement of charged amino acid residues across the membrane. The molecular mechanism by which the gating charges are stabilized in the hydrophobic transmembrane environment and the catalytic mechanism through which they are transported across the membrane in response to changes in membrane potential are the subjects of intense research efforts.Progress has been made in determining high-resolution structures of voltage sensors of K_V and Na_V channels in activated states (17–20). However, high-resolution structures of resting and intermediate states of voltage sensors are unknown. The majority of evidence supports a sliding helix model of the voltage-dependent gating in which the gating charge-carrying arginines in S4 are proposed to sequentially form ion pairs with negatively charged residues in S1–S3 segments during activation of the channel (9–11, 21). However, the structural basis for stabilization of the gating charges in the membrane and catalysis of their movement through the hydrophobic membrane environment remain uncertain. Here, we have integrated bioinformatics analysis of Na_V and K_V channel families using the HHPred homology detection server (22–24), high-resolution structural modeling using the Rosetta Membrane (25–27) and Rosetta Symmetry methods (28), the X-ray structures of the K_v1.2-K_v2.1 chimeric channel and NavAb with activated VSDs (19, 20) and the MlotiK1 CNG channel in the resting state (29), and experimental data showing sequential state-dependent interactions between gating charges in S4 and negatively charged residues in S1–S3 (this work and refs. 30–33). Predictions of the resulting voltage-sensing model are confirmed in this work by disulfide-locking studies and mutant cycle analysis of the interactions of hydrophobic residues in the S4 segment. This model reveals structural details of the voltage-dependent conformational changes in the VSD that stabilize and catalyze gating charge movement and are coupled to opening and closing of the intracellular activation gate of the ion-conducting pore.