Association of auxiliary subunits (β1-4 and γ1-4) with the pore-forming α subunit of the calcium- and voltage-activated potassium (BK) channel provides functional diversity. γ1 promotes a significant leftward shift in the voltage-activation curve, ensuring proper function of secretory glands and allowing BK channels to release K+ at the cell's resting Ca2+ concentration. Given its physiological importance, it is crucial to elucidate the mechanisms of γ1 action. However, structural and functional studies have yielded conflicting conclusions regarding the modulation of BK channels by γ1. Here, using macroscopic, single-channel, and gating-current measurements, we demonstrate that at zero mV, γ1 increases 106-fold the equilibrium constant for the closed-open transition by destabilizing the channel's closed state and enhancing coupling between the voltage sensor and the pore domain, without affecting voltage-sensor activation. These results suggest that γ1 not only increases the energetic coupling between the voltage sensors and the pore but also primarily enhances the channel-opening reaction.

The molecular and physical communication within the microscopic world underpins the entire web of life as we know it. However, how organisms, such as bacteria, amoebae, and nematodes-all ubiquitous-interact to sustain their ecological niches, particularly how their associations generate and influence behavior, remains largely unknown. In this study, we developed a framework to examine long-term interactions between microbes and animals. From soil samples collected in a temperate, semi-arid climate, we isolated culturable bacterial genera, including Comamonas, Stenotrophomonas, Chryseobacterium, and Rhodococcus, as well as the amoeba, Tetramitus. This microbial ensemble was fed to the nematode C. elegans in experiments spanning over 20 nematode generations to assess developmental rate, dauer entry, fertility, and feeding behavior. Our findings reveal that microbes and nematodes create a stable environment where no species are exhausted, and where nematodes enter diapause after several generations. We have termed this phenomenon dauer formation on naturally derived ensembles (DaFNE). DaFNE occurs across a range of optimal temperatures, from 15°C to 25°C, and is dependent on the nematode's pheromone biosynthesis pathway. The phenomenon intensifies with each passing generation, exhibiting both strong intergenerational and transgenerational effects. Moreover, the RNA interference (RNAi) pathway-both systemic and cell-autonomous-is essential for initiating DaFNE, while heritable RNAi effectors are required for its transgenerational effects. These findings indicate that RNA-mediated communication plays a critical role in bacterially induced behaviors in natural environments.IMPORTANCEMicroscopic nematodes are the most abundant multicellular animals on Earth, which implies they have evolved highly successful relationships with their associated microbiota. However, little is known about how nematode behavior is influenced within complex ecosystems where multiple organisms interact. In this study, we used four bacteria and an amoeba from a natural ecosystem to explore behavioral responses in the nematode Caenorhabditis elegans over an 8 week period. The most striking finding was the nematodes' commitment to a form of hibernation known as diapause. We have termed this phenomenon dauer formation on naturally derived ensembles (DaFNE). Our results suggest that nematodes in nature may frequently enter hibernation as a result of communication with their microbial partners. DaFNE requires the production of nematode pheromones, as well as the RNA interference pathway, indicating that the RNA communication between nematodes and their microbiota may play a critical role. Interestingly, at higher temperatures, fewer animals are needed to trigger DaFNE, suggesting that a mild increase in temperature may promote diapause in natural environments without causing stress to the animals.

Do behavioral choices depend on animals’ microbiota? To answer this question, we studied how different bacterial assemblies impact the life-history traits of the bacterivore nematode C. elegans using isolated bacteria found in association with wild nematodes in Chilean soil. We identified the first isolate, Iso1, as a novel species of Stenotrophomonas and isolate Iso2 as Bacillus pumilus .

Bacterivore nematodes are the most abundant animals in the biosphere, largely contributing to global biogeochemistry. The effect of environmental microbes as source of associated microbiota and natural diet on their life history traits of nematodes is likely to impact the general health of the biosphere. Caenorhabditis elegans is a unique model to study the behavioral and physiological outputs of different available microbial diets. Nonetheless, most studies are on monoaxenic cultures of laboratory bacteria while the effect of natural microbiota isolates has only recently started to be reported. Here, we quantified physiological, phenotypical and behavioral traits of worms feeding on two bacteria that co-isolated with wild nematodes and tested how combinations of these isolates with other bacteria affected the traits measured. These bacteria were identified as a putative novel species of Stenotrophomonas denominated Stenotrophomonas sp. Iso1 and a strain of Bacillus pumilus designated Iso2. The isolates induced distinctive behaviors and development patterns that changed in mixes of the two bacteria and/or the pathogen Salmonella enterica . Focusing on the degeneration rate of the touch circuit of C. elegans we show that B. pumilus alone is protective while the mix with Stenotrophomonas sp. is degenerative. The analysis of the metabolite content of each isolate and their combination identified NAD+ as potentially neuroprotective. In vivo supplementation shows that NAD+ restores neuroprotection to the mixes and also to individual non-protective bacteria. The results highlight the need to study the physiological effects of bacteria resembling native diets in a multicomponent scenario rather than using single isolates. Importance The behavioral decisions of animals depend on their microbiota. In nature it is unknown how this interaction affects the health of the biosphere. To study how the nematode-bacteria relationship impacts the life history traits of these animals, we isolated bacteria found in association with wild nematodes and tested their influence as single species and consortia, in the life history traits of the model C. elegans . We identify metabolites from wild bacteria that change these traits. The bacteria isolated were identified a Stenotrophomonas sp and a B. pumilus . We find that all traits depend on the biota composition. For example, B. pumilus is neuroprotective to degenerating neurons of the touch circuit of C. elegans needed to sense and escape from predators in the wild. The co-culture with Stenotrophomonas sp . eliminates the protection. We identified NAD+ as the metabolite lost in the mix, and show that NAD+ by itself is neuroprotective.

Nucleosome DNA unwrapping and its disassembly into hexasomes and tetrasomes is necessary for genomic access and plays an important role in transcription regulation. Previous single-molecule mechanical nucleosome unwrapping revealed a low- and a high-force transitions, and force-FRET pulling experiments showed that DNA unwrapping is asymmetric, occurring always first from one side before the other. However, the assignment of DNA segments involved in these transitions remains controversial. Here, using high-resolution optical tweezers with simultaneous single-molecule FRET detection, we show that the low-force transition corresponds to the undoing of the outer wrap of one side of the nucleosome (∼27 bp), a process that can occur either cooperatively or noncooperatively, whereas the high-force transition corresponds to the simultaneous unwrapping of ∼76 bp from both sides. This process may give rise stochastically to the disassembly of nucleosomes into hexasomes and tetrasomes whose unwrapping/rewrapping trajectories we establish. In contrast, nucleosome rewrapping does not exhibit asymmetry. To rationalize all previous nucleosome unwrapping experiments, it is necessary to invoke that mechanical unwrapping involves two nucleosome reorientations: one that contributes to the change in extension at the low-force transition and another that coincides but does not contribute to the high-force transition.

The endosomal sorting complex required for transport (ESCRT) proteins drive membrane scission of various biological processes such as multivesicular body biogenesis, autophagosome closure, and cytokinesis. The downstream ESCRT-III proteins are responsible for membrane constriction that leads to scission. Recruitment of spastin and VPS4, members of the AAA+ ATPase family, by ESCRTs promote fission; however, its exact role in this process is still unknown. In particular, if recruitment of spastin serves as an ESCRT localized microtubule (MT) severing enzyme or whether it plays a direct role in ESCRT disassembly.

The endosomal sorting complexes required for transport (ESCRT) system is an ancient and ubiquitous membrane scission machinery that catalyzes the budding and scission of membranes. ESCRT-mediated scission events, exemplified by those involved in the budding of HIV-1, are usually directed away from the cytosol (‘reverse-topology’), but they can also be directed towards the cytosol (‘normal-topology’). Of the ESCRT complexes 0-III, ESCRT-III is most directly implicated in membrane severing. Various subunits of ESCRT-III recruit the AAA + ATPase VPS4, which is essential for ESCRT disassembly and reverse topology membrane scission. The ESCRT-III subunits CHMP1B and IST1 can coat and constrict positively curved membrane tubes, suggesting that these subunits could catalyze normal topology membrane severing, perhaps in conjunction with a AAA + ATPase. CHMP1B and IST1 bind and recruit the microtubule-severing AAA + ATPase spastin, a close relative of VPS4, suggesting that spastin could have a VPS4-like role in normal topology membrane scission. In order to determine whether CHMP1B and IST1 are capable of membrane severing on their own or in concert with VPS4 or spastin, we sought to reconstitute the process in vitro using membrane nanotubes pulled from giant unilamellar vesicles (GUVs) using an optical trap. CHMP1B and IST1 copolymerize on membrane nanotubes, forming stable scaffolds that constrict the tubes, but do not, on their own, lead to scission. However, CHMP1B-IST1-scaffolded tubes were severed when an additional extensional force was applied, consistent with a friction-driven scission mechanism. Spastin colocalized with CHMP1B enriched sites but did not disassemble the CHMP1B-IST1 coat from the membrane. VPS4 resolubilized CHMP1B and IST1 but did not lead to scission. These data show that the CHMP1B and IST1 tubular coat contributes to membrane scission. Constriction alone is insufficient for scission. However, the dynamical extension of the coated tube does lead to scission. Finally, we find that in the normal topology setting analyzed here, scission is independent of VPS4 and spastin. These observations show that the CHMP1B-IST1 ESCRT-III combination is capable of severing membranes by a friction-driven mechanism.

Ring ATPases that translocate disordered polymers possess lock-washer architectures that they impose on their substrates during transport via a hand-over-hand mechanism. Here, we investigate the operation of ring motors that transport ordered, helical substrates, such as the bacteriophage ϕ29 dsDNA packaging motor. This pentameric motor alternates between an ATP loading dwell and a hydrolysis burst wherein it packages one turn of DNA in four steps. When challenged with DNA-RNA hybrids and dsRNA, the motor matches its burst to the shorter helical pitches, keeping three power strokes invariant while shortening the fourth. Intermittently, the motor loses grip on the RNA-containing substrates, indicating that it makes optimal load-bearing contacts with dsDNA. To rationalize these observations, we propose a helical inchworm translocation mechanism in which, during each cycle, the motor increasingly adopts a lock-washer structure during the ATP loading dwell and successively regains its planar form with each power stroke during the burst.

Ring ATPases that translocate disordered polymers possess lock-washer architectures that they impose on their substrates during transport via a hand-over-hand mechanism. Here, we investigate the operation of ring motors that transport substrates possessing a preexisting helical structure, such as the bacteriophage ϕ29 dsDNA packaging motor. During each cycle, this pentameric motor tracks one helix strand (the ‘tracking strand’), and alternates between two segregated phases: a dwell in which it exchanges ADP for ATP and a burst in which it packages a full turn of DNA in four steps. We challenge this motor with DNA-RNA hybrids and dsRNA substrates and find that it adapts the size of its burst to the corresponding shorter helical pitches by keeping three of its power strokes invariant while shortening the fourth. Intermittently, the motor loses grip when the tracking strand is RNA, indicating that it makes load-bearing contacts with the substrate that are optimal with dsDNA. The motor possesses weaker grip when ADP-bound at the end of the burst. To rationalize all these observations, we propose a helical inchworm translocation mechanism in which the motor increasingly adopts a lock-washer structure during the ATP loading dwell and successively regains its planar form with each power stroke during the burst.

The DNA packaging motor of the bacteriophage phi29 is a powerful molecular machine that couples the free energy of ATP hydrolysis with DNA translocation in order to complete the production of new viral particles. The active part of this motor is a pentameric ring ATPase which mechanochemical cycle has been described with exquisite detail using hi-resolution optical tweezers. It was described that in each turn of the cycle, the motor packages DNA taking discrete steps of 10 base pairs (bp) each, in what is called a burst of translocation. At the same time it was shown that this 10 bp burst is composed of four 2.5 bp sub-steps, presumably reflecting the power stroke of the individual ATPases. Several models can explain what is the origin of the burst size: the helical pitch of B-form DNA is 10.5 bp/turn of the double helix, suggesting that the structure of the substrate is what determines the burst size; however, the non-integer nature of the sub-steps within the burst allows to hypothesize that is the ATPase’s conformational change what sets the burst size. Yet another possibility is that the DNA packaging motor switches the local conformation of the DNA substrate from B-form to A-form during packaging (DNA scrunching). To test the above hypotheses we challenged the phi29 DNA packaging motor with a double-stranded RNA substrate (that adopts the A-form of nucleic acids) and measured the packaging activity using optical tweezers.

Significance The φ29 DNA packaging motor can be thought of as a five-cylinder engine, where each cylinder (subunit) catalyzes ATP hydrolysis to power DNA translocation. The motor operates in two phases: first, the subunits load fuel (ATP) sequentially one at a time; second, all subunits rapidly hydrolyze ATP in sequential fashion. How the subunits coordinate their activities remained unknown. Here, we show that the subunits use regulatory features observed in signaling pathways, a very different biological context, to coordinate their activities. Moreover, one of the subunits is stimulated by a contact with the DNA, transforming it into a “timer” element that paces the activity of the remaining cylinders. Similar mechanisms are likely present in other molecular machines.

Castillo, Juan P., Sánchez-Rodríguez, Jorge E., Hyde, H. Clark, Zaelzer, Cristian A., Aguayo, Daniel, Sepúlveda, Romina V., Luk, Louis Yu Pan, Kent, Stephen B. H., Gonzalez-Nilo, Fernando D., Bezanilla, Francisco and Latorre, Ramón 2016. 1 subunit-induced structural rearrangements of β the Ca2+and voltage-activated (BK) channel. Proceedings of the National Academy of Sciences of the United States of America 113 (23) , E3231-E3239. 10.1073/pnas.1606381113 file

Large-conductance Ca(2+)- and voltage-activated K(+) (BK) channels are involved in a large variety of physiological processes. Regulatory β-subunits are one of the mechanisms responsible for creating BK channel diversity fundamental to the adequate function of many tissues. However, little is known about the structure of its voltage sensor domain. Here, we present the external architectural details of BK channels using lanthanide-based resonance energy transfer (LRET). We used a genetically encoded lanthanide-binding tag (LBT) to bind terbium as a LRET donor and a fluorophore-labeled iberiotoxin as the LRET acceptor for measurements of distances within the BK channel structure in a living cell. By introducing LBTs in the extracellular region of the α- or β1-subunit, we determined (i) a basic extracellular map of the BK channel, (ii) β1-subunit-induced rearrangements of the voltage sensor in α-subunits, and (iii) the relative position of the β1-subunit within the α/β1-subunit complex.

Mammals maintain homeostatic control of their body temperature. Therefore, these organisms are expected to have adaptations that confer the ability to detect and react to both self and ambient temperature. Temperature-activated ion channels have been discovered to be the primary molecular determinants of thermosensation. The most representative group of these determinants constitutes members of the transient receptor potential superfamily, TRP, which are activated by either low or high temperatures covering the whole range of physiologically relevant temperatures. This review makes a critical assessment of existing analytical methods of temperature-activated TRP channel mechanisms using the cold-activated TRPM8 channel as a paradigm.

Regulatory β and γ subunits are responsible for conferring functional diversity to BK channels but little is known about the detailed way that accessory subunits modulate the structure of the pore forming α subunit. It is known that the γ1 subunit produces a large leftward shift of the open probability vs. voltage curve in the absence of internal Ca2+ (Yan and Aldrich, 2010). To explore the external architecture of α subunit in the presence of γ1 subunit, we used lanthanide-based resonance energy transfer (LRET) as a molecular ruler to measure intra- and inter-molecular distances. We introduced a genetically encoded lanthanide binding tag (LBT) that binds Tb3+ (LRET donor) with high affinity at different positions in the α subunit (N-terminal, S0, S1 and S2) and γ1 subunit. Fluorescent probe BODIPY linked to a scorpion toxin was used as LRET acceptor. LRET sensitized emission (SE) decays were analysed using a nano-positioning system that determines the position of LBT-tagged sites with respect to the fixed acceptor near the pore axis. Interestingly, the external architecture of the BK α subunit is modified when co-expressed with the regulatory γ1 subunit indicating a conformational change of the BK voltage sensor domain of the BK channel. The largest changes was in S1 position (∼25 Angstroms) followed by S0. In addition, all γ1-LBT positions were found peripherally positioned with respect to the α subunit. Fondecyt Grant 1110430 (To R. L.)

Expressed in somatosensory neurons of the dorsal root and trigeminal ganglion, the transient receptor potential melastatin 8 (TRPM8) channel is a Ca2+-permeable cation channel activated by cold, voltage, PIP2 and menthol. Although TRPM8 channel gating has been characterized at the single channel and macroscopic current levels, there is currently no consensus regarding the extent to which temperature and voltage sensors couple to the conduction gate. In the present study we extended the range of voltages at which the TRPM8-induced ionic currents were measured and made careful measurements of the maximum open probability the channel can attain at different temperatures by means of fluctuation analysis. The first direct measurements of TRPM8 channel temperature-driven conformational rearrangements provided here suggest that temperature alone is able to open the channel and that the opening reaction is voltage-independent. Voltage is a partial activator of TRPM8 channels, since absolute open probability values measured with fully activated voltage sensors are less than 1 and they decrease as temperature rises. By unveiling the fast temperature-dependent deactivation process, we show that TRPM8 channel deactivation is well described by a double exponential time course. The fast and slow deactivation processes are temperature-dependent with enthalpy changes of 27.2 kcalmol-1 and 30.8 kcalmol-1. The overall Q10 for the closing reaction is about 33. A three-tiered allosteric model containing four voltage sensors and four temperature sensors can account for the complex deactivation kinetics and coupling between voltage and temperature sensor activation and channel opening.

The Na + /K + -ATPase restores sodium (Na + ) and potassium (K + ) electrochemical gradients dissipated by action potentials and ion-coupled transport processes. As ions are transported, they become transiently trapped between intracellular and extracellular gates. Once the external gate opens, three Na + ions are released, followed by the binding and occlusion of two K + ions. While the mechanisms of Na + release have been well characterized by the study of transient Na + currents, smaller and faster transient currents mediated by external K + have been more difficult to study. Here we show that external K + ions travelling to their binding sites sense only a small fraction of the electric field as they rapidly and simultaneously become occluded. Consistent with these results, molecular dynamics simulations of a pump model show a wide water-filled access channel connecting the binding site to the external solution. These results suggest a mechanism of K + gating different from that of Na + occlusion.

The Na+/K+ pump is a membrane protein which plays a fundamental role in maintaining the Na+ and K+ electrochemical gradients in animal cells. When internal and external Na+ is absent the pump can only undergo K+ translocation reactions. At equilibrium, the distribution of the different protein conformations depends on the rate constants of each step leading to K+ binding and unbinding. If some of these rate constants are voltage-dependent, sudden changes in membrane electric potential will shift the binding-unbinding equilibrium. In those translocation reactions, K+ has to travel a fraction of the membrane electric field generating a transient current signal. Here, K+ pump currents were measured under voltage clamp conditions using the giant axon of the Humboldt squid, which due to its large diameter (1 -1.5 mm) allows the detection of these charge movements. By using H2DTG, a reversible inhibitor of the squid Na+/K+ pump, we were able to obtain H2DTG-sensitive transient currents in response to voltage jumps in K+/K+ conditions. Kinetics of these transient currents shows two main components, that in contrast to their Na+ counterpart, appeared to be uncoupled. The origin of the fast component appears to be the movement of ions along an access channel that it is always open, suggesting that the gate that occlude K ions is deep in the permeation pathway. On the other hand, charge displacement distribution and rate constants of the slow component show a clear dependence on the K+ external concentration revealing that the entrance of the K+ to the Na+/K+ pump from the external side is a voltage-dependent step. Supported by FIRCA grant R03 TW008351 and U54GM087519, GM030376, NS64259, HL36783 and the Intramural Program of the NINDS/NIH and FONDECYT 1110430.

Expressed in somatosensory neurons of the dorsal root and trigeminal ganglion, the transient receptor potential melastatin 8 (TRPM8) channel is a Ca2+-permeable cation channel activated by cold, voltage, phosphatidylinositol 4,5-bisphosphate, and menthol. Although TRPM8 channel gating has been characterized at the single channel and macroscopic current levels, there is currently no consensus regarding the extent to which temperature and voltage sensors couple to the conduction gate. In this study, we extended the range of voltages where TRPM8-induced ionic currents were measured and made careful measurements of the maximum open probability the channel can attain at different temperatures by means of fluctuation analysis. The first direct measurements of TRPM8 channel temperature-driven conformational rearrangements provided here suggest that temperature alone is able to open the channel and that the opening reaction is voltage-independent. Voltage is a partial activator of TRPM8 channels, because absolute open probability values measured with fully activated voltage sensors are less than 1, and they decrease as temperature rises. By unveiling the fast temperature-dependent deactivation process, we show that TRPM8 channel deactivation is well described by a double exponential time course. The fast and slow deactivation processes are temperature-dependent with enthalpy changes of 27.2 and 30.8 kcal mol(-1). The overall Q(10) for the closing reaction is about 33. A three-tiered allosteric model containing four voltage sensors and four temperature sensors can account for the complex deactivation kinetics and coupling between voltage and temperature sensor activation and channel opening.

BKCa channels are involved in a large variety of physiological processes and regulatory β subunits are one of the mechanisms responsible of creating BKCa channel diversity fundamental to the adequate function of many tissues. Regardless the proven importance of this channel little is known about its detailed structure. Here we disclose the external architectural intimacies of BKCa channels using Lanthanide based Resonance Energy Transfer (LRET) as a molecular ruler to measure intra and intermolecular distances. We introduced a genetically encoded lanthanide binding tag (LBT that binds Tb3+ with high affinity) at several positions of the external loops of the α and β1 subunits, and constructed a fluorescent molecule of BODIPY-FL linked to a scorpion toxin, iberiotoxin (Bodipy FL-IbTX), that was used as an acceptor for the LRET interaction with Tb3+. These functional LBT-BKCa constructs were expressed in Xenopus laevis oocytes that were voltage clamped with two microelectrodes to obtain simultaneously electrical and LRET recordings under physiological ionic conditions. Sensitized emission (SE) recordings from different LBT-BKCa positions had different kinetics indicating different relative positions for each construct. We analyzed SE records with a novel method developed by our group that determines the position of LBT-tagged sites of BKCa to obtain an external structural map, including the β1 subunit. Interestingly, when the BKCa α subunit was co-expressed with the regulatory β1 subunit, SE becomes slower, indicating a large conformational change of the BKCa channel structure. The methodology presented here gives us the first glimpses to the BKCa channel external surface structure in its different functional states with and without the β1 subunit. Supported by Fondecyt grant 1110430 and NIH grants U54GM087519 and GM030376. CINV is a Scientific Millennium Institute.