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Connexin-50 (Cx50) is among the most frequently mutated genes associated with congenital cataracts. While most of these disease-linked variants cause loss-of-function due to misfolding or aberrant trafficking, others directly alter channel properties. The mechanistic bases for such functional defects are mostly unknown. We investigated the functional and structural properties of a cataract-linked mutant, Cx50T39R (T39R), in the Xenopus oocyte system. T39R exhibited greatly enhanced hemichannel currents with altered voltage-gating properties compared to Cx50 and induced cell death. Co-expression of mutant T39R with wild-type Cx50 (to mimic the heterozygous state) resulted in hemichannel currents whose properties were indistinguishable from those induced by T39R alone, suggesting that the mutant had a dominant effect. Co-expression with Cx46 also produced channels with altered voltage-gating properties, particularly at negative potentials. All-atom molecular dynamics simulations indicate that the R39 substitution can form multiple electrostatic salt-bridge interactions between neighboring subunits that could stabilize the open-state conformation of the N-terminal domain, while also neutralizing the voltage-sensing residue D3 as well as residue E42 which participates in loop-gating. Together, these results suggest T39R acts as a dominant gain-of-function mutation that produces leaky hemichannels that may cause cytotoxicity in the lens and lead to development of cataracts.Statement of significanceWe investigated the functional and structural properties of a cataract-linked mutant, Cx50T39R (T39R), in the Xenopus oocyte system and showed that T39R exhibited greatly enhanced hemichannel currents with altered voltage-gating properties compared to Cx50 and induced cell death. Consistent with our experimental findings, all-atom equilibrium state molecular dynamics (MD) simulations of T39R show that R39 stabilized the open-state configuration of the N-terminal (NT) domain from an adjacent subunit. These results suggest that T39R causes disease by preventing the hemichannels from closing when present in the plasma membrane in the undocked state and provide an atomistic rationalization for the Cx50 disease-linked phenotype. They also expand our understanding of how connexin hemichannel channel gating is controlled.

In the embryonic heart development of mammals and birds, a straight initial heart tube undergoes left-handed helical looping, which is a remarkable and puzzling event. We are interested in the mechanism of this chiral helical looping. Recently, observations were reported that myocardial cells in the embryonic chick heart show intrinsic chirality of rotation. The chirality of myocardial cells, via anisotropic polarization of Golgi inside the cells, leads to a left-right (LR) asymmetry of cell shape. On cell boundaries of LR asymmetric cells, phosphorylated myosin and N-cadherin are enriched. Such LR asymmetric cellular circumstances lead to a large-scale three-dimensional chiral structure, the left-handed helical loop. However, the physical mechanism of this looping is unclear. Computer simulations were performed using a cell-based three-dimensional mathematical model assuming an anterior-rightward-biased contractile force of the cell boundaries on the ventral surface of the heart (orientation of a clock hand pointing to 10 to 11 o'clock). An initially straight heart tube was successfully remodeled to the left-handed helical tube via frequent convergent extension (CE) of collective cells, which corresponds to the previously reported observations of chick heart development. Although we assumed that the biased boundary contractile force was uniform all over the ventral side, orientations of the CEs became position specific on the anterior, posterior, right, and left regions on the ventral tube. Such position-specific CEs produced the left-handed helical loop. In addition, our results suggest the loop formation process consists of two distinct phases of preparation and explicit looping. Intrinsic cell properties of chirality in this investigation were discussed relating to extrinsic factors investigated by other researches. Finally, because CE is generally exerted in the axial developmental process across different animal species, we discussed the contribution of CE to the chiral heart structure across species of chick, mouse, Xenopus, and zebrafish.

Calcium (Ca2+) is a universal second messenger that participates in the regulation of innumerous physiological processes. The way in which local elevations of the cytosolic Ca2+ concentration spread in space and time is key for the versatility of the signals. Ca2+ diffusion in the cytosol is hindered by its interaction with proteins that act as buffers. Depending on the concentrations and the kinetics of the interactions, there is a large range of values at which Ca2+ diffusion can proceed. Having reliable estimates of this range, particularly of its highest end, which corresponds to the ions free diffusion, is key to understand how the signals propagate. In this work, we present the first experimental results with which the Ca2+-free diffusion coefficient is directly quantified in the cytosol of living cells. By means of fluorescence correlation spectroscopy experiments performed in Xenopus laevis oocytes and in cells of Saccharomyces cerevisiae, we show that the ions can freely diffuse in the cytosol at a higher rate than previously thought.

Cryptochrome proteins are activated by the absorption of blue light, leading to the formation of radical pairs through electron transfer in the active site. Recent experimental studies have shown that once some of the amino acid residues in the active site of Xenopus laevis cryptochrome DASH are mutated, radical-pair formation is still observed. In this study, we computationally investigate electron-transfer pathways in the X. laevis cryptochrome DASH by extensively equilibrating a previously established homology model using molecular dynamics simulations and then mutating key amino acids involved in the electron transfer. The electron-transfer pathways are then probed by using tight-binding density-functional theory. We report the alternative electron-transfer pathways resolved at the molecular level and, through comparison of amino acid sequences for cryptochromes from different species, we demonstrate that one of these alternative electron-transfer pathways could be general for all cryptochrome DASH proteins.

Recent work has introduced a new fluorescent voltage sensor, ASAP1, which can monitor rapid trains of action potentials in cultured neurons. This indicator is based on the Gallus gallus voltage-sensitive phosphatase with the phosphatase domain removed and a circularly permuted GFP placed in the S3-S4 linker. However, many of the biophysical details of this indicator remain unknown. In this work, we study the biophysical properties of ASAP1. Using the cut-open voltage clamp technique, we have simultaneously recorded fluorescence signals and gating currents from Xenopus laevis oocytes expressing ASAP1. Gating charge movement and fluorescence kinetics track closely with each other, although ASAP1 gating currents are significantly faster than those of Ciona intestinalis voltage-sensitive phosphatase. Altering the residue before the first gating charge removes a split in the ASAP1 QV curve, but preserves the accelerated kinetics that allow for the faithful tracking of action potentials in neurons.

The ectoderm of the Xenopus embryo is permeated by a network of channels that appear in histological sections as interstitial gaps. We characterized this interstitial space by measuring gap sizes, angles formed between adjacent cells, and curvatures of cell surfaces at gaps. From these parameters, and from surface-tension values measured previously, we estimated the values of critical mechanical variables that determine gap sizes and shapes in the ectoderm, using a general model of interstitial gap mechanics. We concluded that gaps of 1–4 μ m side length can be formed by the insertion of extracellular matrix fluid at three-cell junctions such that cell adhesion is locally disrupted and a tension difference between cell-cell contacts and the free cell surface at gaps of 0.003 mJ/m 2 is generated. Furthermore, a cell hydrostatic pressure of 16.8 ± 1.7 Pa and an interstitial pressure of 3.9 ± 3.6 Pa, relative to the central blastocoel cavity of the embryo, was found to be consistent with the observed gap size and shape distribution. Reduction of cell adhesion by the knockdown of C-cadherin increased gap volume while leaving intracellular and interstitial pressures essentially unchanged. In both normal and adhesion-reduced ectoderm, cortical tension of the free cell surfaces at gaps does not return to the high values characteristic of the free surface of the whole tissue.

Fluorescence recovery after photobleaching (FRAP) is a well-established experimental technique to study binding and diffusion of molecules in cells. Although a large number of analytical and numerical models have been developed to extract binding and diffusion rates from FRAP recovery curves, active transport of molecules is typically not included in the existing models that are used to estimate these rates. Here we present a validated numerical method for estimating diffusion, binding/unbinding rates, and active transport velocities using FRAP data that captures intracellular dynamics through partial differential equation models. We apply these methods to transport and localization of mRNA molecules in Xenopus laevis oocytes, where active transport processes are essential to generate developmental polarity. By providing estimates of the effective velocities and diffusion, as well as expected run times and lengths, this approach can help quantify dynamical properties of localizing and nonlocalizing RNA. Our results confirm the distinct transport dynamics in different regions of the cytoplasm, and suggest that RNA movement in both the animal and vegetal directions may influence the timescale of RNA localization in Xenopus oocytes. We also show that model initial conditions extracted from FRAP postbleach intensities prevent underestimation of diffusion, which can arise from the instantaneous bleaching assumption. The numerical and modeling approach presented here to estimate parameters using FRAP recovery data is a broadly applicable tool for systems where intracellular transport is a key molecular mechanism.

Organisms rely on many signaling molecules to control how their cells grow, divide and heal. For example, when the cell membrane is damaged, two signaling proteins, Rho and Cdc42, are recruited to wounds and activated to promote repair. Active Rho and active Cdc42 form two concentric rings at the membrane to direct the closure of the wound. Rho and Cdc42 belong to the RhoGTPase family, a group of proteins that act as molecular switches and alternate between active and inactive forms. At the level of the cell, RhoGTPases are only active in the tiny patches of the membrane where they bind. However, individual proteins hop on and off membranes in a matter of seconds, only staying bound for short periods. This mechanism is controlled by a regulatory protein known as RhoGDI, and it allows RhoGTPases to form precise patterns of activity at membranes – such as the rings that surround a wound site. However, it was not known exactly how RhoGDI regulates the activity of RhoGTPases over space and time, partly because it is difficult to study these proteins in the laboratory. To fill this knowledge gap, Golding, Visco et al. developed new fluorescent probes to track Rho and Cdc42 in wounded cells from frogs and on artificial membranes. The experiments showed that pools of inactive Cdc42 accumulated on membranes, alongside the active form of the protein. RhoGDI removed both active and inactive RhoGTPases from artificial and frog cell membranes. In fact, removing active Rho and Cdc42 proteins from the cell membrane was necessary to form the spatial patterns of RhoGTPase activity observed in wounded frog cells. The findings of Golding, Visco et al. help to understand how RhoGDI proteins regulate RhoGTPases and provide new tools to further study these proteins. In humans, mutations in either RhoGDI or Cdc42 are responsible for severe conditions such as Nephrotic Syndrome Type 8 or Takenouchi-Kosaki syndrome. In the future, this work may aid the development of treatments and cures for these conditions.

Protein filament networks are structures crucial for force generation and cell shape. A central open question is how collective filament dynamics emerges from interactions between individual network constituents. To address this question we study a minimal but generic model for a nematic network where filament sliding is driven by the action of motor proteins. Our theoretical analysis shows how the interplay between viscous drag on filaments and motor-induced forces governs force propagation through such interconnected filament networks. We find that the ratio between these antagonistic forces establishes the range of filament interaction, which determines how the local filament velocity depends on the polarity of the surrounding network. This force propagation mechanism implies that the polarity-independent sliding observed in Xenopus egg extracts, and in-vitro experiments with purified components, is a consequence of a large force propagation length. We suggest how our predictions can be tested by tangible in vitro experiments whose feasibility is assessed with the help of simulations and an accompanying theoretical analysis.

G protein-coupled receptors (GPCRs) mediate many signal transduction processes in the body. The discovery that these receptors are voltage-sensitive has changed our understanding of their behavior. The M2 muscarinic acetylcholine receptor (M2R) was found to exhibit depolarization-induced charge movement-associated currents, implying that this prototypical GPCR possesses a voltage sensor. However, the typical domain that serves as a voltage sensor in voltage-gated channels is not present in GPCRs, making the search for the voltage sensor in the latter challenging. Here, we examine the M2R and describe a voltage sensor that is comprised of tyrosine residues. This voltage sensor is crucial for the voltage dependence of agonist binding to the receptor. The tyrosine-based voltage sensor discovered here constitutes a noncanonical by which membrane proteins may sense voltage.

The 1,2,4-benzothiadiazine 1,1-dioxide type of positive allosteric modulators of the ionotropic glutamate receptor A2 (GluA2) are promising lead compounds for the treatment of cognitive disorders, e.g., Alzheimer's disease. The modulators bind in a cleft formed by the interface of two neighboring ligand binding domains and act by stabilizing the agonist-bound open-channel conformation. The driving forces behind the binding of these modulators can be significantly altered with only minor substitutions to the parent molecules. In this study, we show that changing the 7-fluorine substituent of modulators BPAM97 ( 2 ) and BPAM344 ( 3 ) into a hydroxyl group (BPAM557 ( 4 ) and BPAM521 ( 5 ), respectively), leads to a more favorable binding enthalpy (ΔH, kcal/mol) from −4.9 ( 2 ) and −7.5 ( 3 ) to −6.2 ( 4 ) and −14.5 ( 5 ), but also a less favorable binding entropy (−TΔS, kcal/mol) from −2.3 ( 2 ) and −1.3 ( 3 ) to −0.5 ( 4 ) and 4.8 ( 5 ). Thus, the dissociation constants (K d , μ M) of 4 (11.2) and 5 (0.16) are similar to those of 2 (5.6) and 3 (0.35). Functionally, 4 and 5 potentiated responses of 10 μ M L-glutamate at homomeric rat GluA2( Q ) i receptors with EC 50 values of 67.3 and 2.45 μ M, respectively. The binding mode of 5 was examined with x-ray crystallography, showing that the only change compared to that of earlier compounds was the orientation of Ser-497 pointing toward the hydroxyl group of 5 . The favorable enthalpy can be explained by the formation of a hydrogen bond from the side-chain hydroxyl group of Ser-497 to the hydroxyl group of 5 , whereas the unfavorable entropy might be due to desolvation effects combined with a conformational restriction of Ser-497 and 5 . In summary, this study shows a remarkable example of enthalpy-entropy compensation in drug development accompanied with a likely explanation of the underlying structural mechanism.

Auxiliary Kv channel-interacting proteins 1–4 (KChIPs1–4) coassemble with pore-forming Kv4 α-subunits to form channel complexes underlying somatodendritic subthreshold A-type current that regulates neuronal excitability. It has been hypothesized that different KChIPs can competitively bind to Kv4 α-subunit to form variable channel complexes that can exhibit distinct biophysical properties for modulation of neural function. In this study, we use single-molecule subunit counting by total internal reflection fluorescence microscopy in combinations with electrophysiology and biochemistry to investigate whether different isoforms of auxiliary KChIPs, KChIP4a, and KChIP4bl, can compete for binding of Kv4.3 to coassemble heteromultimeric channel complexes for modulation of channel function. To count the number of photobleaching steps solely from cell membrane, we take advantage of a membrane tethered k-ras-CAAX peptide that anchors cytosolic KChIP4 proteins to the surface for reduction of background noise. Single-molecule subunit counting reveals that the number of KChIP4 isoforms in Kv4.3-KChIP4 complexes can vary depending on the KChIP4 expression level. Increasing the amount of KChIP4bl gradually reduces bleaching steps of KChIP4a isoform proteins, and vice versa. Further analysis of channel gating kinetics from different Kv4-KChIP4 subunit compositions confirms that both KChIP4a and KChIP4bl can modulate the channel complex function upon coassembly. Taken together, our findings show that auxiliary KChIPs can heteroassemble with Kv4 in a competitive manner to form heteromultimeric Kv4-KChIP4 channel complexes that are biophysically distinct and regulated under physiological or pathological conditions.

KV11.1 voltage-gated K+ channels are noted for unusually slow activation, fast inactivation, and slow deactivation kinetics, which tune channel activity to provide vital repolarizing current during later stages of the cardiac action potential. The bulk of charge movement in human ether-a-go-go-related gene (hERG) is slow, as is return of charge upon repolarization, suggesting that the rates of hERG channel opening and, critically, that of deactivation might be determined by slow voltage sensor movement, and also by a mode-shift after activation. To test these ideas, we compared the kinetics and voltage dependence of ionic activation and deactivation with gating charge movement. At 0 mV, gating charge moved ∼threefold faster than ionic current, which suggests the presence of additional slow transitions downstream of charge movement in the physiological activation pathway. A significant voltage sensor mode-shift was apparent by 24 ms at +60 mV in gating currents, and return of charge closely tracked pore closure after pulses of 100 and 300 ms duration. A deletion of the N-terminus PAS domain, mutation R4AR5A or the LQT2-causing mutation R56Q gave faster-deactivating channels that displayed an attenuated mode-shift of charge. This indicates that charge movement is perturbed by N- and C-terminus interactions, and that these domain interactions stabilize the open state and limit the rate of charge return. We conclude that slow on-gating charge movement can only partly account for slow hERG ionic activation, and that the rate of pore closure has a limiting role in the slow return of gating charges.

Transporters of the SLC34 family (NaPi-IIa,b,c) catalyze uptake of inorganic phosphate (Pi) in renal and intestinal epithelia. The transport cycle requires three Na(+) ions and one divalent Pi to bind before a conformational change enables translocation, intracellular release of the substrates, and reorientation of the empty carrier. The electrogenic interaction of the first Na(+) ion with NaPi-IIa/b at a postulated Na1 site is accompanied by charge displacement, and Na1 occupancy subsequently facilitates binding of a second Na(+) ion at Na2. The voltage dependence of cotransport and presteady-state charge displacements (in the absence of a complete transport cycle) are directly related to the molecular architecture of the Na1 site. The fact that Li(+) ions substitute for Na(+) at Na1, but not at the other sites (Na2 and Na3), provides an additional tool for investigating Na1 site-specific events. We recently proposed a three-dimensional model of human SLC34a1 (NaPi-IIa) including the binding sites Na2, Na3, and Pi based on the crystal structure of the dicarboxylate transporter VcINDY. Here, we propose nine residues in transmembrane helices (TM2, TM3, and TM5) that potentially contribute to Na1. To verify their roles experimentally, we made single alanine substitutions in the human NaPi-IIa isoform and investigated the kinetic properties of the mutants by voltage clamp and (32)P uptake. Substitutions at five positions in TM2 and one in TM5 resulted in relatively small changes in the substrate apparent affinities, yet at several of these positions, we observed significant hyperpolarizing shifts in the voltage dependence. Importantly, the ability of Li(+) ions to substitute for Na(+) ions was increased compared with the wild-type. Based on these findings, we adjusted the regions containing Na1 and Na3, resulting in a refined NaPi-IIa model in which five positions (T200, Q206, D209, N227, and S447) contribute directly to cation coordination at Na1.

One of the main tasks in the analysis of models of biomolecular networks is to characterize the domain of the parameter space that corresponds to a specific behavior. Given the large number of parameters in most models, this is no trivial task. We use a model of the embryonic cell cycle to illustrate the approaches that can be used to characterize the domain of parameter space corresponding to limit cycle oscillations, a regime that coordinates periodic entry into and exit from mitosis. Our approach relies on geometric construction of bifurcation sets, numerical continuation, and random sampling of parameters. We delineate the multidimensional oscillatory domain and use it to quantify the robustness of periodic trajectories. Although some of our techniques explore the specific features of the chosen system, the general approach can be extended to other models of the cell cycle engine and other biomolecular networks.

Models of biological diffusion-reaction systems require accurate classification of the underlying diffusive dynamics (e.g., Fickian, subdiffusive, or superdiffusive). We use a renormalization group operator to identify the anomalous (non-Fickian) diffusion behavior from a short trajectory of a single molecule. The method provides quantitative information about the underlying stochastic process, including its anomalous scaling exponent. The classification algorithm is first validated on simulated trajectories of known scaling. Then it is applied to experimental trajectories of microspheres diffusing in cytoplasm, revealing heterogeneous diffusive dynamics. The simplicity and robustness of this classification algorithm makes it an effective tool for analysis of rare stochastic events that occur in complex biological systems.

Intracellular cargo transport frequently involves multiple motor types, either having opposite directionality or having the same directionality but different speeds. Although significant progress has been made in characterizing kinesin motors at the single-molecule level, predicting their ensemble behavior is challenging and requires tight coupling between experiments and modeling to uncover the underlying motor behavior. To understand how diverse kinesins attached to the same cargo coordinate their movement, we carried out microtubule gliding assays using pairwise mixtures of motors from the kinesin-1, -2, -3, -5, and -7 families engineered to have identical run lengths and surface attachments. Uniform motor densities were used and microtubule gliding speeds were measured for varying proportions of fast and slow motors. A coarse-grained computational model of gliding assays was developed and found to recapitulate the experiments. Simulations incorporated published force-dependent velocities and run lengths, along with mechanical interactions between motors bound to the same microtubule. The simulations show that the force-dependence of detachment is the key parameter that determines gliding speed in multimotor assays, while motor compliance, surface density, and stall force all play minimal roles. Simulations also provide estimates for force-dependent dissociation rates, suggesting that kinesin-1 and the mitotic motors kinesin-5 and -7 maintain microtubule association against loads, whereas kinesin-2 and -3 readily detach. This work uncovers unexpected motor behavior in multimotor ensembles and clarifies functional differences between kinesins that carry out distinct mechanical tasks in cells.

The concentration of glutamate within the glutamatergic synapse is tightly regulated by the excitatory amino-acid transporters (EAATs). In addition to their primary role of clearing extracellular glutamate, the EAATs also possess a thermodynamically uncoupled Cl− conductance. Several crystal structures of an archaeal EAAT homolog, GltPh, at different stages of the transport cycle have been solved. In a recent structure, an aqueous cavity located at the interface of the transport and trimerization domains has been identified. This cavity is lined by polar residues, several of which have been implicated in Cl− permeation. We hypothesize that this cavity opens during the transport cycle to form the Cl− channel. Residues lining this cavity in EAAT1, including Ser-366, Leu-369, Phe-373, Arg-388, Pro-392, and Thr-396, were mutated to small hydrophobic residues. Wild-type and mutant transporters were expressed in Xenopus laevis oocytes and two-electrode voltage-clamp electrophysiology, and radiolabeled substrate uptake was used to investigate function. Significant alterations in substrate-activated Cl− conductance were observed for several mutant transporters. These alterations support the hypothesis that this aqueous cavity at the interface of the transport and trimerization domains is a partially formed Cl− channel, which opens to form a pore through which Cl− ions pass. This study enhances our understanding as to how glutamate transporters function as both amino-acid transporters and Cl− channels.

The inverted emulsion method is used to prepare giant liposomes by pushing water-in-oil droplets through the oil/water interface into an aqueous medium. Due to the high encapsulation efficiency of proteins under physiological conditions and the simplicity of the protocol, it has been widely used to prepare various cell models. However, the lamellarity of liposomes prepared by this method has not been evaluated quantitatively. Here, we prepared liposomes that were partially stained with a fluorescent dye, and analyzed their fluorescence intensity under an epifluorescence microscope. The fluorescence intensities of the membranes of individual liposomes were plotted against their diameter. The plots showed discrete distributions, which were classified into several groups. The group with the lowest fluorescence intensity was determined to be unilamellar by monitoring the exchangeability of the inner and the outer solutions of the liposomes in the presence of the pore-forming toxin α-hemolysin. Increasing the lipid concentration dissolved in oil increased the number of liposomes ∼100 times. However, almost all the liposomes were unilamellar even at saturating lipid concentrations. We also investigated the effects of lipid composition and liposome content, such as highly concentrated actin filaments and Xenopus egg extracts, on the lamellarity of the liposomes. Remarkably, over 90% of the liposomes were unilamellar under all conditions examined. We conclude that the inverted emulsion method can be used to efficiently prepare giant unilamellar liposomes and is useful for designing cell models.

The meiotic spindle is a bipolar molecular machine that is designed to segregate duplicated chromosomes toward the opposite poles of the cell. The size and shape of the spindle are considered to be maintained by a balance of forces produced by molecular motors and microtubule assembly dynamics. Several studies have probed how mechanical perturbations of the force balance affect the spindle structure. However, the spindle’s response to a stretching force acting at the spindle pole and along its long axis, i.e., the direction in which chromosomes are segregated, has not been examined. Here, we describe a method to apply a stretching force to the metaphase spindle assembled in Xenopus egg extracts and measure the relationship between the force and the three-dimensional deformation of the spindle. We found that the spindle behaves as a Zener-type viscoelastic body when forces are applied at the spindle pole, generating a restoring force for several minutes. In addition, both the volume of the spindle and the tubulin density are conserved under the stretching force. These results provide insight into how the spindle size is maintained at metaphase.

Pentameric ligand gated ion channels (pLGIC) mediate fast synaptic transmission in animals, where they are known as Cys-loop receptors. Prokaryotic homologues of these proteins have been found in bacteria, although these do not possess the eponymous Cys-loop. Each receptor is constituted of five subunits which surround a central pore. A detailed analysis of sequences of all these subunits reveals that only the Pro in the Cys-loop is absolutely conserved; this has led to the proposal (which has not been widely adopted) that these receptors should be renamed ‘Pro-loop’ receptors. In addition to this Pro, there are 3 other Pros that are largely conserved and have been shown to play important but different roles. Here we combine old and new data to compare the characteristics of these Pro residues that allow them to have these distinct roles. Pro has a unique chemistry in proteins because it lacks a backbone hydrogen bond, and has a stronger cis-bias, and greater steric bulk at the amine than other amino acids. Determining which of these properties is essential at which location in Cys-loop receptors can only be accomplished using proline analogues, i.e non-conventional amino acids. These were inserted using nonsense suppression into nACh and 5-HT_3 receptors and receptor characteristics were determined using two electrode voltage clamp in Xenopus oocytes. The data reveal some similarities but also some differences in the roles of Pro at equivalent positions, and identify differences in the specific Pro properties that allow these two related Cys-loop receptors to function.

Measurements of inter- and intramolecular distances are important for monitoring structural changes and understanding protein interaction networks. Fluorescence resonance energy transfer and functionalized chemical spacers are the two predominantly used strategies to map short-range distances in living cells. Here, we describe the development of a hybrid approach that combines the key advantages of spectroscopic and chemical methods to estimate dynamic distance information from labeled proteins. Bifunctional spectroscopic probes were designed to make use of adaptable-anchor and length-varied spacers to estimate molecular distances by exploiting short-range collisional electron transfer. The spacers were calibrated using labeled polyproline peptides of defined lengths and validated by molecular simulations. This approach was extended to estimate distance restraints that enable us to evaluate the resting-state model of the Shaker potassium channel.

Connexin hemichannels display two distinct forms of voltage-dependent gating, corresponding to the operation of Vj- or fast gates and loop- or slow gates. The carboxyl terminus (CT) of connexin 32 has been reported to be required for the operation of the Vj (fast) gates, but this conclusion was inferred from the loss of a fast kinetic component in macroscopic currents of CT-truncated intercellular channels elicited by transjunctional voltage. Such inferences are complicated by presence of both fast and slow gates in each hemichannel and the serial head-to-head arrangement of these gates in the intercellular channel. Examination of voltage gating in undocked hemichannels and Vj gate polarity reversal by a negative charge substitution (N2E) in the amino terminal domain allow unequivocal separation of the two gating processes in a Cx32 chimera (Cx32∗43E1). This chimera expresses currents as an undocked hemichannel in Xenopus oocytes and provides a model system to study the molecular determinants and mechanisms of Cx32 voltage gating. Here, we demonstrate that both Vj- and loop gates are operational in a truncation mutation that removes all but the first four CT residues (ACAR219) of the Cx32∗43E1 hemichannel. We conclude that an operational Cx32 Vj (fast) gate does not require CT residues 220–283, as reported previously by others.

Low pH depolarizes the voltage-dependence of cardiac voltage-gated sodium (NaV1.5) channel activation and fast inactivation and destabilizes the fast-inactivated state. The molecular basis for these changes in protein behavior has not been reported. We hypothesized that changes in the kinetics of voltage sensor movement may destabilize the fast-inactivated state in NaV1.5. To test this idea, we recorded NaV1.5 gating currents in Xenopus oocytes using a cut-open voltage-clamp with extracellular solution titrated to either pH 7.4 or pH 6.0. Reducing extracellular pH significantly depolarized the voltage-dependence of both the QON/V and QOFF/V curves, and reduced the total charge immobilized during depolarization. We conclude that destabilized fast-inactivation and reduced charge immobilization in NaV1.5 at low pH are functionally related effects.

Interactions between millimeter waves (MMWs) and biological systems have received increasing attention due to the growing use of MMW radiation in technologies ranging from experimental medical devices to telecommunications and airport security. Studies have shown that MMW exposure alters cellular function, especially in neurons and muscles. However, the biophysical mechanisms underlying such effects are still poorly understood. Due to the high aqueous absorbance of MMW, thermal mechanisms are likely. However, nonthermal mechanisms based on resonance effects have also been postulated. We studied MMW stimulation in a simplified preparation comprising Xenopus laevis oocytes expressing proteins that underlie membrane excitability. Using electrophysiological recordings simultaneously with 60 GHz stimulation, we observed changes in the kinetics and activity levels of voltage-gated potassium and sodium channels and a sodium-potassium pump that are consistent with a thermal mechanism. Furthermore, we showed that MMW stimulation significantly increased the action potential firing rate in oocytes coexpressing voltage-gated sodium and potassium channels, as predicted by thermal terms in the Hodgkin-Huxley model of neurons. Our results suggest that MMW stimulation produces significant thermally mediated effects on excitable cells via basic thermodynamic mechanisms that must be taken into account in the study and use of MMW radiation in biological systems.

This work presents experimental results combined with model-dependent predictions regarding the osmotic-permeability regulation of human aquaporin 1 (hAQP1) expressed in Xenopus oocyte membranes. Membrane elastic properties were studied under fully controlled conditions to obtain a function that relates internal volume and pressure. This function was used to design a model in which osmotic permeability could be studied as a pressure-dependent variable. The model states that hAQP1 closes with membrane-tension increments. It is important to emphasize that the only parameter of the model is the initial osmotic permeability coefficient, which was obtained by model-dependent fitting. The model was contrasted with experimental records from emptied-out Xenopus laevis oocytes expressing hAQP1. Simulated results reproduce and predict volume changes in high-water-permeability membranes under hypoosmotic gradients of different magnitude, as well as under consecutive hypo- and hyperosmotic conditions. In all cases, the simulated permeability coefficients are similar to experimental values. Predicted pressure, volume, and permeability changes indicate that hAQP1 water channels can transit from a high-water-permeability state to a closed state. This behavior is reversible and occurs in a cooperative manner among monomers. We conclude that hAQP1 is a constitutively open channel that closes mediated by membrane-tension increments. Fil: Ozu, Marcelo. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Fisiología y Biofísica Bernardo Houssay; Universidad de Buenos Aires. Facultad de Medicina. Departamento de Ciencias Fisiológicas. Laboratorio de Biomembranas; Argentina; Fil: Dorr, Ricardo Alfredo. Universidad de Buenos Aires. Facultad de Medicina. Departamento de Ciencias Fisiológicas; Argentina; Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina; Fil: Gutiérrez, Facundo. Universidad de Buenos Aires. Facultad de Medicina. Departamento de Ciencias Fisiológicas. Laboratorio de Biomembranas; Argentina; Fil: Politi, María Teresa. Universidad de Buenos Aires. Facultad de Medicina. Departamento de Ciencias Fisiológicas. Laboratorio de Biomembranas; Argentina; Fil: Toriano, Roxana Mabel. Universidad de Buenos Aires. Facultad de Medicina. Departamento de Ciencias Fisiológicas. Cátedra de Fisiología; Argentina; Universidad de Buenos Aires. Facultad de Medicina. Departamento de Ciencias Fisiológicas. Laboratorio de Biomembranas; Argentina; Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina

The Na

The bilaterian voltage-gated sodium channel (Nav) would have evolved from the voltage-gated calcium channel (Cav). This hypothesis is supported by the existence of channels such as the Drosophila sodium channel 1 (DSC1) which features a DEEA selectivity sequence, an intermediate between the selectivity sequence of Cav and Nav channels.Initially, the DSC1 channel was classified as a calcium permeable sodium channel. Further phylogenetic investigations classified DSC1 and its homologs as Nav2 channels since their sequence would be more similar to known Nav channels although insect proteins investigated were more permeable to calcium than sodium.Here, we report the cloning, the molecular and biophysical characterization of the honeybee (Apis mellifera) DSC1 ortholog. The cloning of this channel revealed several sequence variations. When expressed in Xenopus oocytes, the channel exhibits slow activation and inactivation kinetics reminiscent of Cav channels. Furthermore, the channel is not inhibited by tetrodotoxin but is blocked by cadmium. Our results also show that the channel is calcium selective and is permeable to barium as well as marginally permeable to lithium. Magnesium and sodium did not yield measurable currents.Based on these properties, the existence of the DEEA selectivity filter and of an inactivation gate motif, we propose to classify DSC1 homologs as Cav4 rather than Nav2. Indeed, channels which feature the DEEA selectivity sequence are likely to feature the same properties as the honeybee's channel i.e. calcium as the most permeable physiological cation, slow activation and inactivation kinetics and small single channel conductance. Furthermore, we speculate that Cav channels might have evolved toward ancestral Nav channels in response to pressure to acquire an inactivation mechanism as Cav4 are calcium selective, but feature the inactivation gate motif which is characteristic of Nav channels.

Background: Metabotropic glutamate type 2 receptor (mGlu2R) is a Class C G-protein coupled receptor (GPCR), widely distributed in the brain where it mainly functions to limit excess glutamate release. mGlu2R positive allosteric modulators (PAMs) provide a novel way to regulate glutamatergic function since they bind to the transmembrane domain (TMD) and enhance responses of mGlu2R to extracellular orthosteric ligands via modulation of affinity and/or efficacy. Compared to orthosteric agonists, PAMs have the unique ability to modulate glutamate release in a ‘state-dependent’ manner that helps fine-tune physiological responses.Methods: We docked different PAMs to a mGlu2R homology model based on the mGlu1R X-ray structure and performed 300 ns molecular dynamics (MD) simulations on receptor-ligand complexes. Mutagenesis and voltage-clamp experiments are being used to test the model's predictions on the effects of PAMs on wild-type and mutant mGlu2R expressed in the Xenopus laevis oocyte heterologous expression system.Results and Conclusions: Preliminary computational results indicate that PAMs can induce allosteric conformational changes in mGlu2R TMD helices and intracellular loop regions that accompany G-protein binding and/or activation, and thus enhance formation of an active mGlu2R conformation. In addition, our simulation results also show that PAM binding can affect the interaction between the pre-TMH1 and the extracellular loop 1, which may influence the orthosteric ligand binding in the extracellular Venus flytrap domain. These predictions are being validated experimentally. Our simulations suggest that mGlu2R PAM binding alone produces a unique PAM-bound conformation that may resemble an intermediate conformation formed on the pathway to full receptor activation. These results provide insight into the mechanisms of allosteric modulation of mGlu2R, a potential therapeutic target for a variety of neuropsychiatric disorders.

Influenza A (IAV) M2 proton channel mutations in the primary target site for amantadine cause resistance to its anti-viral drug effect. Though there is still some debate about how this mutation has caused this resistance, it is clear that novel M2 blockers are needed that are effective against the ubiquitous S31N M2 mutation. Divalent copper has previously been shown to have anti-IAV properties using in vitro assays involving block of wild type M2 channels in transfected Xenopus laevis oocytes(1) and binds to the His37 imidazoles according to solid state NMR(2). In this research, the anti-IAV property of divalent copper has been extended to organic complexes of copper. We have synthesized and purified a set of novel copper complexes of derivatized M2 blockers. We have identified a set that are stable in water, purified them of residual free copper through precipitation or crystallization as validated indirectly with inductively coupled plasma mass spec, and tested their stability in typical buffers using UV and Vis absorption spectroscopy. In two-electrode voltage clamp of Xenopus laevis oocytes transfected with A/Udorn/72 M2 S31N, some of these compounds show excellent, poorly reversible block of acid-induced inward currents with low EC50's. In addition, liposome and miniplaque assays, which were previously successful with such compounds(3), are under investigation for the refined set.1. Gandhi et al. 1999. JBC 274:5474-5482.2. Su et al. 2012. JACS 134:8693-8702.3. McGuire et al, 2015. BJ 108:582a.

Voltage-gated potassium channels undergo slow (C-type) inactivation upon persistent voltage stimulation; channel opening leads to a molecular rearrangement of the selectivity filter, shutting down potassium conduction. An important question is how the peripherally located voltage-sensor domain (VSD) can affect the inactivation of the pore domain, and conversely, how the pore domain can affect the VSD with respect to C-type inactivation. We studied the Shaker K+ channel expressed in Xenopus oocytes by the two-electrode voltage clamp technique. We showed experimentally that pulling residue 416 in the pore domain towards the VSD by a Cd2+ bridge accelerates C-type inactivation. In line with this, molecular dynamics simulations show that such pulling widens the selectivity filter, a hallmark for C-type inactivation. Experimentally induced rotations of the voltage sensor S4 and an engineered Cd2+ bridge within the VSD also affects C-type inactivation; conversely, a pore domain mutation affects VSD gating charge movement. Finally, experimental data suggests that C-type inactivation is caused by a concerted action of distant amino acid residues in the pore domain. These data suggest a reciprocal communication between the pore domain and the VSD in the extracellular portion of the channel.

Pentameric ligand-gated ion channels (pLGICs) are membrane proteins involved in fast synaptic transmission, and have been implicated in a number of neurodegenerative and psychological diseases in humans. These proteins have been the focus of extensive biochemical investigation, but high-resolution structures of mammalian receptors have only recently become available. The bacterial pLGIC Gloeobacter violaceus ligand-gated ion channel (GLIC), whose structure was first published in 2008, has served as a valuable model. There are now over 40 published structures obtained under various conditions, and these suggest specific conformational transitions that may occur during channel activation. If these gating motions are conserved throughout the pLGIC family, a detailed understanding of the process holds promise for the development of strategies to tune receptor activity. Here we discuss recent work in which we explore the role of prolines in the gating transitions of GLIC. Receptors were expressed in Xenopus oocytes, and examined using whole-cell electrophysiology. We first probed the importance of each proline residue in the receptor with an alanine scan. Pro119 in the Cys-loop, Pro198 and Pro203 in the M1 helix, and Pro299 in the M4 helix were sensitive to substitution. At these positions, we then used in vivo non-canonical amino acid mutagenesis with a range of proline analogs to determine specific structural requirements. The data revealed unique requirements at each position, and show that Pro119, Pro203 and Pro299 obey previously observed phenotypes for cis-preferring, bulge-inducing, and kink-inducing prolines respectively. However, an unusual phenotype was displayed by Pro198, a non-conserved site, and possible interpretations will be discussed.

The human ether-a-go-go (EAG) family of voltage-gated K+ channels, including hERG, hEAG, and hELK exhibit variable biophysical and physiological properties. Ginsenoside Rg3 (Rg3), a steroid glycoside is a potent hERG channel activator. Here we characterize the mechanisms of action of Rg3 on three EAG-family representative channels, including hERG1, hEAG1 and hELK1 heterologously expressed in Xenopus laevis oocytes. Rg3 enhanced hERG1 channel current via a negative shift in the half-point for the voltage dependence of activation (V0.5act) that saturated at −14 mV, and a profound slowing in the rate of deactivation (EC50 = 242 nM). Rg3 had no effect on the rate of activation or the voltage dependence of hERG1 inactivation gating. For hEAG1 channels, Rg3 induced a −28 mV maxiumal shift in V0.5act (EC50 = 1.18 µM) and accelerated the rate of activation (∼20-fold faster). The maximum shift in V0.5act for hELK1 channels was much greater (>-100 mV; EC50 = 176 nM). The onset of Rg3 effects upon extracellular application was very fast and rapidly reversible upon washout for all 3 channel types, indicating this large molecule (MW = 785) binds to an extracellular region. Rg3 at 3 µM had no effect on Kv1.5 channels and only affects other Kv channels at much higher concentrations. Understanding the mechanisms of action of Rg3 may facilitate the development of more potent and selective gensenosides for therapeutic use.

The mutation from serine to asparagine at residue 31 of M2, which is found in the majority of influenza strains circulating in humans, inhibits amantadine (ADA-NH3+) block, at least in part due to increased exit rate (1), but from simulations it is unclear whether the inhibition results from weak binding (2) or incomplete block (3). We addressed the question of whether there is amantadine binding without block of proton conductance experimentally. Xenopus laevis oocytes were transfected with A/Udorn/72 S31N mRNA. Two-electrode voltage clamp was used to measure amantadine block of M2 proton conductance at unusually high drug concentrations. Results showed that the S31N M2 channel is completely blocked at 10 mM, proving that there is no “binding without block” for amantadine in the influenza M2 channel. More surprisingly, we found that rimantadine (ADA-CH(NH3+)-CH3), which blocks the wild-type M2 S31 channel better than amantadine, achieves a maximal, modest level of block in the S31N M2 channel at 1 mM, with no further increase in block at 10 mM drug. We conclude that rimantadine binds without block in the S31N M2 channel. These findings can further constrain simulations aimed at evaluating the mechanism of block.1. Stouffer, A. L. et al. (2008) Structure 16:1067-1076.2. Gleed, M.L. et al (2015) JPCB 119:11548-59.3. Gleed, M.L and Busath, D.D. (2015) JPCB 119:1225-31.

The actin cytoskeleton plays a major role during the initial stages of embryonic development. In particular, the actin cytoskeleton can switch, in a cell-cycle dependent manner, into a contractile state and exhibit large scale flows which are essential for the organization and the establishment of polarity in early embryos. For example, myosin-driven contractile flows are essential for the initial cortical polarization in many species, while bulk actin network contraction can drive directional transport in large oocytes. We developed a reconstituted model system to study the onset of contraction in actomyosin networks, and emulate these processes in artificial cells. We encapsulate Xenopus cell extracts in cell-sized water-in-oil emulsions, and introduce different types of actin nucleators at the interface or in bulk, to reconstitute cortical or bulk actin networks, respectively. Importantly, the presence of dynamic turnover in our system allows these networks to attain a dynamic steady state characterized by contractile actin flows which can persist for hours. We add single wall carbon nanotubes as inert fluorescent probes to map the network dynamics at high spatial and temporal resolution, and uncover the self-organized dynamics that lead to the formation of these steady state contractile networks.

In recent years, cholesterol emerged as a major regulator of ion channel function. The most common effect of cholesterol on ion channels is a decrease in channel activity. We have recently shown that unexpectedly cholesterol enrichment up-regulates G-protein gated inwardly rectifying potassium (GIRK or Kir3) activity in atrial myocytes.Here we focus on neuronal Kir3 channels, and determine the effect of cholesterol on their function. Several Kir3 subunits are expressed in the hippocampus: Kir3.1, Kir3.2a, Kir3.2c and Kir3.3. Among these, Kir3.2 may be expressed as a homomer or as a heteromer with Kir3.1 and/or Kir3.3, both of which do not express as homomers in the plasma membrane.We first studied the effect of cholesterol on a pore mutant of Kir3.2 that increases its membrane expression and activity, Kir3.2∧ (Kir3.2_E152D). We show that when Kir3.2∧ was expressed in Xenopus oocytes, the resulting current was up-regulated by in vitro cholesterol enrichment of the oocyte membrane. Next, using planar lipid bilayers we demonstrate that cholesterol significantly increased the open probability of the Kir3.2∧ channels. No change was observed in the unitary conductance. Most importantly, we show that also in vitro cholesterol enrichment of freshly isolated hippocampal pyramidal neurons resulted in a strong increase in physiological Kir3 currents. These findings indicate that cholesterol plays a critical role in modulating neuronal Kir3 channels.

Voltage-gated sodium channels (Nav) play an essential role in the generation and propagation of action potentials. Mammalian Nav alpha subunits are large single polypeptide chains organized in four different domains (DI-DIV). Each domain is composed of 6 transmembrane segments (S1-S6) in which S1-S4 constitute the voltage sensing domain (VSD) and S5 and S6 form the pore. Although Nav function has been studied comprehensively, the precise structural basis for the gating mechanisms has not been fully elucidated. Recently, the crystal structures of several prokaryotic sodium channels have been solved, but they are homo-tetrameric proteins in contrast to the mammalian Navs and these studies only provide a single snapshot of the channels in one of many possible conformational states. Therefore, techniques that provide dynamic structural information are needed. In this work we have used Lanthanide-based resonance energy transfer (LRET) to obtain structural information and conformational dynamics of the VSD from each domain of the rat skeletal muscle sodium channel (Nav1.4). We prepared Nav1.4 constructs with a genetically encoded lanthanide binding tag (LBT), which binds a lanthanide (Tb3+) ion with high affinity, inserted in several strategic places at the top of the S4 segment in each domain. Two new conotoxin analogs were synthesized to function as LRET acceptors: KIIIA-Bodipy and GIIIA-Bodipy. We calculated multiple distances from each one of the domains to the pore region where the labeled toxin binds in both resting and slow inactivated states in voltage-clamped Xenopus laevis oocytes expressing our Nav1.4 constructs, which remained functionally active. The results give a geometrical map of the S4 positions of each domain in the mammalian Nav channels and provide evidence for voltage-dependent structural changes. Support: 13POST14800031 (AHA), MOP-10053 (CIHR), GM68044-07, U54GM087519 and GM030376.

Background: Dysregulation of human cardiac voltage-gated Na+ channel (hNaV1.5) inactivation predisposes patients to sudden death. hNaV1.5 comprises a requisite α-subunit that contains four domains (DI-DIV); each of these contains a voltage sensing domain (VSD). We have shown that prolonged depolarizing pulses uniquely immobilize the DIII-VSD. However, the significance of this immobilization and its potential connection to inactivation has not been explored. Here, we observe activation of the VSDs in the presence of mutations that disrupt inactivation.Methods: Previously, we created four DNA constructs with a cysteine introduced into the DI-DIV VSDs. Channels encoded by these constructs were expressed in Xenopus oocytes, and cysteine-labeled with TAMRA-MTS fluorophores. Ionic current and fluorescence emission that reflected changes in VSD conformation were simultaneously recorded using the cut-open oocyte configuration.Results: A hydrophobic motif (IFM) on the DIII-DIV linker is essential for inactivation, and we disrupted it with the F1486Q mutation. With F1486Q, activation of the DI-, DII- and DIII-VSDs was unaffected, while DIV-VSD activation was shifted to depolarized potentials (DIV-VSD WT-V1/2=-61.8±3.5 mV, IQM-V1/2=-48.9±3.5 mV, p=0.03), stabilizing its rested stated. Previously, we observed that DIII-VSD deactivation was significantly slowed after prolonged depolarizing pulses (t10-90%=11.6±1.9 ms after 1 ms, 18.6±1.6 ms after 200 ms, p=0.02). In contrast, F1486Q caused faster DIII-VSD deactivation (t10-90%=16.3±2.2 ms after 1 ms, 8.8±0.9 ms after 200 ms, p=0.01), implying the IFM motif also stabilizes the DIII-VSD activated state, but only after prolonged pulses.Conclusions: Our results show a clear interaction of the DIII-VSD with the IFM motif in the 100 ms time domain, which is essential for regulating late Na+ current (INa,L). Our results indicate that INa,L, whose enhancement predisposes patients with heart-failure, diabetes and ischemia to arrhythmia, might be regulated by therapeutically targeting the DIII-VSD.

Intracellular trafficking of organelles often involves cytoskeletal track switching. Organelles such as melanosomes are transported by multiple motors including kinesin-2, dynein, and myosin-V, which drive switching between microtubules and actin filaments during dispersion and aggregation. Here, we used optical trapping to determine the unitary and ensemble forces of kinesin-2, and to reconstitute cargo switching at cytoskeletal intersections in a minimal system with kinesin-2 and myosin-V motors bound to beads. Single kinesin-2 motors exerted forces up to ∼5 pN, similar to kinesin-1. However, kinesin-2 motors were more likely to detach at submaximal forces, and the duration of force maintenance was short as compared to kinesin-1. In multimotor assays, force increased with kinesin-2 density but was not affected by the presence of myosin-V. In crossed filament assays, switching frequencies of motor-bound beads were dependent on the starting track. At equal average forces, beads tended to switch from microtubules onto overlying actin filaments consistent with the relatively faster detachment of kinesin-2 at near-maximal forces. Thus, in addition to relative force, switching probability at filament intersections is determined by the dynamics of motor-filament interaction, such as the quick detachment of kinesin-2 under load. This may enable fine-tuning of filament switching in the cell.

Changes in voltage-dependent gating represent a common pathogenetic mechanism for genetically inherited channelopathies, such as benign familial neonatal seizures or peripheral nerve hyperexcitability caused by mutations in neuronal K(v)7.2 channels. Mutation-induced changes in channel voltage dependence are most often inferred from macroscopic current measurements, a technique unable to provide a detailed assessment of the structural rearrangements underlying channel gating behavior; by contrast, gating currents directly measure voltage-sensor displacement during voltage-dependent gating. In this work, we describe macroscopic and gating current measurements, together with molecular modeling and molecular-dynamics simulations, from channels carrying mutations responsible for benign familial neonatal seizures and/or peripheral nerve hyperexcitability; K(v)7.4 channels, highly related to K(v)7.2 channels both functionally and structurally, were used for these experiments. The data obtained showed that mutations affecting charged residues located in the more distal portion of S(4) decrease the stability of the open state and the active voltage-sensing domain configuration but do not directly participate in voltage sensing, whereas mutations affecting a residue (R4) located more proximally in S(4) caused activation of gating-pore currents at depolarized potentials. These results reveal that distinct molecular mechanisms underlie the altered gating behavior of channels carrying disease-causing mutations at different voltage-sensing domain locations, thereby expanding our current view of the pathogenesis of neuronal hyperexcitability diseases.