Molecular determinants of PI3Kγ-mediated activation downstream of G-protein–coupled receptors (GPCRs)

Phosphoinositide 3-kinase gamma (PI3Kγ) has profound roles downstream of G-protein–coupled receptors in inflammation, cardiac function, and tumor progression. To gain insight into how the enzyme’s activity is shaped by association with its p101 adaptor subunit, lipid membranes, and Gβγ heterodimers, we mapped these regulatory interactions using hydrogen–deuterium exchange mass spectrometry. We identify residues in both the p110γ and p101 subunits that contribute critical interactions with Gβγ heterodimers, leading to PI3Kγ activation. Mutating Gβγ-interaction sites of either p110γ or p101 ablates G-protein–coupled receptor-mediated signaling to p110γ/p101 in cells and severely affects chemotaxis and cell transformation induced by PI3Kγ overexpression. Hydrogen–deuterium exchange mass spectrometry shows that association with the p101 regulatory subunit causes substantial protection of the RBD-C2 linker as well as the helical domain of p110γ. Lipid interaction massively exposes that same helical site, which is then stabilized by Gβγ. Membrane-elicited conformational change of the helical domain could help prepare the enzyme for Gβγ binding. Our studies and others identify the helical domain of the class I PI3Ks as a hub for diverse regulatory interactions that include the p101, p87 (also known as p84), and p85 adaptor subunits; Rab5 and Gβγ heterodimers; and the β-adrenergic receptor kinase.The phosphoinositide 3-kinase γ (PI3Kγ) has far-reaching roles in the processes of mammalian biology, including inflammation, cell migration, cardiac function, response to pathogens, wound healing, olfaction, nociception, and tumor progression. Activation of p110γ produces the lipid second messenger phosphatidylinositol (3,4,5)-trisphosphate (PIP₃), which in turn recruits downstream effectors, such as protein kinase B that bears PIP₃-recognizing PH domains.PI3Kγ plays a critical role in inflammation, with mice lacking the Pik3cg gene for p110γ having reduced inflammatory responses (1–3), increased protection from anaphylaxis (4), and protection from sepsis (5). Aberrant activation of the PI3K pathway is one of the most common events in cancer (6). Overexpression of p110γ induces cell transformation (7). Pharmacological inhibition of p110γ can prevent tumor growth and spreading by blocking myeloid-derived tumor inflammation and by suppressing breast cancer cell invasion (8–10). Depletion of p110γ or its regulatory subunit p101 inhibited primary tumor formation and metastasis by murine epithelial carcinoma cells (11). In pancreatic cancer, it has been proposed that p110γ is an important component of disease progression (12). PI3Kγ (together with PI3Kδ) is strictly required for development and maintenance of T-cell lymphoblastic leukemia that is driven by PTEN loss (13). Recently, it was also shown that PI3Kγ plays an essential role in formation of sarcomas induced by a viral GPCR (vGPCR) encoded by Kaposi’s sarcoma herpes virus and that p110γ-deficient mice were completely resistant to vGPCR-induced sarcomagenesis (14). PI3Kγ also has major functions in the heart, where it regulates cardiac contractility downstream of the β-adrenergic receptor (βAR) (15). In addition to its function as a lipid kinase, p110γ also plays a scaffolding/kinase-independent role in the heart (16–19). These results have established PI3Kγ as a target for the treatment of inflammation and cardiac diseases.The PI3Kγ functions depend on direct, transient associations with various regulators, such as Gβγ heterodimers (Gβγ), Ras, βAR kinase, PKA, and PP2A. Central to its many roles is the activation of PI3Kγ downstream of G-protein–coupled receptors (GPCRs) via direct binding to Gβγ. Although p110γ was among the first PI3Ks cloned, the mechanisms of p110γ’s regulation by Gβγ heterodimers and by its regulatory subunits are still not clear. In contrast to other class I PI3Ks, p110γ uniquely associates with a p101 or a p87 (also called p84) regulatory subunit (20–23). PI3Kγ shares with the class I_A PI3K p110β the ability to be directly activated by Gβγ heterodimers (24–26). The p110γ catalytic subunit on its own can be activated only to a limited extent by Gβγ heterodimers, whereas maximal activation requires association with the p101 subunit and with Ras (21, 25, 27, 28). In cells, p101 is required for membrane translocation and activation of p110γ in response to ligand-induced G-protein activation (28). However, a p110γ that is constitutively localized to the plasma membrane is still sensitive to G-protein activation (28, 29).In an effort to understand the molecular basis of PI3Kγ activation, we have investigated the structural determinants of the p110γ/p101 interaction with Gβγ heterodimers on membranes. Although there is a crystal structure of p110γ (30), no structural information or even domain organization for the p101 regulatory subunit is known, and similarly, there is no structural information about PI3Kγ interaction with membranes. Hydrogen–deuterium exchange mass spectrometry (HDX-MS) is a useful tool for analyzing interactions of proteins with membranes (31), and it has been important for understanding how PI3K complexes become activated on lipid membranes (24). The method also provides insight into protein dynamics that is difficult or impossible to obtain with other tools (32–35). Using HDX-MS, we have determined the interactions and conformational changes in p110γ that accompany binding to p101, to membranes, and to Gβγ. Our studies show that the helical domain of p110γ is a critical element in the regulation of p110γ activity. We also identified mutations in p110γ and p101 that specifically affect PI3Kγ activation downstream of GPCRs and impair its cellular functions.     

α-Synuclein assembles into higher-order multimers upon membrane binding to promote SNARE complex formation

Physiologically, α-synuclein chaperones soluble NSF attachment protein receptor (SNARE) complex assembly and may also perform other functions; pathologically, in contrast, α-synuclein misfolds into neurotoxic aggregates that mediate neurodegeneration and propagate between neurons. In neurons, α-synuclein exists in an equilibrium between cytosolic and membrane-bound states. Cytosolic α-synuclein appears to be natively unfolded, whereas membrane-bound α-synuclein adopts an α-helical conformation. Although the majority of studies showed that cytosolic α-synuclein is monomeric, it is unknown whether membrane-bound α-synuclein is also monomeric, and whether chaperoning of SNARE complex assembly by α-synuclein involves its cytosolic or membrane-bound state. Here, we show using chemical cross-linking and fluorescence resonance energy transfer (FRET) that α-synuclein multimerizes into large homomeric complexes upon membrane binding. The FRET experiments indicated that the multimers of membrane-bound α-synuclein exhibit defined intermolecular contacts, suggesting an ordered array. Moreover, we demonstrate that α-synuclein promotes SNARE complex assembly at the presynaptic plasma membrane in its multimeric membrane-bound state, but not in its monomeric cytosolic state. Our data delineate a folding pathway for α-synuclein that ranges from a monomeric, natively unfolded form in cytosol to a physiologically functional, multimeric form upon membrane binding, and show that only the latter but not the former acts as a SNARE complex chaperone at the presynaptic terminal, and may protect against neurodegeneration.α-Synuclein is an abundant presynaptic protein that physiologically acts to promote soluble NSF attachment protein receptor (SNARE) complex assembly in vitro and in vivo (1–3). Point mutations in α-synuclein (A30P, E46K, H50Q, G51D, and A53T) as well as α-synuclein gene duplications and triplications produce early-onset Parkinson''s disease (PD) (4–10). Moreover, α-synuclein is a major component of intracellular protein aggregates called Lewy bodies, which are pathological hallmarks of neurodegenerative disorders such as PD, Lewy body dementia, and multiple system atrophy (11–14). Strikingly, neurotoxic α-synuclein aggregates propagate between neurons during neurodegeneration, suggesting that such α-synuclein aggregates are not only intrinsically neurotoxic but also nucleate additional fibrillization (15–18).α-Synuclein is highly concentrated in presynaptic terminals where α-synuclein exists in an equilibrium between a soluble and a membrane-bound state, and is associated with synaptic vesicles (19–22). The labile association of α-synuclein with membranes (23, 24) suggests that binding of α-synuclein to synaptic vesicles, and its dissociation from these vesicles, may regulate its physiological function. Membrane-bound α-synuclein assumes an α-helical conformation (25–32), whereas cytosolic α-synuclein is natively unfolded and monomeric (refs. 25, 26, 31, and 32; however, see refs. 33 and 34 and Discussion for a divergent view). Membrane binding by α-synuclein is likely physiologically important because in in vitro experiments, α-synuclein remodels membranes (35, 36), influences lipid packing (37, 38), and induces vesicle clustering (39). Moreover, membranes were found to be important for the neuropathological effects of α-synuclein (40–44).However, the relation of membrane binding to the in vivo function of α-synuclein remains unexplored, and it is unknown whether α-synuclein binds to membranes as a monomer or oligomer. Thus, in the present study we have investigated the nature of the membrane-bound state of α-synuclein and its relation to its physiological function in SNARE complex assembly. We found that soluble monomeric α-synuclein assembles into higher-order multimers upon membrane binding and that membrane binding of α-synuclein is required for its physiological activity in promoting SNARE complex assembly at the synapse.     

Elasticity,friction, and pathway of γ-subunit rotation in FoF1-ATP synthase

We combine molecular simulations and mechanical modeling to explore the mechanism of energy conversion in the coupled rotary motors of F_oF₁-ATP synthase. A torsional viscoelastic model with frictional dissipation quantitatively reproduces the dynamics and energetics seen in atomistic molecular dynamics simulations of torque-driven γ-subunit rotation in the F₁-ATPase rotary motor. The torsional elastic coefficients determined from the simulations agree with results from independent single-molecule experiments probing different segments of the γ-subunit, which resolves a long-lasting controversy. At steady rotational speeds of ∼1 kHz corresponding to experimental turnover, the calculated frictional dissipation of less than k_BT per rotation is consistent with the high thermodynamic efficiency of the fully reversible motor. Without load, the maximum rotational speed during transitions between dwells is reached at ∼1 MHz. Energetic constraints dictate a unique pathway for the coupled rotations of the F_o and F₁ rotary motors in ATP synthase, and explain the need for the finer stepping of the F₁ motor in the mammalian system, as seen in recent experiments. Compensating for incommensurate eightfold and threefold rotational symmetries in F_o and F₁, respectively, a significant fraction of the external mechanical work is transiently stored as elastic energy in the γ-subunit. The general framework developed here should be applicable to other molecular machines.F_oF₁-ATP synthase is essential for life. From bacteria to human, this protein synthesizes ATP from ADP and inorganic phosphate P_i in its F₁ domain, powered by an electrochemical proton gradient that drives the rotation of its membrane-embedded F_o domain (1–5). Its two rotary motors, F₁ and F_o, are coupled through the γ-subunit forming their central shaft (2). ATP synthase is a fully reversible motor, in which the rotational direction switches according to different sources of energy (2, 6). In hydrolysis mode, the F₁ motor pumps protons against an electrochemical gradient across the membrane-embedded F_o part, converting ATP to ADP and P_i (7, 8).F₁ has a symmetric ring structure composed of three αβ-subunits with the asymmetric γ-subunit sitting inside the ring (9, 10). Each αβ-subunit has a catalytic site located at the αβ-domain interface. The F₁ ring has a pseudothreefold symmetry with the three αβ-subunits taking three different conformations, E (empty), TP (ATP-bound), and DP (ADP bound) (9–11). The F_o part is composed of a c ring and an a subunit (3, 12). Driven by protons passing through the interface of the c ring and the a subunit, the c ring rotates together with the γ-subunit (rotor) relative to the a subunit, which is connected to the F₁ ring through the peripheral stalk of the b subunit (stator) (12). Interestingly, in nature, one finds a large variation in the number of subunits in the c ring. In animal mitochondria, one finds c₈ rings, requiring a minimal number of eight proton translocations for the synthesis of three ATP, at least 20% fewer protons than in bacteria and plant chloroplasts with c₁₀–c₁₅ rings (13, 14). The resulting symmetry mismatches between F₁ and F_o (15–17) clearly distinguish the biomolecular motor from macroscopic machines.Key open questions concern the detailed rotational pathway of the two coupled rotary motors, the impact of the rotational symmetry mismatch between the F_o and F₁ motors on the motor mechanics, the resulting need for transient energy storage, the role of frictional dissipation, and the molecular elements associated with stepping of the F₁ motor (18–24). Here we explore these questions by building a dissipative mechanical model of the F₁ motor on the basis of atomistic molecular dynamics (MD) simulations. Friction and torsional elasticity of the γ-subunit are central to the efficient function of the coupled F_oF₁ nanomotors (15, 25, 26). For γ-subunits cross-linked with the α₃β₃-ring, estimates have been obtained by monitoring thermal angle fluctuations in single-molecule experiments (16, 27) and MD simulations (28). To probe the elastic and frictional properties under mechanical load over broad ranges of rotation angles and angular velocities, we induce torque-driven γ-subunit rotation in MD simulations (20, 29). From the resulting mechanical deformation and energy dissipation, we construct a fully quantitative viscoelastic model. We account for the torsional elasticity and friction by describing the rotational motion of the γ-subunit as overdamped Langevin dynamics on a 2D harmonic free energy surface. The model quantifies the magnitude of transient elastic energy storage compensating for the incommensurate rotational symmetries of the F_o and F₁ motors (30). The resulting energetic constraints allow us to map out a detailed pathway for their coupled rotary motions, and to rationalize the finer stepping of the mammalian F₁ motor seen in recent experiments (31), with only eight c subunits in the corresponding F_o motor. By quantifying the frictional dissipation, we identify a key contributor to the high thermodynamic efficiency of the F₁ motor. The general framework developed here for F₁ should be applicable also to other molecular machines.     

Interacting cytoplasmic loops of subunits a and c of Escherichia coli F1F0 ATP synthase gate H+ transport to the cytoplasm

H⁺-transporting F₁F₀ ATP synthase catalyzes the synthesis of ATP via coupled rotary motors within F₀ and F₁. H⁺ transport at the subunit a–c interface in transmembranous F₀ drives rotation of a cylindrical c₁₀ oligomer within the membrane, which is coupled to rotation of subunit γ within the α₃β₃ sector of F₁ to mechanically drive ATP synthesis. F₁F₀ functions in a reversible manner, with ATP hydrolysis driving H⁺ transport. ATP-driven H⁺ transport in a select group of cysteine mutants in subunits a and c is inhibited after chelation of Ag⁺ and/or Cd⁺² with the substituted sulfhydryl groups. The H⁺ transport pathway mapped via these Ag⁺(Cd⁺²)-sensitive Cys extends from the transmembrane helices (TMHs) of subunits a and c into cytoplasmic loops connecting the TMHs, suggesting these loop regions could be involved in gating H⁺ release to the cytoplasm. Here, using select loop-region Cys from the single cytoplasmic loop of subunit c and multiple cytoplasmic loops of subunit a, we show that Cd⁺² directly inhibits passive H⁺ transport mediated by F₀ reconstituted in liposomes. Further, in extensions of previous studies, we show that the regions mediating passive H⁺ transport can be cross-linked to each other. We conclude that the loop-regions in subunits a and c that are implicated in H⁺ transport likely interact in a single structural domain, which then functions in gating H⁺ release to the cytoplasm.The F₁F₀-ATP synthase of oxidative phosphorylation uses the energy of a transmembrane electrochemical gradient of H⁺ or Na⁺ to mechanically drive the synthesis of ATP via two coupled rotary motors in the F₁ and F₀ sectors of the enzyme (1). H⁺ transport through the transmembrane F₀ sector is coupled to ATP synthesis or hydrolysis in the F₁ sector at the surface of the membrane. Homologous ATP synthases are found in mitochondria, chloroplasts, and many bacteria. In Escherichia coli and other eubacteria, F₁ consists of five subunits in an α₃β₃γδε stoichiometry. F₀ is composed of three subunits in a likely ratio of a₁b₂c₁₀ in E. coli and Bacillus PS3 (2, 3) or a₁b₂c₁₁ in the Na⁺ translocating Ilyobacter tartaricus ATP synthase (1, 4) and may contain as many as 15 c subunits in other bacterial species (5). Subunit c spans the membrane as a hairpin of two α-helices, with the first transmembrane helix (TMH) on the inside and the second TMH on the outside of the c ring (1, 4). The binding of Na⁺ or H⁺ occurs at an essential, membrane-embedded Glu or Asp on cTMH2. High-resolution X-ray structures of both Na⁺- and H⁺-binding c-rings have revealed the details and variations in the cation binding sites (4–8). In the H⁺-translocating E. coli enzyme, Asp-61 at the center of cTMH2 is thought to undergo protonation and deprotonation, as each subunit of the c ring moves past the stationary subunit a. In the functioning enzyme, the rotation of the c ring is thought to be driven by H⁺ transport at the subunit a/c interface. Subunit γ physically binds to the cytoplasmic surface of the c-ring, which results in the coupling of c-ring rotation with rotation of subunit γ within the α₃β₃ hexamer of F₁ to mechanically drive ATP synthesis (1).E. coli subunit a folds in the membrane with five TMHs and is thought to provide aqueous access channels to the H⁺-binding cAsp-61 residue (9, 10). Interaction of the conserved Arg-210 residue in aTMH4 with cTMH2 is thought to be critical during the deprotonation–protonation cycle of cAsp-61 (1, 11, 12). At this time, very limited biophysical or crystallographic information is available on the 3D arrangement of the TMHs in subunit a. TMHs 2–5 of subunit a pack in a four-helix bundle, which was initially defined by cross-linking (13), but now, such a bundle, packing at the periphery of the c-ring, has been viewed directly by high-resolution cryoelectron microscopy in the I. tartaricus enzyme (14). Previously published cross-linking experiments support the identification of aTMH4 and aTMH5 packing at the periphery of the c-ring and the identification of aTHM2 and aTMH3 as the other components of the four-helix bundle seen in these images (13, 15, 16). More recently, published cross-linking experiments identify the N-terminal α-helices of two b subunits, one of which packs at one surface of aTMH2 with close enough proximity to the c-ring to permit cross-linking (17). The other subunit b N-terminal helix packs on the opposite peripheral surface of aTMH2 in a position where it can also be cross-linked to aTMH3 (17). The last helix density shown in Hakulinen and colleagues (14) packs at the periphery of the c-ring next to aTMH5 and is very likely to be aTMH1.The aqueous accessibility of Cys residues introduced into the five TMHs of subunit a has been probed on the basis of their reactivity with and inhibitory effects of Ag⁺ and other thiolate-reactive agents (18–20). Two regions of aqueous access were found with distinctly different properties. One region in TMH4, extending from Asn-214 and Arg-210 at the center of the membrane to the cytoplasmic surface, contains Cys substitutions that are sensitive to inhibition by both N-ethylmaleimide (NEM) and Ag⁺ (18–20; Fig. 1). These NEM- and Ag⁺-sensitive residues in TMH4 pack at or near the peripheral face and cytoplasmic side of the modeled four-helix bundle (11, 13). A second set of Ag⁺-sensitive substitutions in subunit a mapped to the opposite face and periplasmic side of aTMH4 (18, 19), and Ag⁺-sensitive substitutions were also found in TMHs 2, 3, and 5, where they extend from the center of the membrane to the periplasmic surface (19, 20). The Ag⁺-sensitive substitutions on the periplasmic side of TMHs 2–5 cluster at the interior of the four-helix bundle predicted by cross-linking and could interact to form a continuous aqueous pathway extending from the periplasmic surface to the central region of the lipid bilayer (11, 13, 19, 20). We have proposed that the movement of H⁺ from the periplasmic half-channel and binding to the single ionized Asp61 in the c-ring is mediated by a swiveling of TMHs at the a–c subunit interface (16, 21–24). This gating is thought to be coupled with ionization of a protonated cAsp61 in the adjacent subunit of the c-ring and with release of the H⁺ into the cytoplasmic half-channel at the subunit a–c interface. The route of aqueous access to the cytoplasmic side of the c subunit packing at the a–c interface has also been mapped by the chemical probing of Cys substitutions and, more recently, by molecular dynamics simulations (22, 25, 26).Open in a separate windowFig. 1.The predicted topology of subunit a in the E. coli inner membrane. The location of the most Ag⁺-sensitive Cys substitutions are highlighted in red (>85% inhibition) or orange (66–85% inhibition). The five proposed TMHs are shown in boxes, each with a span of 21 amino acids, which is the minimum length required to span the hydrophobic core of a lipid bilayer. The α-helical segments shown in loops 1–2 and 3–4 are consistent with the predictions of TALOS, based on backbone chemical shifts seen by NMR (29). Others have also predicted extensive α-helical regions in these loops (12, 30), but the possible positions remain largely speculative. aArg210 is highlighted in green. Figure is modified from those shown previously (21, 23, 24, 27).We have also reported Ag⁺-sensitive Cys substitutions in two cytoplasmic loops of subunit a (27) and, more recently, in the cytoplasmic loop of subunit c (28). The mechanism by which Ag⁺ inhibited F₁F₀-mediated H⁺ transport was uncertain. Several of these substitutions were also sensitive to inhibition by Cd⁺², and these substitutions provided a means of testing whether Cd⁺² directly inhibits passive H⁺ transport through F₀ (28). In the case of two subunit c loop substitutions, Cd⁺² was shown to directly inhibit passive F₀-mediated transport activity. In this study, we have extended the survey to Cd⁺²-sensitive Cys substitutions in cytoplasmic loops of subunit a. We report four loop substitutions in which Cd⁺² inhibits passive F₀-mediated H⁺ transport. Further, in two cases, we show cross-linking between pairs of Cys substitutions, which lie in subunits a and c, respectively, and which individually mediate passive H⁺ transport activity. These results suggest that the a and c loops, which gate H⁺ release to the cytoplasm, fold into a single domain at the surface of F₀.     

Helical arrays of U-shaped ATP synthase dimers form tubular cristae in ciliate mitochondria

F₁F_o-ATP synthases are universal energy-converting membrane protein complexes that synthesize ATP from ADP and inorganic phosphate. In mitochondria of yeast and mammals, the ATP synthase forms V-shaped dimers, which assemble into rows along the highly curved ridges of lamellar cristae. Using electron cryotomography and subtomogram averaging, we have determined the in situ structure and organization of the mitochondrial ATP synthase dimer of the ciliate Paramecium tetraurelia. The ATP synthase forms U-shaped dimers with parallel monomers. Each complex has a prominent intracrista domain, which links the c-ring of one monomer to the peripheral stalk of the other. Close interaction of intracrista domains in adjacent dimers results in the formation of helical ATP synthase dimer arrays, which differ from the loose dimer rows in all other organisms observed so far. The parameters of the helical arrays match those of the cristae tubes, suggesting the unique features of the P. tetraurelia ATP synthase are directly responsible for generating the helical tubular cristae. We conclude that despite major structural differences between ATP synthase dimers of ciliates and other eukaryotes, the formation of ATP synthase dimer rows is a universal feature of mitochondria and a fundamental determinant of cristae morphology.F₁F_o-ATP synthases are ubiquitous, highly conserved energy-converting membrane protein complexes. ATP synthases produce ATP from ADP and inorganic phosphate (P_i) by rotary catalysis (1, 2) using the energy stored in a transmembrane electrochemical gradient. The ∼600-kDa monomer of the mitochondrial ATP synthase is composed of a soluble F₁ subcomplex and a membrane-bound F_o subcomplex (3). The main components of the F₁ subcomplex are the (αβ)₃ hexamer and the central stalk (4). The F_o subcomplex includes a rotor ring of 8–15 hydrophobic c subunits (5), the peripheral stalk, and several small hydrophobic stator subunits. Protons flowing through the membrane part of the F_o subcomplex drive the rotation of the c-ring (6–9). The central stalk transmits the torque generated by c-ring rotation to the catalytic head of the F₁ subcomplex, where it induces conformational changes of the α and β subunits that result in phosphate bond formation and the generation of ATP. The catalytic (αβ)₃ hexamer is held stationary relative to the membrane region by the peripheral stalk (10, 11). Several high-resolution structures of the F₁/rotor ring complexes have been solved by X-ray crystallography (12–16), and the structure of the complete assembly has been determined by cryoelectron microscopy (cryo-EM) (10, 17–20).In mitochondria, the ATP synthase forms dimers in the inner membrane. In fungi, plants, and metazoans, the dimers are V-shaped and associate into rows along the highly curved ridges of lamellar cristae (19–22). F_o subcomplexes of the two monomers in the dimer interact in the lipid bilayer via a number of hydrophobic stator subunits (20, 23–25). Coarse-grained molecular dynamics simulations have suggested that the V-shape of the ATP synthase dimers induces local membrane curvature, which in turn drives the association of ATP synthase dimers into rows (20). The exact role of the dimer rows is unclear, however rows of ATP synthase dimers have been proposed to promote the formation of lamellar cristae in yeast (20, 26).So far, all rows of ATP synthase dimers observed by electron cryotomography have been more or less straight (19–22, 27). However, an earlier deep-etch freeze-fracture study of mitochondria from the ciliate Paramecium multimicronucleatum revealed double rows of interdigitating 10-nm particles on helical tubular cristae (28). These particles were interpreted as ATP synthases, which, if correct, would suggest that the mitochondrial ATP synthase can assemble into rows that differ significantly from the standard geometry found in lamellar cristae (19, 21, 22).To investigate the helical rows in more detail, we performed electron cryotomography of isolated mitochondrial membranes from Paramecium tetraurelia. Using subtomogram averaging, we show that these helical rows do indeed consist of ATP synthase molecules, as suggested by Allen et al. (28). However, unlike the V-shaped dimers of metazoans, the ATP synthase of this species forms U-shaped dimers, which have new and unusual structural features. When assembled into the helical rows, the ATP synthase monomers interdigitate, whereas the U-shaped dimers align side by side. Thus, rows of ATP synthase dimers seem to be a universal feature of all mitochondria. We propose that the particular shape of the P. tetraurelia ATP synthase dimer induces its assembly into helical rows, which in turn cause the formation of the helical tubular cristae of ciliates.     

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.     

Anatomy of F1-ATPase powered rotation

F₁-ATPase, the catalytic complex of the ATP synthase, is a molecular motor that can consume ATP to drive rotation of the γ-subunit inside the ring of three αβ-subunit heterodimers in 120° power strokes. To elucidate the mechanism of ATPase-powered rotation, we determined the angular velocity as a function of rotational position from single-molecule data collected at 200,000 frames per second with unprecedented signal-to-noise. Power stroke rotation is more complex than previously understood. This paper reports the unexpected discovery that a series of angular accelerations and decelerations occur during the power stroke. The decreases in angular velocity that occurred with the lower-affinity substrate ITP, which could not be explained by an increase in substrate-binding dwells, provides direct evidence that rotation depends on substrate binding affinity. The presence of elevated ADP concentrations not only increased dwells at 35° from the catalytic dwell consistent with competitive product inhibition but also decreased the angular velocity from 85° to 120°, indicating that ADP can remain bound to the catalytic site where product release occurs for the duration of the power stroke. The angular velocity profile also supports a model in which rotation is powered by Van der Waals repulsive forces during the final 85° of rotation, consistent with a transition from F₁ structures 2HLD1 and 1H8E (Protein Data Bank).The purified F₁-ATPase is a molecular motor that can hydrolyze ATP to drive counterclockwise (CCW) rotation of the γ-subunit within the (αβ)₃-ring (Fig. 1A). In most living organisms, the F_o component of the F_oF₁ complex uses energy derived from a proton-motive force across a membrane to power F₁-dependent synthesis of ATP from ADP and P_i. Consumption of an ATP at each F₁ catalytic site, primarily composed of a β-subunit, correlates with a 120° rotational power stroke of the γ-subunit separated by a catalytic dwell with an 8-ms duration in Escherichia coli enzyme (1). A second “ATP-binding” dwell can occur after the γ-subunit has rotated ∼30° to 40° from the catalytic at limiting substrate concentrations (2, 3). Thus, three successive catalytic events that include power strokes and dwells are required to complete one revolution of the γ-subunit. Once bound to F₁, ATP is retained for 240° (3) such that the ADP and P_i generated are released two catalytic events later.Open in a separate windowFig. 1.Structural components of the F₁-ATPase molecular motor. (A) Top (from membrane) and side views of F₁ composed of the ring of α (orange) and β (purple) subunits surrounding the γ-subunit rotor (blue and green). (B) Open (β_E) and closed (β_D) conformations of the catalytic site composed of the catalytic domain (tan ribbon) and the lever domain (purple) relative to the γ-subunit coiled-coil (green) and foot (blue) domains. In the Gibbons et al. (32) F₁ structure (PDB ID code 1E79) used here, the γ-subunit foot domain is rotated 7° CCW from the structure determined by Menz et al. (4).The γ-subunit is composed of coiled-coil and globular “foot” domains where the former extends through the core of the (αβ)₃-ring (Fig. 1B). The β-subunits contain a catalytic domain and a C-terminal “lever” domain that is extended or open when the catalytic site is devoid of nucleotide and contracted (closed) when nucleotide is bound. In most F₁ crystal structures (4, 5), the coiled-coil faces the β-subunit with the open lever (β_E) whereas the foot domain extends over the lever domains of catalytic sites that usually contain bound ADP (β_D) and ATP (β_T). Although crystal structures provide excellent pictures of the subunit conformations at one rotational position, the rotational movement of the γ-subunit between these static structures and the mechanism in which ATP fuels this movement occurs remain major unresolved questions.Consensus is currently lacking regarding the relationship of nucleotide occupancy at the three catalytic sites to the catalytic dwell and ATP-binding dwell despite the intense scrutiny this question has received since Boyer and coworkers (6, 7) showed that F₁ operates via an alternating site mechanism. The catalytic dwell includes ATP hydrolysis and is believed to be terminated by the release of phosphate (3). Some single-molecule experiments support a mechanism whereby ATP binding and ADP release are concurrent during the ATP-binding dwell (3). As a result, only two catalytic sites are occupied the majority of the time such that three-site occupancy occurs transiently during the ATP-binding dwell. However, these results are inconsistent with the F₁ structure that contains transition state analogs and has three-site nucleotide occupancy (4). Nucleotide binding studies also strongly support a mechanism in which all three sites must be occupied (8, 9) and are consistent with other single-molecule studies that support alternative three-site mechanisms (10–12). At this time there is no consistent evidence that correlates any of the crystal structures to the prevailing rotational mechanism.The β-subunit lever domain is positioned to push against the γ-foot and the γ–coiled-coil as it opens and closes, respectively (Fig. 1B). The asymmetry of the γ-subunit at these interfaces resembles a camshaft that would be consistent with CCW directionality in response to lever movement. The energy for a 120° power stroke has been proposed to derive from the binding affinity of ATP that is used as ATP binding-induced closing of the β-lever (13) and is supported by experiments in which the lever was truncated (14).Based on single-molecule measurements, it was concluded that F₁ is nearly 100% efficient (15). A necessary outcome of this conclusion is that the 120° power strokes must occur at a constant angular velocity (13). Although a number of simulation studies have modeled rotation of the F₁-ATPase γ-subunit (16–19), only one of these (19) has provided a result showing that the angular velocity should vary during a power stroke. The claim of 100% efficiency (15) that serves as the energetic basis of this power stroke mechanism is unwarranted because the magnitude of the viscous flow coupling to the surface was unknown owing to technical limitations, and the authors erroneously used the value of ΔG° in lieu of ΔG in their calculation. The technical handicap was subsequently overcome by Junge and coworkers (20, 21), who relied on elastic probe curvature instead of rotation speed to calculate the average torque of the power stroke. Similar average torque values were subsequently obtained using rotation under limiting drag conditions, when the drag on the probe was measured directly (22).Based on 100% enzyme efficiency, it was difficult to explain how the energy from ATP binding was able to power 120° of rotation when the catalytic dwell interrupts rotation 80° after ATP binds. It was hypothesized that the remaining binding energy needed to power the final 40° of rotation until the next ATP binds is stored as elastic energy in the closed β-lever, which upon product release pushes on the γ-foot as it opens (13). However, to date, experimental evidence that tests these hypotheses is lacking owing to the inability to measure the rotary motion under conditions where the angular velocity is limited by the internal mechanism of the motor.Here we have resolved the angular velocity as a function of the rotational position using an assay that provides 10-μs time resolution. The results clearly show that the angular velocity is not constant during a power stroke, but undergoes a series of accelerations and decelerations as a function of rotational position. The slower angular velocity observed with the lower-affinity substrate ITP provides direct evidence that substrate binding affinity provides energy to power rotation. The correlation of the angular velocity profile of the final 85° of rotation presented here to the profile resulting from the simulations of Pu and Karplus (19) strongly supports a model in which ATP binding-dependent closure of the lever applies force to the γ-subunit. This also provides evidence that associates the F₁ structures of Kabaleeswaran et al. (23) and Menz et al. (4) with the protein conformations at 35° and 120° because they were used as the reference structures for the simulations of Pu and Karplus (19). The data also show that elevated ADP concentrations increase dwells at ∼35° and decrease the angular velocity between 85° and 120°. This indicates that ADP can remain bound subsequent to the ATP-binding dwell consistent with a three-site mechanism.     

IRBIT regulates CaMKIIα activity and contributes to catecholamine homeostasis through tyrosine hydroxylase phosphorylation

Inositol 1,4,5-trisphosphate receptor (IP₃R) binding protein released with IP₃ (IRBIT) contributes to various physiological events (electrolyte transport and fluid secretion, mRNA polyadenylation, and the maintenance of genomic integrity) through its interaction with multiple targets. However, little is known about the physiological role of IRBIT in the brain. Here we identified calcium calmodulin-dependent kinase II alpha (CaMKIIα) as an IRBIT-interacting molecule in the central nervous system. IRBIT binds to and suppresses CaMKIIα kinase activity by inhibiting the binding of calmodulin to CaMKIIα. In addition, we show that mice lacking IRBIT present with elevated catecholamine levels, increased locomotor activity, and social abnormalities. The level of tyrosine hydroxylase (TH) phosphorylation by CaMKIIα, which affects TH activity, was significantly increased in the ventral tegmental area of IRBIT-deficient mice. We concluded that IRBIT suppresses CaMKIIα activity and contributes to catecholamine homeostasis through TH phosphorylation.Inositol 1,4,5-trisphosphate receptor (IP₃R) binding protein released with IP₃ (IRBIT) was originally identified as a molecule that interacts with the intracellular calcium channel, IP₃R. IRBIT binds to and suppresses IP₃R activity in the resting state by blocking IP₃ access to IP₃R (1, 2). Our group and others have reported that IRBIT contributes to electrolyte transport, mRNA processing, and the maintenance of genomic integrity (3–9) through its interaction with multiple targets. However, little is known about the physiological role of IRBIT in the brain, where it is most highly expressed (1).Calcium calmodulin (CaM) dependent kinase II alpha (CaMKIIα) is a Ser/Thr kinase that is abundant in the central nervous system and is activated by the binding of Ca²⁺–CaM. CaMKIIα phosphorylates various target proteins and is involved in the regulation of synaptic transmission and plasticity (10, 11). CaMKIIα is expressed in the hippocampus, neocortex, thalamus, hypothalamus, olfactory bulb, cerebellum, and basal ganglia (12, 13). Many studies involving mutant mice and also pharmacological studies have indicated that CaMKIIα activity is essential for the acquisition of memory and learning (14, 15). In addition, the appropriate regulation of CaMKIIα is required for cognitive function and mood control (16–18). Thus, aberrant CaMKIIα activity is associated with several neuronal disorders such as schizophrenia, autism spectrum disorder, attention-deficit hyperactivity disorder (ADHD), and drug addiction, in which hyperactivity and social abnormalities are frequently observed (19–23). However, the precise mechanism linking CaMKIIα dysregulation and mental disorders is poorly understood.Recent behavioral studies using knockout (KO) mouse models or pharmacological approaches have revealed that the dysregulation of dopamine (DA) systems is correlated with a hyperactive phenotype and social abnormalities (24–26). The catecholamines, DA and norepinephrine (NE) are biosynthesized from the amino acids phenylalanine and tyrosine. The sequence of steps starts with the enzyme, tyrosine hydroxylase (TH). Thus, TH is the rate-limiting enzyme for both DA and NE synthesis. The appropriate regulation of TH activity is important for the maintenance of normal brain function and mental state (27).In this study, we identified CaMKIIα as an IRBIT-interacting molecule in the central nervous system. IRBIT binds to and suppresses the kinase activity of CaMKIIα by inhibiting the binding of CaM to CaMKIIα. In addition, we found that mice deficient in IRBIT present with hyperactivity and social abnormalities. In IRBIT KO mice, we observed increased catecholamine levels and hyperphosphorylation of Ser19 on TH, which is known to enhance TH activity and increase the biosynthesis of DA and NE (27, 28). Thus, we have concluded that IRBIT regulates CaMKIIα activity and contributes to catecholamine homeostasis through TH phosphorylation.     

Computational redesign of the lipid-facing surface of the outer membrane protein OmpA

Advances in computational design methods have made possible extensive engineering of soluble proteins, but designed β-barrel membrane proteins await improvements in our understanding of the sequence determinants of folding and stability. A subset of the amino acid residues of membrane proteins interact with the cell membrane, and the design rules that govern this lipid-facing surface are poorly understood. We applied a residue-level depth potential for β-barrel membrane proteins to the complete redesign of the lipid-facing surface of Escherichia coli OmpA. Initial designs failed to fold correctly, but reversion of a small number of mutations indicated by backcross experiments yielded designs with substitutions to up to 60% of the surface that did support folding and membrane insertion.The β-barrel membrane proteins comprise one of the two structural classes of integral membrane proteins. They are found within the outer membranes of bacteria, mitochondria, and chloroplasts, where they perform a range of structural, transport, and catalytic functions (1). In addition to their biological interest they are increasingly relevant to biotechnology, serving as scaffolds for bacterial surface display (2, 3) and atomically precise pores for nanopore-based DNA sequencing. Although the suitability of natural β-barrel membrane proteins for biotechnology has been improved by protein engineering (3–10), the ability to design membrane proteins de novo would deliver tools customized to meet the demands of each application.De novo design provides a stringent test of our understanding of the determinants of protein folding and stability. Protein design software [e.g., Rosetta (11, 12)] has made tremendous strides in addressing the design problem for small water-soluble proteins (13–15), and design of simplified model α-helical membrane proteins including single transmembrane helices and small bundles (16–20) has also been accomplished. In contrast, a designed β-barrel membrane protein has yet to be reported, perhaps as a consequence of the unique design challenges presented by the folding pathway and architecture of these proteins. Unlike the α-helical membrane proteins, nascent β-barrel membrane proteins must transit the periplasm to the outer membrane, where folding and membrane insertion are thought to occur in concert (21, 22). An extensive network of chaperones maintains the solubility of the unfolded barrel and guides membrane insertion. The C-terminal β-strand is known to interact with the BAM chaperone complex (23–25), which assists the folding of all β-barrel membrane proteins. However, despite recent progress (26–30), we do not fully understand how interactions between chaperones and transiting membrane proteins are directed by sequence-encoded information.Further complicating design is the inside-out architecture of β-barrel membrane proteins. In place of a hydrophobic core is either a central water-filled pore or a solid core composed of polar side chains. The lipid bilayer becomes increasingly hydrophobic at greater depths within the membrane (31), and this environmental anisotropy is reflected in the amino acid composition of the barrel surface. Aliphatic side chains are prevalent toward the center of the membrane, and aromatic side chains are common in the lipid head group regions, where they encircle the barrel in external- and periplasmic-side girdles (32).Recently we developed E_zβ, a membrane depth-dependent, residue-level potential calculated from an ensemble of experimentally determined outer membrane protein structures (33, 34). E_zβ can be used to estimate energetics of membrane insertion to predict transmembrane protein orientation within the bilayer, and to detect oligomerization sites on β-barrel surfaces (34). E_zβ and related statistical functions (35, 36) can recapitulate properties of natural outer membrane proteins (37, 38) and predict the effects of mutations on protein stability and oligomerization (39). Similar potentials have driven computational approaches that have fully redesigned α-helical membrane protein surfaces to convert membrane proteins into water-soluble ones (40–42).Here, we considered whether the complete redesign of the lipid-facing surface of an outer membrane protein using a statistical potential such as E_zβ preserves its structure and function. This approach allowed us to investigate whether membrane insertion requires only a lipid-facing surface composed of depth-appropriate hydrophobic residues, or whether folding requires sequence-specific interstrand interactions, chaperone-recruiting sequences, evolutionarily optimized aromatic girdles, folding nucleation sites, or other design features lost during the population averaging inherent in parameter fitting of statistical potentials.Previous studies have explored the sensitivity of the β-barrel fold and its chaperone recognition mechanisms to mutations. The canonical eight-stranded β-barrel membrane protein OmpA tolerates a limited number of mutations to the lipid-facing surface, provided hydrophobicity is maintained (43, 44). More radically, the eight-stranded barrel OmpX has been duplicated to form a 16-stranded barrel capable of membrane insertion (45). However, the lipid-facing residues of transmembrane β-strands are conserved across homologous β-barrel membrane proteins beyond the extent expected from hydrophobicity alone (46, 47), implying a functional role that has yet to be elucidated.To explore the sequence constraints on β-barrel membrane proteins, we extensively redesigned the lipid-facing surface of E. coli OmpA. We created a series of OmpA variants with entirely or partially redesigned lipid-facing surfaces and tested their ability to insert into the outer membrane of E. coli. Our results indicate that the surfaces of β-barrel membrane proteins are amenable to large-scale redesign, provided that energetically destabilizing substitutions are avoided.     

Individual IKs channels at the surface of mammalian cells contain two KCNE1 accessory subunits

KCNE1 (E1) β-subunits assemble with KCNQ1 (Q1) voltage-gated K⁺ channel α-subunits to form I_Kslow (I_Ks) channels in the heart and ear. The number of E1 subunits in I_Ks channels has been an issue of ongoing debate. Here, we use single-molecule spectroscopy to demonstrate that surface I_Ks channels with human subunits contain two E1 and four Q1 subunits. This stoichiometry does not vary. Thus, I_Ks channels in cells with elevated levels of E1 carry no more than two E1 subunits. Cells with low levels of E1 produce I_Ks channels with two E1 subunits and Q1 channels with no E1 subunits—channels with one E1 do not appear to form or are restricted from surface expression. The plethora of models of cardiac function, transgenic animals, and drug screens based on variable E1 stoichiometry do not reflect physiology.Voltage-gated potassium (K_V) channels include four α-subunits that form a single, central ion conduction pathway with four peripheral voltage sensors (1–3). Incorporation of accessory β-subunits modifies the function of K_V channels to suit the diverse requirements of different tissues. KCNE genes encode minK-related peptides (MiRPs) (4–6), β-subunits with a single transmembrane span that assemble with a wide array of K_V α-subunits (7, 8) to control surface expression, voltage dependence, and kinetics of gating transitions, unitary conductance, ion selectivity, and pharmacology of the resultant channel complexes (4, 9–15). I_Kslow (I_Ks) channels in the heart and inner ear are formed by the α-subunit encoded by KCNQ1 (called Q1, K_VLQT1, K_V7.1, or KCNQ1) and the β-subunit encoded by KCNE1 (called E1, mink, or KCNE1) (16, 17). Inherited mutations in Q1 and E1 are associated with cardiac arrhythmia and deafness.The number of E1 subunits in I_Ks channels has been a longstanding matter of disagreement. We first argued for two E1 subunits per channel based on the suppression of current by an E1 mutant (18). Subsequently, we reached the same conclusion by determining the total number of channels using radiolabeled charybdotoxin (CTX), a scorpion toxin that blocks channels when one molecule binds in the external conduction pore vestibule, and an antibody-based luminescence assay to tally E1 subunits (19). Morin and Kobertz (20) used iterative chemical linkage between CTX in the pore and E1, and they also assigned two accessory subunits to >95% of I_Ks channels without gathering evidence for variation in subunit valence. Furthermore, when we formed I_Ks channels from separate E1 and Q1 subunits and compared them with channels enforced via genetic encoding to contain two or four E1 subunits (19), we observed the natural I_Ks channels to have the same gating attributes, small-molecule pharmacology, and CTX on and off rates (a reflection of pore vestibule structure) as channels encoded with two E1 subunits but not those with four. These findings support the conclusion that two E1 subunits are necessary, sufficient, and the normal number in I_Ks channels.In contrast, others have argued that I_Ks channels have variable stoichiometry with one to four E1 subunits, or even more (21–24). Recently, Nakajo et al. (25) applied single-particle spectroscopy to the question; this powerful “gold-standard” tool has been a valuable strategy to assess the subunit composition of ion channels (26–28) and should be expected to improve on prior investigations conducted on populations of I_Ks channels and subject, therefore, to the simplifying assumptions that attend macroscopic studies (29). Nakajo et al. (25) reported a variable number of E1 subunits, from one to four, in I_Ks channels studied in Xenopus laevis oocytes. The impact of this result has been striking because it has engendered new models of cardiac physiology, altered models of I_Ks channel biosynthesis and function, stimulated the use of transgenic animals artificially enforced to express I_Ks channels with four E1 subunits (by expression of a fused E1–Q1 subunit), and prompted cardiac drug design based on the assumption that I_Ks channels can form with one E1 subunit (23, 30–32).We were concerned that the conclusions of Nakajo et al. (25) were in error because they appraised only a limited fraction of particles that were immobile in the oocyte membrane; counted E1 and Q1 asynchronously rather than simultaneously (increasing the risk that particles moved into or out of the field of view); and studied Q1 and E1 appended not only with the fluorescent proteins (FP) required to count subunits by photobleaching but also with a common trafficking motif that suppressed channel mobility by interacting with an overexpressed anchoring protein, thereby risking nonnatural aggregation of subunits.Here, to resolve mobility problems and obviate the need for modification of subunits with targeting motifs, we describe and perform single-fluorescent-particle photobleaching at the surface of live mammalian cells, demonstrating three spectroscopic counting approaches: standard, asynchronous subunit counting; simultaneous, two-color subunit counting; and toxin-directed, simultaneous, two-color photobleaching. To analyze the data, we use two statistical approaches—one to assess the degree of colocalization of objects in dual-color images (33) and the other to infer stoichiometry from single-molecule photobleaching (34). These methods also allow determination of the surface density of assemblies of defined subunit composition and are therefore useful to assess the formation and life cycle of membrane protein complexes.We report that single I_Ks channels at the surface of mammalian cells contain two E1 subunits—no more and no less. This finding refutes the single-particle studies of Nakajo et al. (25) in oocytes and macroscopic studies (21–24, 30–32), arguing that forcing cells to express excess E1 produces I_Ks channels containing more than two E1 subunits and that low levels of E1 yields I_Ks channels with less than two E1 subunits. Not once did we observe an I_Ks channel with three or four E1 subunits. Moreover, simultaneous, two-color subunit counting revealed that low amounts of E1 relative to Q1 [ratios like those reported in human cardiac ventricle (35, 36)] produced two types of channels on the cell surface: I_Ks channels (with two E1 subunits) and Q1 channels (with no E1 subunits). Finally, E1 was shown to increase in I_Ks channel surface expression threefold, as we predicted based on assessment of I_Ks channel unitary conductance (11), whereas few E1 subunits were on the surface outside of I_Ks channels, even when E1 was expressed alone. This finding indicates that E1 does not travel to the surface readily on its own, that two E1 subunits facilitate I_Ks channel trafficking to the surface (or enhance surface residence time compared with Q1 channels), and that I_Ks channels with only one E1 subunit do not form, do not reach the surface, or are rapidly recycled.     

Contribution of reactive oxygen species to cerebral amyloid angiopathy,vasomotor dysfunction,and microhemorrhage in aged Tg2576 mice

Cerebral amyloid angiopathy (CAA) is characterized by deposition of amyloid β peptide (Aβ) within walls of cerebral arteries and is an important cause of intracerebral hemorrhage, ischemic stroke, and cognitive dysfunction in elderly patients with and without Alzheimer’s Disease (AD). NADPH oxidase-derived oxidative stress plays a key role in soluble Aβ-induced vessel dysfunction, but the mechanisms by which insoluble Aβ in the form of CAA causes cerebrovascular (CV) dysfunction are not clear. Here, we demonstrate evidence that reactive oxygen species (ROS) and, in particular, NADPH oxidase-derived ROS are a key mediator of CAA-induced CV deficits. First, the NADPH oxidase inhibitor, apocynin, and the nonspecific ROS scavenger, tempol, are shown to reduce oxidative stress and improve CV reactivity in aged Tg2576 mice. Second, the observed improvement in CV function is attributed both to a reduction in CAA formation and a decrease in CAA-induced vasomotor impairment. Third, anti-ROS therapy attenuates CAA-related microhemorrhage. A potential mechanism by which ROS contribute to CAA pathogenesis is also identified because apocynin substantially reduces expression levels of ApoE—a factor known to promote CAA formation. In total, these data indicate that ROS are a key contributor to CAA formation, CAA-induced vessel dysfunction, and CAA-related microhemorrhage. Thus, ROS and, in particular, NADPH oxidase-derived ROS are a promising therapeutic target for patients with CAA and AD.Cerebral amyloid angiopathy (CAA) is characterized by amyloid deposition within walls of leptomeningeal and cortical arterioles. Among the several types of amyloid proteins causing CAA, fibrillar amyloid β (Aβ) is by far the most common (1). This pathological form of Aβ is also the major constituent of neuritic plaques in patients with Alzheimer’s disease (AD) (2). Aβ is a 39- to 43-amino acid peptide that is produced from the amyloid precursor protein (APP) via sequential proteolytic cleavage processed by β- and γ-secretases (3, 4). Aβ₄₀ is the predominant Aβ species present in CAA whereas Aβ₄₂ is the major Aβ species present in neuritic plaques. CAA is a very common disorder, pathologically affecting about one-third of all elderly patients (>60 y of age) and about 90% of patients with AD (5, 6). CAA is a well-recognized cause of intracerebral hemorrhage (7, 8). It is also a major contributor to ischemic stroke and dementia (2, 9–12)—two conditions in which CAA-induced impairment in cerebral arteriole function is likely to play a fundamental role (13).Multiple lines of evidence indicate that soluble Aβ monomers and insoluble Aβ fibrils in the form of CAA cause significant cerebrovascular (CV) impairment. Ex vivo studies with isolated cerebral arterioles show that synthetic Aβ₄₀ (and to a lesser degree Aβ₄₂) induces direct vessel constriction, enhanced response to vasoconstrictors, and reduced response to vasodilators (14–22). Similar results have been demonstrated with synthetic Aβ₄₀ topically applied to the cerebral cortex (23, 24), results that are generally supported by in vivo studies (20, 23, 25). For example, Iadecola and coworkers have shown that young APP transgenic mice (Tg2576) exposed to elevated levels of Aβ₄₀ and Aβ₄₂ (but no CAA) have reduced baseline cerebral blood flow (CBF) and decreased CBF responses to topical vasodilators (23, 24, 26). We have shown similar CV deficits in young Tg2576 mice (13). Moreover, we provided the most direct evidence to date that endogenous soluble Aβ plays a causal role in these CV deficits when we found that depletion of soluble Aβ via γ-secretase inhibition restores CV function in young Tg2576 mice (13).Fibrillar Aβ in the form of CAA produces even greater degrees of CV impairment. Evidence for this notion comes from several experimental studies from different laboratories that show reduced pial arteriole responses (27) and diminished CBF responses (27, 28) to a variety of vasodilatory stimuli in aged APP mice with CAA vs. young APP mice without CAA. Our past work examining pial arteriole function in young vs. aged Tg2576 mice shows similar age-dependent CV deficits (13). Moreover, multiple additional observations from our study show that CAA (and not prolonged exposure to soluble Aβ and/or mutant APP) is the principle cause of the severe CV dysfunction noted in aged Tg2576 mice: (i) The severity of the vasomotor deficits noted in these mice is dependent on the presence and extent of CAA; (ii) even small amounts of CAA are associated with profound vasomotor impairment; and (iii) the CV dysfunction noted in CAA-ladened arteries is poorly responsive to depletion of soluble Aβ via γ-secretase inhibition (13).Regarding the mechanism of soluble Aβ-induced CV deficits, increased reactive oxygen species (ROS) are strongly implicated. Cerebral arterioles exposed to exogenous Aβ₄₀ develop significant oxidative stress (29), and various anti-ROS strategies have been shown to improve Aβ₄₀-induced vessel dysfunction (16, 23). Similarly, cerebral arterioles of young APP mice producing elevated levels of endogenous Aβ₄₀ and Aβ₄₂ (but no CAA) display oxidative stress (19), and the CV deficits found in these mice can be attenuated by both genetic and pharmacologic anti-ROS interventions (15, 19, 20, 30). In particular, ROS derived from NADPH oxidase—one of two major sources of ROS in the cerebrovasculature (31–33)—have been implicated (28–30, 34, 35).Regarding the mechanism of CAA-induced CV deficits, far less is known; however, three recent findings suggest that ROS may play a role. First, CAA-affected vessels were shown to have significantly greater oxidative stress than CAA-free vessels of aged Tg2576 mice (36). Second, genetic knockdown of mitochondrial superoxide dismutase 2 (SOD2)—which increases mitochondria-derived ROS—was shown to exacerbate CAA pathology in aged APP mice (37). Third, genetic depletion of the catalytic subunit Nox2 of NADPH oxidase was shown to reduce oxidative stress and improve CV function in aged Tg2576 mice (28, 35). Importantly, the latter studies did not examine for the presence of CAA, nor did they assess for the effect of CAA on cerebral arteriole function (28, 35). To address this critical knowledge gap, we examined the effect of the NADPH oxidase inhibitor, apocynin, and the nonspecific ROS scavenger, tempol, on CAA-induced CV dysfunction in aged Tg2576 mice. The effect of these agents on CAA formation and CAA-related microhemorrhage was also examined.     

Hydrogen sulphide pathway contributes to the enhanced human platelet aggregation in hyperhomocysteinemia

Homocysteine is metabolized to methionine by the action of 5,10 methylenetetrahydrofolate reductase (MTHFR). Alternatively, by the transulfuration pathway, homocysteine is transformed to hydrogen sulphide (H₂S), through multiple steps involving cystathionine β-synthase and cystathionine γ-lyase. Here we have evaluated the involvement of H₂S in the thrombotic events associated with hyperhomocysteinemia. To this purpose we have used platelets harvested from healthy volunteers or patients newly diagnosed with hyperhomocysteinemia with a C677T polymorphism of the MTHFR gene (MTHFR++). NaHS (0.1–100 µM) or l-cysteine (0.1–100 µM) significantly increased platelet aggregation harvested from healthy volunteers induced by thrombin receptor activator peptide–6 amide (2 µM) in a concentration-dependent manner. This increase was significantly potentiated in platelets harvested from MTHFR++ carriers, and it was reversed by the inhibition of either cystathionine β-synthase or cystathionine γ-lyase. Similarly, in MTHFR++ carriers, the content of H₂S was significantly higher in either platelets or plasma compared with healthy volunteers. Interestingly, thromboxane A₂ production was markedly increased in response to both NaHS or l-cysteine in platelets of healthy volunteers. The inhibition of phospholipase A₂, cyclooxygenase, or blockade of the thromboxane receptor markedly reduced the effects of H₂S. Finally, phosphorylated–phospholipase A₂ expression was significantly higher in MTHFR++ carriers compared with healthy volunteers. In conclusion, the H₂S pathway is involved in the prothrombotic events occurring in hyperhomocysteinemic patients.Hyperhomocysteinemia (HHcy) is a risk factor for neurovascular and cardiovascular disease associated with endothelial dysfunction and accelerated atherosclerosis (1–3). Many clinical and epidemiological studies have demonstrated a positive correlation between homocysteine (Hcy) plasma levels and cardiovascular disorders (4, 5), leading to the general conclusion that Hcy is a prothrombotic factor (6–8). However, the mechanism(s) through which elevated circulating levels of Hcy promote vascular disease and thrombosis is still unclear (9). Hcy has two primary fates: conversion through a reaction catalyzed by 5,10 methylenetetrahydrofolate reductase (MTHFR) into l-methionine or conversion to l-cysteine (l-Cys) via a transulfuration pathway (10, 11). The transulfuration pathway relies upon cystathionine β-synthase (CBS) to transform Hcy in cysthathionine, which is converted by cystathionine γ-lyase (CSE) into l-Cys. Thereafter, both enzymes convert l-Cys to generate hydrogen sulphide (H₂S) (12, 13). H₂S has been recognized as the third member of the family of gaseous transmitters (14), and it is present in human blood at micromolar concentrations (10–100 μM) (15). It rapidly travels through cell membranes without using any specific receptor/transporter or intracellular signaling proteins. CBS and CSE are differentially expressed in cardiovascular as well as in several other body districts (13). The physiological functions of H₂S are mediated by a variety of molecular targets, including ion channels and signaling proteins (16–18). Alterations in H₂S metabolism contribute to an array of cardiovascular disorders such as hypertension, atherosclerosis, heart failure, and diabetes (19). Nevertheless, the influence of H₂S on platelet function and, in turn, on blood clotting has been poorly explored. We hypothesized that H₂S could be involved in the thrombotic events associated with HHcy. To address this issue, we used human platelets harvested either from healthy volunteers or from patients with a C677T polymorphism of the MTHFR gene (MTHFR++) that is linked to HHcy (20–22).     

Structural insights into the interaction of IL-33 with its receptors

Interleukin (IL)-33 is an important member of the IL-1 family that has pleiotropic activities in innate and adaptive immune responses in host defense and disease. It signals through its ligand-binding primary receptor ST2 and IL-1 receptor accessory protein (IL-1RAcP), both of which are members of the IL-1 receptor family. To clarify the interaction of IL-33 with its receptors, we determined the crystal structure of IL-33 in complex with the ectodomain of ST2 at a resolution of 3.27 Å. Coupled with structure-based mutagenesis and binding assay, the structural results define the molecular mechanism by which ST2 specifically recognizes IL-33. Structural comparison with other ligand–receptor complexes in the IL-1 family indicates that surface-charge complementarity is critical in determining ligand-binding specificity of IL-1 primary receptors. Combined crystallography and small-angle X-ray–scattering studies reveal that ST2 possesses hinge flexibility between the D3 domain and D1D2 module, whereas IL-1RAcP exhibits a rigid conformation in the unbound state in solution. The molecular flexibility of ST2 provides structural insights into domain-level conformational change of IL-1 primary receptors upon ligand binding, and the rigidity of IL-1RAcP explains its inability to bind ligands directly. The solution architecture of IL-33–ST2–IL-1RAcP complex from small-angle X-ray–scattering analysis resembles IL-1β–IL-1RII–IL-1RAcP and IL-1β–IL-1RI–IL-1RAcP crystal structures. The collective results confer IL-33 structure–function relationships, supporting and extending a general model for ligand–receptor assembly and activation in the IL-1 family.Interleukin (IL)-33 has important roles in initiating a type 2 immune response during infectious, inflammatory, and allergic diseases (1–5). It was initially identified as a nuclear factor in endothelial cells and named NF-HEV (nuclear factor from high endothelial venules) (6, 7). In 2005, it was rediscovered as a new member of the IL-1 family and an extracellular ligand for the orphan IL-1 receptor family member ST2 (8). As an extracellular cytokine, IL-33 is involved in the polarization of Th2 cells and activation of mast cells, basophils, eosinophils, and natural killer cells (1–3). Recent studies also discovered that the type 2 innate lymphoid cells (ILC2s) are major target cells of IL-33 (9, 10). ILC2s express a high level of ST2 and secrete large amounts of Th2 cytokines, most notably IL-5 and IL-13, when stimulated with IL-33 (11–13). Activation of ILC2s is essential in the initiation of the type 2 immune response against helminth infection and during allergic diseases such as asthma (9, 10).IL-33 does not have a signal peptide and is synthesized with an N-terminal propeptide upstream of the IL-1–like cytokine domain. It is preferentially and constitutively expressed in the nuclei of structural and lining cells, particularly in epithelial and endothelial cells (14, 15). Tissue damage caused by pathogen invasion or allergen exposure may lead to the release of IL-33 into extracellular environment from necrotic cells, which functions as an endogenous danger signal or alarmin (14, 16). Full-length human IL-33 consists of 270 residues and is biologically active (17, 18). It is also a substrate of serine proteases released by inflammatory cells recruited to the site of injury (18, 19). The proteases elastase, cathespin G, and proteinase 3 cleave full-length IL-33 to release N-terminal–truncated mature forms containing the IL-1–like cytokine domain: IL-33_95–270, l-33_99–270, and IL-33_109–270 (18). These mature IL-33 forms process a 10-fold greater potency to activate ST2 than full-length IL-33 (18). Caspase-1 was also suggested to cleave IL-33 to generate an active IL-33_112–270 that is the commercially available mature IL-33 form (8). However, it was later demonstrated that this cleavage site does not exist and cleavage by caspases at other sites actually inactivates IL-33 (17, 20, 21).The signaling of IL-33 depends on its binding to the primary receptor ST2 and subsequent recruitment of accessory receptor IL-1RAcP (8, 22, 23). The ligand-binding–induced receptor heterodimerization results in the juxtaposition of the intracellular toll/interleukin-1 receptor (TIR) domains of both receptors, which is necessary and sufficient to activate NF-κB and MAPK pathways in the target cells (24). Previously, we determined the complex structure of IL-1β with its decoy receptor IL-1RII and accessory receptor IL-1RAcP (25). Based on this structure and other previous studies, we proposed a general structural model for the assembly and activation of IL-1 family of cytokines with their receptors (25). In this model, ligand recognition relies on interaction of IL-1 cytokine with its primary receptor: IL-1α and IL-1β with IL-1RI; IL-33 with ST2; IL-18 with IL-18Rα; and IL-36α, IL-36β, and IL-36γ with IL-1Rrp2 (26–28). The binding forms a composite surface to recruit accessory receptor IL-1RAcP shared by IL-1α, IL-1β, IL-33, IL-36α, IL-36β, and IL-36γ and IL-18Rβ by IL-18 (26, 27). This general structural model is further supported by the subsequent structural determination of IL-1β with IL-1RI and IL-1RAcP (29). However, there are still many key missing parts in the general structural model of ligand–receptor interaction in the IL-1 family. For example, the structural basis for specific recognition of IL-33 by ST2 and IL-18 by IL-18Rα, and promiscuous recognition of IL-36α, IL-36β, and IL-36γ by IL-1Rrp2 remains elusive. The proposed general model also needs further confirmation from structural studies of other signaling complexes in the IL-1 family. To address these issues, we studied the interaction of IL-33 with its receptors by a combination of X-ray crystallography and small-angle X-ray–scattering (SAXS) methods.     

Cysteine cathepsins are essential in lysosomal degradation of α-synuclein

A cellular feature of Parkinson’s disease is cytosolic accumulation and amyloid formation of α-synuclein (α-syn), implicating a misregulation or impairment of protein degradation pathways involving the proteasome and lysosome. Within lysosomes, cathepsin D (CtsD), an aspartyl protease, is suggested to be the main protease for α-syn clearance; however, the protease alone only generates amyloidogenic C terminal-truncated species (e.g., 1–94, 5–94), implying that other proteases and/or environmental factors are needed to facilitate degradation and to avoid α-syn aggregation in vivo. Using liquid chromatography–mass spectrometry, to our knowledge, we report the first peptide cleavage map of the lysosomal degradation process of α-syn. Studies of purified mouse brain and liver lysosomal extracts and individual human cathepsins demonstrate a direct involvement of cysteine cathepsin B (CtsB) and L (CtsL). Both CtsB and CtsL cleave α-syn within its amyloid region and circumvent fibril formation. For CtsD, only in the presence of anionic phospholipids can this protease cleave throughout the α-syn sequence, suggesting that phospholipids are crucial for its activity. Taken together, an interplay exists between α-syn conformation and cathepsin activity with CtsL as the most efficient under the conditions examined. Notably, we discovered that CtsL efficiently degrades α-syn amyloid fibrils, which by definition are resistant to broad spectrum proteases. This work implicates CtsB and CtsL as essential in α-syn lysosomal degradation, establishing groundwork to explore mechanisms to enhance their cellular activity and levels as a potential strategy for clearance of α-syn.A presynaptic neuronal protein, α-synuclein (α-syn), is linked genetically and neuropathologically to Parkinson’s disease (PD) (1). The protein exists in a multitude of conformations that likely dictates the physiological function of α-syn (2–6). Upon membrane association, α-syn adopts an α-helical structure (7), whereas in solution, the protein is disordered (3, 6, 8). Elucidation of molecular mechanisms underlying the transformation of α-syn to a disease-associated species is still the subject of intense research. However, the deposition of insoluble α-syn fibrils rich in β-sheet structure, generally referred to as amyloid is a hallmark of the disease (9, 10).Understanding the cellular pathways that control α-syn aggregation is critical in establishing ways to circumvent the progression of PD. A primary cause leading to PD is associated with impaired α-syn turnover (11–13). Hence, boosting the degradation of α-syn could be an effective way to alleviate this burden. Both in vitro and in vivo data support the involvement of the proteasome and lysosome in α-syn degradation (14, 15). The proteasome pathway is generally considered to be responsible for removal of soluble α-syn, whereas the lysosome eliminates aggregation-prone species or excess levels of α-syn. Dysfunction of either system has been shown to increase α-syn levels (16).α-syn enters via two main autophagic pathways (17), either through macroautophagy (14) or chaperone-mediated autophagy (18) into the lysosome, where the protein is degraded. The main class of lysosomal proteases is the cathepsins (Cts), which are subdivided based on the active-site amino acids that confer catalytic activity. These are cysteine (CtsB, CtsC, CtsF, CtsH, CtsK, CtsL, CtsS, CtsV, and CtsX), serine (CtsA and CtsG), and aspartyl (CtsD and CtsE) proteases (19). Activity of cathepsins is optimal in acidic pH, and most are inactive at neutral pH. CtsD (20) is the only protease implicated to date in the lysosomal degradation of α-syn (21–24).Although prior literature supports CtsD involvement, the question of whether other lysosomal protease(s) are involved is also raised. For example, a strong correlation exists in vivo between overexpression of CtsD and α-syn levels as well as the neurotoxic potential of overexpressed α-syn (21, 22). However, in vitro CtsD activity yields incomplete proteolysis of α-syn and generates truncated C-terminal (α-synΔC) species (24). More concerning is that both α-synΔC peptides, such as residues 1–87 (10), 1–103 (25), and 1–119 (26) of α-syn (residues 1–140), and the acidic environment of the lysosomal lumen (27) are known to enhance amyloid formation. Interestingly, human tissue samples contain an abundance of α-synΔC species under normal physiological conditions (28). Some of these α-syn fragments (1–115, 1–119, 1–122, 1–133, and 1–135) have also been isolated from Lewy bodies (29), classic PD hallmarks. The significance of these truncations remain ill-defined, but one could speculate the involvement of CtsD and partial lysosomal degradation of α-syn (24).Here, we sought to identify other lysosomal cathepsins and environmental factors needed to facilitate α-syn degradation and to avoid aggregation. Direct interaction between α-syn and lysosomal proteases was shown by purified mouse brain and liver lysosomal extracts as well as purified human cathepsins and was characterized by peptide mapping using liquid chromatography–mass spectrometry (LC-MS). We found that mouse brain and liver lysosomal extracts harbor mostly cysteine cathepsin activity (namely CtsB and CtsL) that efficiently degrades recombinant α-syn. Lysosomal and individual cathepsin activities on soluble, membrane-bound, and fibrillar α-syn as well as their impact on α-syn amyloid formation have been evaluated.     

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.