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Presynaptic active zones (AZs) contain many molecules essential for neurotransmitter release and are assembled in a highly organized manner. A network of adaptor proteins known as cytomatrix at the AZ (CAZ) is important for shaping the structural characteristics of AZ. Rab3-interacting molecule (RIM)-binding protein (RBP) family are binding partners of the CAZ protein RIM and also bind the voltage-gated calcium channels (VGCCs) in mice and flies. Here, we investigated the physiological roles of RIMB-1, the homolog of RBPs in the nematode Caenorhabditis elegans RIMB-1 is expressed broadly in neurons and predominantly localized at presynaptic sites. Loss-of-function animals of rimb-1 displayed slight defects in motility and response to pharmacological inhibition of synaptic transmission, suggesting a modest involvement of rimb-1 in synapse function. We analyzed genetic interactions of rimb-1 by testing candidate genes and by an unbiased forward genetic screen for rimb-1 enhancer. Both analyses identified the RIM homolog UNC-10 that acts together with RIMB-1 to regulate presynaptic localization of the P/Q-type VGCC UNC-2/Cav2. We also find that the precise localization of RIMB-1 to presynaptic sites requires presynaptic UNC-2/Cav2. RIMB-1 has multiple FN3 and SH3 domains. Our transgenic rescue analysis with RIMB-1 deletion constructs revealed a functional requirement of a C-terminal SH3 in regulating UNC-2/Cav2 localization. Together, these findings suggest a redundant role of RIMB-1/RBP and UNC-10/RIM to regulate the abundance of UNC-2/Cav2 at the presynaptic AZ in C. elegans, depending on the bidirectional interplay between CAZ adaptor and channel proteins.SIGNIFICANCE STATEMENT Presynaptic active zones (AZs) are highly organized structures for synaptic transmission with characteristic networks of adaptor proteins called cytomatrix at the AZ (CAZ). In this study, we characterized a CAZ protein RIMB-1, named for RIM-binding protein (RBP), in the nematode Caenorhabditis elegans Through systematic analyses of genetic interactions and an unbiased genetic enhancer screen of rimb-1, we revealed a redundant role of two CAZ proteins RIMB-1/RBP and UNC-10/RIM in regulating presynaptic localization of UNC-2/Cav2, a voltage-gated calcium channel (VGCC) critical for proper neurotransmitter release. Additionally, the precise localization of RIMB-1/RBP requires presynaptic UNC-2/Cav2. These findings provide new mechanistic insight about how the interplay among multiple CAZ adaptor proteins and VGCC contributes to the organization of presynaptic AZ.

Generation of amyloid-β peptides (Aβs) by proteolytic cleavage of the amyloid-β protein precursor (AβPP), especially increased production of Aβ42/Aβ43 over Aβ40, and their aggregation as oligomers and plaques, represent a characteristic feature of Alzheimer's disease (AD). In familial AD (FAD), altered Aβ production originates from specific mutations of AβPP or presenilins 1/2 (PS1/PS2), the catalytic subunits of γ-secretase. In sporadic AD, the origin of altered production of Aβs remains unknown. We hypothesize that the 'human chemical exposome' contains products able to favor the production of Aβ42/Aβ43 over Aβ40 and shorter Aβs. To detect such products, we screened a library of 3500 + compounds in a cell-based assay for enhanced Aβ42/Aβ43 production. Nine pyrazole insecticides were found to induce a β- and γ-secretase-dependent, 3-10-fold increase in the production of extracellular Aβ42 in various cell lines and neurons differentiated from induced pluripotent stem cells derived from healthy and FAD patients. Immunoprecipitation/mass spectrometry analyses showed increased production of Aβs cleaved at positions 42/43, and reduced production of peptides cleaved at positions 38 and shorter. Strongly supporting a direct effect on γ-secretase activity, pyrazoles shifted the cleavage pattern of another γ-secretase substrate, alcadeinα, and shifted the cleavage of AβPP by highly purified γ-secretase toward Aβ42/Aβ43. Focusing on fipronil, we showed that some of its metabolites, in particular the persistent fipronil sulfone, also favor the production of Aβ42/Aβ43 in both cell-based and cell-free systems. Fipronil administered orally to mice and rats is known to be metabolized rapidly, mostly to fipronil sulfone, which stably accumulates in adipose tissue and brain. In conclusion, several widely used pyrazole insecticides enhance the production of toxic, aggregation prone Aβ42/Aβ43 peptides, suggesting the possible existence of environmental "Alzheimerogens" which may contribute to the initiation and propagation of the amyloidogenic process in sporadic AD.

The quantal nature of synaptic transmission requires a mechanism to transport neurotransmitter into synaptic vesicles without promoting non-vesicular efflux across the plasma membrane. Indeed, the vesicular transport of most classical transmitters involves a mechanism of H(+) exchange, which restricts flux to acidic membranes such as synaptic vesicles. However, vesicular transport of the principal excitatory transmitter glutamate depends primarily on membrane potential, which would drive non-vesicular efflux, and the role of protons is unclear. Adapting electrophysiology to record currents associated with the vesicular glutamate transporters (VGLUTs), we characterize a chloride conductance that is gated by lumenal protons and chloride and supports glutamate uptake. Rather than coupling stoichiometrically to glutamate flux, lumenal protons and chloride allosterically activate vesicular glutamate transport. Gating by protons serves to inhibit what would otherwise be substantial non-vesicular glutamate efflux at the plasma membrane, thereby restricting VGLUT activity to synaptic vesicles.

[F.sub.o][F.sub.1]-ATP synthase ([F.sub.o][F.sub.1]) is a motor enzyme that couples ATP synthesis/hydrolysis with a transmembrane proton translocation. [F.sub.1], a water-soluble ATPase portion of [F.sub.o][F.sub.1], rotates by repeating ATP-waiting dwell, 80[degrees] substep rotation, catalytic dwell, and 40[degrees]- substep rotation. Compared with [F.sub.1], rotation of [F.sub.o][F.sub.1] has yet been poorly understood, and, here, we analyzed ATP-driven rotations of [F.sub.o][F.sub.1]. Rotation was probed with an 80-nm bead attached to the ring of c subunits in the immobilized [F.sub.o][F.sub.1] and recorded with a submillisecond fast camera. The rotation rates at various ATP concentrations obeyed the curve defined by a Km of [approximately equal to]30 [micro]M and a Vmax of [approximately equal to]350 revolutions per second (at 37[degrees]C). At low ATP, ATP-waiting dwell was seen and the [k.sub.on]-ATP was estimated to be 3.6 x [10.sup.7] [M.sup.-1] x [s.sup.-1]. At high ATP, fast, poorly defined stepwise motions were observed that probably reflect the catalytic dwells. When a slowly hydrolyzable substrate, adenosine 5'-[[gamma]-thio]triphosphate, was used, the catalytic dwells consisting of two events were seen more clearly at the angular position of [approximately equal to]80[degrees]. The rotational behavior of [F.sub.o][F.sub.1] resembles that of [F.sub.1]. This finding indicates that 'friction' in [F.sub.o] motor is negligible during the ATP-driven rotation. Tributyltin chloride, a specific inhibitor of proton translocation, slowed the rotation rate by 96%. However, dwells at clearly defined angular positions were not observed under these conditions, indicating that inhibition by tributyltin chloride is complex. ATP hydrolysis | binding change mechanism | membrane protein | single-molecule imaging

In a rotary motor [F.sub.o][F.sub.1]-ATP synthase that couples [H.sup.+] transport with ATP synthesis/hydrolysis, it is thought that an [F.sub.o]c subunit oligomer ring (c-ring) in the membrane rotates as protons pass through [F.sub.o] and a 120[degrees] rotation produces one ATP at [F.sub.1]. Despite several structural studies, the copy number of [F.sub.o]c subunits in the c-ring has not been determined for any functional [F.sub.o][F.sub.1]. Here, we have generated and isolated thermophilic Bacillus [F.sub.o][F.sub.1], each containing genetically fused 2-mer-14-mer c ([c.sub.2]-[c.sub.14]). Among them, [F.sub.o][F.sub.1] containing [c.sub.2], [c.sub.5], or [c.sub.10] showed ATP-synthesis and other activities. When [F.sub.1] was removed, [F.sub.o] containing [c.sub.10] worked as an [H.sup.+] channel but [F.sub.o]S containing [c.sub.9], [c.sub.11] or [c.sub.12] did not. Thus, the c-ring of functional [F.sub.o][F.sub.1] of this organism is a decamer. The inevitable consequence of this finding is noninteger ratios of rotation step sizes of [F.sub.1]/[F.sub.o] (120[degrees]/36[degrees]) and of [H.sup.+]/ATP (10:3). This step-mismatch necessitates elastic twisting of the rotor shaft (and/or the side stalk) during rotation and permissive coupling between unit rotations by [H.sup.+] transport at [F.sub.o] and elementary events in catalysis at [F.sub.1].