A novel neuron-specific regulator of the V-ATPase in Drosophila

The V-ATPase is a highly conserved enzymatic complex that ensures appropriate levels of organelle acidification in virtually all eukaryotic cells. While the general mechanisms of this proton pump have been well studied, little is known about the specific regulations of neuronal V-ATPase. Here, we studied CG31030, a previously uncharacterized Drosophila protein predicted from its sequence homology to be part of the V-ATPase family. In contrast to its ortholog ATP6AP1/VhaAC45 which is ubiquitous, we observed that CG31030 expression is apparently restricted to all neurons, and using CRISPR/Cas9-mediated gene tagging, that it is mainly addressed to synaptic terminals. In addition, we observed that CG31030 is essential for fly survival and that this protein co-immunoprecipitates with identified V-ATPase subunits, and in particular ATP6AP2. Using a genetically-encoded pH probe (VMAT-pHluorin) and electrophysiological recordings at the larval neuromuscular junction, we show that CG31030 knock-down induces a major defect in synaptic vesicle acidification and a decrease in quantal size, which is the amplitude of the postsynaptic response to the release of a single synaptic vesicle. These defects were associated with severe locomotor impairments. Overall, our data indicate that CG31030, which we renamed VhaAC45-related protein (VhaAC45RP), is a specific regulator of neuronal V-ATPase in Drosophila that is required for proper synaptic vesicle acidification and neurotransmitter release

Functional Interaction between CFTR and the Sodium-Phosphate Co-Transport Type 2a in Xenopus laevis Oocytes

A growing number of proteins, including ion transporters, have been shown to interact with Cystic Fibrosis Transmembrane conductance Regulator (CFTR). CFTR is an epithelial chloride channel that is involved in Cystic Fibrosis (CF) when mutated; thus a better knowledge of its functional interactome may help to understand the pathophysiology of this complex disease. In the present study, we investigated if CFTR and the sodium-phosphate co-transporter type 2a (NPT2a) functionally interact after heterologous expression of both proteins in Xenopus laevis oocytes.NPT2a was expressed alone or in combination with CFTR in X. laevis oocytes. Using the two-electrode voltage-clamp technique, the inorganic phosphate-induced current (IPi) was measured and taken as an index of NPT2a activity. The maximal IPi for NPT2a substrates was reduced when CFTR was co-expressed with NPT2a, suggesting a decrease in its expression at the oolemna. This was consistent with Western blot analysis showing reduced NPT2a plasma membrane expression in oocytes co-expressing both proteins, whereas NPT2a protein level in total cell lysate was the same in NPT2a- and NPT2a+CFTR-oocytes. In NPT2a+CFTR- but not in NPT2a-oocytes, IPi and NPT2a surface expression were increased upon PKA stimulation, whereas stimulation of Exchange Protein directly Activated by cAMP (EPAC) had no effect. When NPT2a-oocytes were injected with NEG2, a short amino-acid sequence from the CFTR regulatory domain that regulates PKA-dependent CFTR trafficking to the plasma membrane, IPi values and NPT2a membrane expression were diminished, and could be enhanced by PKA stimulation, thereby mimicking the effects of CFTR co-expression.We conclude that when both CFTR and NPT2a are expressed in X. laevis oocytes, CFTR confers to NPT2a a cAMPi-dependent trafficking to the membrane. This functional interaction raises the hypothesis that CFTR may play a role in phosphate homeostasis

Drosophila ammonium transporter Rh50 is required for integrity of larval muscles and neuromuscular system

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Phosphate-induced current in <i>X. laevis</i> oocytes.

<p>Phosphate-induced current (1 mM of Pi added in ND96) was measured at holding potential (Vc) of −50 mV in oocytes expressing NPT2a or CFTR alone or co-expressing NPT2a and CFTR; oocytes injected with water were used as control oocytes. <u>A</u>: Original tracings showing the current induced by the addition of 1 mM Pi (indicated by black bars) in the superfusing medium. The type of oocytes is indicated above the tracings. <u>B</u>: Summary of the results (means ± SEM) of the effect of 1 mM Pi in the different types of oocytes (indicated below the columns). Significance of the results *: P<0.05</p

Effects of agonists of EPAC and PKA pathways on NPT2a function and surface expression.

<p>Phosphate-induced currents (IPi) were evoked in voltage-clamped (–50 mV) cells by the exposure of NPT2a-oocytes (white columns), or NPT2a+CFTR-oocytes (black columns) to 1 mM Pi. As above, IPi was first induced in basal condition, ^basalIPi, then after the activation of a cAMP-dependent signaling pathway, ^exptlIPi, during 10 min (panels A and B). Results are as means ± SEM. IPi were normalized against ^basalIPi from NPT2a-oocytes from the same batch of oocytes. The significance of the difference between ^basalIPi from NPT2a-oocytes and ^basalIPi or ^exptlIPi from NPT2a+CFTR-oocytes was analyzed using unpaired Student’s <i>t</i>-test (*: P < 0.05). The significance of the difference between ^basalIPi and ^exptlIPi within the same type of oocytes was assessed using paired Student’s <i>t</i>-test (#: P < 0.05). <u>A</u>: Effect of para-Chlorophenylthio-2′-O-methyladenosine-3′, 5′-cyclic monophosphate (8-pCPT-2′-O-Me-cAMP, 25 µM), an activator on the EPAC pathway on IPi in NPT2a- and NPT2a+CFTR-oocytes (n  =  9 for each type; N =  2). <u>B</u>: Effect of N⁶-monobutyryladenosine-3′, 5′-cyclic monophosphate (6-MB-cAMP, 25 µM), an activator of the PKA pathway on IPi in NPT2a- and NPT2a+CFTR-oocytes (n  =  19 and n  =  22, respectively; N =  6).</p

Effect of PKA stimulation on NPT2a and CFTR cell surface expression.

<p>Myc-NPT2a- and Myc-NPT2a+CFTR-oocytes from a same batch were kept in control condition or submitted to 25 µM of 6-MB-cAMP for 15 min, as indicated below the panel. Control oocytes were H₂O-injected.<u> A</u>: Effect of PKA stimulation on NPT2a cell surface expression. Left: Cell surface biotinylated proteins were probed with an anti-Myc antibody; the molecular weight of Myc-NPT2a is indicated on the figure. Right: Results from 4 separate experiments, were quantified. The staining intensity of Myc-NPT2a cell surface expression from Myc-NPT2a+CFTR oocytes (black columns) was normalized against the staining intensity of Myc-NPT2a cell surface expression from Myc-NPT2a-oocytes (white columns) in basal condition. The significance of the difference between the results was assessed using unpaired (*: P < 0.05) or paired (#: P < 0.05) Student’s <i>t</i>-test. <u>B</u>: Effect of PKA stimulation on CFTR cell surface expression. Cell surface biotinylated proteins were probed with an anti-CFTR antibody; the molecular weight of CFTR is indicated on the figure. Similar results were obtained in 2 independent experiments.</p

Effect of injecting NEG2 peptide on NPT2a function.

<p>The current induced by 1 mM phosphate (IPi) was measured in voltage-clamped (−50 mV) NPT2a-oocytes and was measured in oocytes injected either with the NEG2 peptide (black column, n  =  12), with a scrambled peptide (sNEG2, grey column, n  =  9), or in non-injected oocytes (white column, n  = 10). IPi was measured in basal condition (^basalIPi, measured in ND96 medium) then under an experimental stimulating condition (^exptlIPi, measured after a 10 min-incubation in ND96 supplemented with forskolin, 1µM, and isobutylmethylxanthine, 100 µM, Forsk+IBMX). Results are presented as means ± SEM from N  =  4 experiments. IPi were normalized against ^basalIPi from control (non injected with a peptide) NPT2a-oocytes from the same batch of oocytes. The significance of the difference was analyzed using unpaired Student’s <i>t</i>-test (*: P < 0.05) or paired Student’s <i>t</i>-test (#: P < 0.05).</p

NPT2a and CFTR expressed in <i>X. laevis</i> oocytes co-immunoprecipitate.

<p>Representative experiment showing the co-immunoprecipitation of NPT2a and CFTR after their expression in <i>X. laevis</i> oocytes. Proteins from oocytes co-expressing Myc-NPT2a and CFTR were immunoprecipitated with MM13-4 Ab, or with IgG1 Ab used as a negative control. They were probed with the anti-Myc antibody. The molecular weight of the detected protein is indicated Similar results were obtained in N  =  3 independent experiments.</p

Kinetic analysis of inorganic phosphate transport in oocytes expressing NPT2a or co-expressing NPT2a and CFTR.

<p>Phosphate-induced currents (IPi) were measured (Vc =  −50 mV) in NPT2a-oocytes (empty circles) and in NPT2a+CFTR-oocytes (filled circles). The dashed lines through the points (means ± SEM from n  =  6 to 14, N  =  5) were calculated using SigmaPlot software (Systat software Inc., San Jose, CA). <u>A</u>: IPi was induced by increasing Pi concentration in ND 96, pH 7.5, as indicated in abscissa and normalized against extrapolated maximal current (IPi_max). Data were fitted to Michaelis-Menten equation. Results, as means ± SD, were as follows: the apparent concentration of Pi substrate (K_m) giving the half IPi_max was not changed by CFTR expression (K_m  =  0.07± 0.02 <i>vs</i> 0.06 ± 0.02 mM in NPT2a- and NPT2a+CFTR-oocytes, respectively), but IPi_max was decreased by 40±3% (P <0.05). <u>B</u>: IPi was induced by 1 mM Pi at increasing Na⁺ concentrations (equimolary substituted by choline⁺, pH 7.5) and normalized against IPi_max. Data were fitted to the modified Hill equation. Results, as means ± SD, were as follows: the apparent K_m for Na⁺ substrate was 40.7 ± 14.6 in NPT2a-oocytes, not different from 41.8 ± 13.4 mM in NPT2a+CFTR-oocytes; IPi_max was decreased by 39±7% in NPT2a+CFTR-oocytes compared to NPT2a-oocytes (P < 0.05). Inset: Effect of varying extracellular pH from 7.0 to 8.0 on the current induced by 1 mM Pi in NPT2a-oocytes (white column) and in NPT2a+CFTR-oocytes (black column). IPi was normalized against the Pi-induced current measured at pH 7.5 in NPT2a-oocytes from the same batch of oocytes. Results are shown as means± SEM, n  =  5 oocytes of each type.</p