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The cystathionine-beta-synthase (CBS) domain is an evolutionarily conserved protein domain that is present in the proteome of archaebacteria, prokaryotes, and eukaryotes. CBS domains usually come in tandem repeats and are found in cytosolic and membrane proteins performing different functions (metabolic enzymes, kinases, and channels). Crystallographic studies of bacterial CBS domains have shown that two CBS domains form an intramolecular dimeric structure (CBS pair). Several human hereditary diseases (homocystinuria, retinitis pigmentosa, hypertrophic cardiomyopathy, myotonia congenital, etc.) can be caused by mutations in CBS domains of, respectively, cystathionine-beta-synthase, inosine 5'-monophosphate dehydrogenase, AMP kinase, and chloride channels. Despite their clinical relevance, it remains to be established what the precise function of CBS domains is and how they affect the structural and/or functional properties of an enzyme, kinase, or channel. Depending on the protein in which they occur, CBS domains have been proposed to affect multimerization and sorting of proteins, channel gating, and ligand binding. However, recent experiments revealing that CBS domains can bind adenosine-containing ligands such ATP, AMP, or S-adenosylmethionine have led to the hypothesis that CBS domains function as sensors of intracellular metabolites.

ATP13A2 (PARK9) is a late endo-lysosomal transporter of unknown function that is genetically implicated in a spectrum of neurodegenerative disorders, including Kufor-Rakeb syndrome, a parkinsonism with dementia 1 and early-onset Parkinson's disease (PD) 2. ATP13A2 offers protection against genetic and environmental risk factors of PD, whereas loss of ATP13A2 compromises lysosomal function 3. The lysosomal transport function of ATP13A2 remained unclear, but here, we establish ATP13A2 as a lysosomal polyamine exporter with highest affinity for spermine. Polyamines stimulate the activity of purified ATP13A2, while disease mutants are functionally impaired to a degree that correlates with the disease phenotype. ATP13A2 promotes cellular polyamine uptake via endocytosis and transports polyamines into the cytosol, which highlights a role for endo-lysosomes in cellular polyamine uptake. At high concentrations, polyamines induce cell toxicity, which is exacerbated by ATP13A2 loss due to lysosomal dysfunction, lysosomal rupture and cathepsin B activation. This phenotype is recapitulated in neurons and nematodes with loss of ATP13A2 or its orthologues. Thus, defective lysosomal polyamine export is a new mechanism for lysosome-dependent cell death that may be implicated in neurodegeneration. Our findings further shed light on the molecular identity of the elusive mammalian polyamine transport system. ATP13A2 is a P5B-ATPase belonging to the family of P-type ATPases, which couple ATP hydrolysis to substrate transport while transiently forming a catalytic phospho-intermediate 4. ATP13A2 is generally described as a heavy metal transporter 5 , but Ca 2+ 6 and the polyamine spermidine (SPD) 7,8 were also proposed. To screen for the transported substrate(s) of ATP13A2, we measured ATPase activity in the presence of various candidate substrates in solubilized microsomal membrane fractions of SH-SY5Y cells that overexpress human ATP13A2 wild type (WT) (WT-OE) or comparable levels of the catalytically dead D508N mutant (D508N-OE) 9,10. ATPase activity of ATP13A2 WT was significantly stimulated by the polyamines SPD and spermine (SPM) (Fig. 1a), whereas SPM had no effect on the D508N mutant (Extended Data Fig. 1a). MnCl2, ZnCl2, FeCl3, CaCl2, diamines, monoamines and amino acids exerted no effect (Extended Data Fig. 1a-3 d). The polyamines SPM, N 1-acetylspermine and SPD were able to stimulate ATPase activity in a concentration-dependent manner (Fig. 1b, Extended Data Fig. 1e) with the highest apparent affinity for SPM (Extended Data Table 1). The catalytic auto-phosphorylation and/or dephosphorylation reactions of P-type ATPases occur in response to binding of the transported substrate 4. ATP13A2 forms a phospho-intermediate on the D508 residue in the absence of SPM supplementation 9,10 , whereas SPM leads to a dose-dependent reduction in ATP13A2 phospho-enzyme levels (Fig. 1c), which is not seen with ornithine (Extended Data Fig. 1f). The dephosphorylation rate following a chase with non-radioactive ATP increased in the presence of...

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This study investigated the volume-regulated anion channel (VRAC) of human cervical cancer SiHa cells under various culture conditions, testing the hypothesis that the progression of the cell cycle is accompanied by differential expression of VRAC activity. Exponentially growing SiHa cells expressed VRACs, as indicated by the presence of large outwardly rectifying currents activated by hypotonic stress with the anion permeability sequence I- > Br- > Cl-. VRACs were potently inhibited by tamoxifen with an IC50 of 4.6 [mu]M. Fluorescence-activated cell sorting (FACS) experiments showed that 59 +/- 0.5, 5 +/- 0.5 and 36 +/- 1.1% of unsynchronized, exponentially growing cervical cancer SiHa cells were in G0/G1, S and G2/M stage, respectively. Treatment with aphidicolin (5 [mu]M) arrested 88 +/- 1.4% of cells at the G0/G1 stage. Arrest of cell growth in the G0/G1 phase was accompanied by a significant decrease of VRAC activity. The normalized hypotonicity-induced current decreased from 48 +/- 5.2 pA pF-1 at +100 mV in unsynchronized cells to 15 +/- 2.6 pA pF-1 at +100 mV in aphidicolin-treated cells. After removal of aphidicolin, culturing in medium containing 10% fetal calf serum triggered a rapid re-entry into the cell cycle and a concomitant recovery of VRAC density. Pharmacological blockade of VRACs by tamoxifen or NPPB caused proliferating cervical cancer cells to arrest in the G0/G1 stage, suggesting that activity of this channel is critical for G1/S checkpoint progression. This study provides new information on the functional significance of VRACs in the cell cycle clock of human cervical cancer cells.

The role of protein tyrosine phosphorylation and of G proteins in the activation of a swelling‐activated Cl− current (ICl,swell) in calf pulmonary artery endothelial (CPAE) cells was studied using the whole‐cell patch clamp technique. ICl,swell was activated by reducing the extracellular osmolality by either 12.5 % (mild hypotonicity) or 25 % (strong hypotonicity). The protein tyrosine kinase (PTK) inhibitors tyrphostin B46, tyrphostin A25 and genistein inhibited ICl,swell with IC50 values of, respectively, 9.2 ± 0.2, 61.4 ± 1.7 and 62.9 ± 1.3μM. Tyrphostin A1, a tyrphostin analogue with little effect on PTK activity, and daidzein, an inactive genistein analogue, were without effect on ICl,swell. The protein tyrosine phosphatase (PTP) inhibitors Na3VO4 (200 μM) and dephostatin (20 μM) potentiated ICl,swell activated by mild hypotonicity by 47 ± 9 and 69 ± 15 %, respectively. Intracellular perfusion with GTPγS (100 μM) transiently activated a Cl− current with an identical biophysical and pharmacological profile to ICl,swell. This current was inhibited by the tested PTK inhibitors and potentiated by the PTP inhibitors. Hypertonicity‐induced cell shrinkage completely inhibited the GTPγS‐activated Cl− current. Intracellular perfusion with GDPβS (1 mM) caused a time‐dependent inhibition of ICl,swell, which was more pronounced when the current was activated by mild hypotonicity. Our results demonstrate that the activity of endothelial swelling‐activated Cl− channels is dependent on tyrosine phosphorylation and suggest that G proteins regulate the sensitivity to cell swelling.

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We combined patch clamp and fura‐2 fluorescence methods to characterize human TRP3 (hTRP3) channels heterologously expressed in cultured bovine pulmonary artery endothelial (CPAE) cells, which do not express the bovine trp3 isoform (btrp3) but express btrp1 and btrp4. ATP, bradykinin and intracellular InsP3 activated a non‐selective cation current (IhTRP3) in htrp3‐transfected CPAE cells but not in non‐transfected wild‐type cells. During agonist stimulation, the sustained rise in [Ca2+]i was significantly higher in htrp3‐transfected cells than in control CPAE cells. The permeability for monovalent cations was PNa > PCs≈PK >> PNMDG and the ratio PCa/PNa was 1·62 ± 0·27 (n= 11). Removal of extracellular Ca2+ enhanced the amplitude of the agonist‐activated IhTRP3 as well as that of the basal current The trivalent cations La3+ and Gd3+ were potent blockers of IhTRP3 (the IC50 for La3+ was 24·4 ± 0·7 μM). The single‐channel conductance of the channels activated by ATP, assessed by noise analysis, was 23 pS. Thapsigargin and 2,5‐di‐tert‐butyl‐1,4‐benzohydroquinone (BHQ), inhibitors of the organellar Ca2+‐ATPase, failed to activate IhTRP3. U‐73122, a phospholipase C blocker, inhibited IhTRP3 that had been activated by ATP and bradykinin. Thimerosal, an InsP3 receptor‐sensitizing compound, enhanced IhTRP3, but calmidazolium, a calmodulin antagonist, did not affect IhTRP3. It is concluded that hTRP3 forms non‐selective plasmalemmal cation channels that function as a pathway for agonist‐induced Ca2+ influx.