Journal: eLife

Article Title: Agonist-specific voltage-dependent gating of lysosomal two-pore Na + channels

doi: 10.7554/eLife.51423

Figure Lengend Snippet: ( A ) Representative basal I TPC2 step currents elicited by a voltage step protocol in the whole-endolysosome (EL) configuration. Voltage steps from −140 to 100 mV with a voltage increment (∆V) of 20 mV for 0.5 s were used to elicit I TPC2 in A-D. HP = 0 mV. Unless otherwise indicated, symmetric (bath/cytosol vs pipette/lumen) Na + (150 mM) solutions were used for all whole-endolysosome recordings, and PI(3,5)P 2 (0.3 µM), LyNa-VA1.2 (100 µM), and LyNA1 (300 µM) were bath- applied to induce I TPC2 . ( B–D ) Representative I TPC2 step currents activated by PI(3,5)P 2 ( B ), LyNa-VA1.2 ( C ), and LyNA1 ( D ). ( E ) Representative normalized I-V plots based on the instantaneous currents activated by various agonists. ( F ) Rectification index, calculated as the ratio of the current amplitudes between +80 and −80 mV, of PI(3,5)P 2 -, LyNa-VA1.2-, and LyNA1- activated I TPC2 . ( G ) The inactivation of I TPC2 at −120 mV was quantified as the ratio of current amplitudes at 10 vs. 500 ms, based on step currents in B , ( C ) and D. ( H ) Voltage steps from −120 to 100 mV (∆V = 20 mV) for 1 s were used to elicit tail currents at −120 mV shown in ( I ) and ( J ). ( I ) The tail currents of PI(3,5)P 2 - evoked whole-endolysosome I TPC2 . ( J ) The tail currents of LyNa-VA1.2- activated whole-endolysosome I TPC2 . Arrows in ( I ) and ( J ) indicate where the currents were measured to calculate the channel conductance (G = I/V). ( K ) Normalized G-V curves of PI(3,5)P 2 - and LyNa-VA1.2- activated I TPC2 . LyNa-VA1.2 activated I TPC2 in a voltage dependent manner with a V 1/2 = −20.3 ± 3.5 mV (n = 5 patches). For panels F and G , individual data and Mean ± S.E.M. are presented. ***, p<0.001. Individual data for ( F ) and ( G ) are presented in . Figure 2—source data 1. The inactivation of I TPC2 and rectification index of TPC2.

Article Snippet: Chemical compound, drug , PI(3,5)P 2 , Echelon Biosciences , Cat. #: P-3508 , .

Techniques: Transferring

Journal: eLife

Article Title: Agonist-specific voltage-dependent gating of lysosomal two-pore Na + channels

doi: 10.7554/eLife.51423

Figure Lengend Snippet: ( A ) The effects of PI(3,5)P 2 (0.3 µM) and LyNa-VA1.2 (100 µM) on whole-endolysosome I TPC2-K204A in TPC2 K204A -transfected HEK293 cells . ( B ) Comparison effects of LyNa-VA1.2 and PI(3,5)P 2 on WT and PI(3,5)P 2 -insensitive K204A mutant TPC2 channels (also see ). ( C ) The synergistic effects of PI(3,5)P 2 (50 nM) and LyNa-VA1.1 on whole-endolysosome I TPC2 and I TPC2-K204A currents. Data are presented as Mean ± S.E.M (n = 3 patches). ( D ) The summary of EC 50 of LyNa-VA1.1 with or without PI(3,5)P 2 for WT and K204A mutant TPC2 channels. ( E, F ) Co-application of PI(3,5)P 2 (0.3 µM) and LyNa-VA1.1 (50 µM) activated whole-endolysome I TPC in WT ( E ) but not TPC1/2 DKO ( F ) HAP1 cells. Note that the endogenous TPCs were more difficult to activate compared to overexpressed TPCs. ( G ) Summary of LyNa-VA1.1 effects on whole-endolysome I TPC in WT and TPC1/2 DKO cells. For panels B, D and F, individual data and Mean ± S.E.M are presented. *, p<0.05; **, p<0.01; ***, p<0.001; N.S., no significance. Individual data for ( B–D ) and ( G ) are presented in . Figure 3—source data 1. Synergistic activation of TPC2 channels by TCAs and PI(3,5)P 2 .

Article Snippet: Chemical compound, drug , PI(3,5)P 2 , Echelon Biosciences , Cat. #: P-3508 , .

Techniques: Transfection, Mutagenesis, Activation Assay

Journal: eLife

Article Title: Agonist-specific voltage-dependent gating of lysosomal two-pore Na + channels

doi: 10.7554/eLife.51423

Figure Lengend Snippet: ( A ) Basal whole-endolysosome I TPC1 (left) or I TPC1-R540I (right) was elicited by voltage steps (−140 to +100 mV with ΔV = 20 mV) in the absence of PI(3,5)P 2 in TPC1-transfected Cos1 cells . Tail currents were elicited at −120 mV. ( B ) The effects of PI(3,5)P 2 (0.3 µM) on I TPC1 (left) and I TPC1-R540I (right) step currents. ( C ) I-V plots of steady-state I TPC1 and I TPC1-R540I step currents shown in B. ( D ) LyNa-VA1.2-induced whole-endolysosome I TPC2-I551R was evoked by a voltage ramp from −120 to +120 mV (duration = 200 ms). ( E ) The basal (right), LyNa-VA1.2 (200 µM)- (middle) and PI(3,5)P 2 (0.3 µM)- (left) activated lysosomal I TPC1-I551R were recorded from the same vacuole. A preconditioning voltage (+80 mV, 0.1 s) was applied before voltage steps starting from −140 to 100 mV (0.5 s, ∆V = 20 mV). HP = 0 mV.

Article Snippet: Chemical compound, drug , PI(3,5)P 2 , Echelon Biosciences , Cat. #: P-3508 , .

Techniques: Transfection

Journal: eLife

Article Title: Agonist-specific voltage-dependent gating of lysosomal two-pore Na + channels

doi: 10.7554/eLife.51423

Figure Lengend Snippet: ( A ) Representative PI(3,5)P 2 -evoked whole-endolysosome I TPC2 elicited by a voltage ramp from −120 to 120 mV. The recordings were performed under a bi-ionic condition with 150 (in mM) Na + in the pipette solution and 150 Na + or 150 K + in the bath solution. PI(3,5)P 2 (0.3 µM) was bath-applied. ( B, C ) Representative PI(3,5)P 2 - evoked I TPC2 elicited with voltage steps (−140 mV to 100 mV with a ∆V = 20 mV, 0.5 s) with 150 mM Na + ( B ) or K + ( C ) in the bath solution. ( D, E ) Representative LyNa-VA1.2-activated I TPC2 step currents. ( F ) Representative I-V plots of LyNa-VA1.2- activated I TPC2 measured from the instantaneous currents in ( D ) and ( E ). Note the reversal potentials (E rev ) of LyNa-VA1.2- activated I TPC2 in the presence of Na + or K + bath solution. ( G ) LyNa-VA1.2- activated I TPC2 under the bi-ionic conditions of bath/cytosolic Na + and pipette/luminal Ca 2+ . 150 Na + solution contained (in mM) 145 NaCl, 5 NaOH, 20 HEPES, (pH 7.2); isotonic (105 mM) Ca 2+ solution contained (in mM) 100 CaCl 2 , 5 Ca(OH) 2 , 20 HEPES (pH 7.2). Right panel zoom-in micrograph shows the E rev of LyNa-VA1.2- activated I TPC2 . ( H–J ) Representative I-V plots of LyNa-VA1.2- evoked I TPC2-N653G ( J ) measured from Na + ( H ) and K + ( I ) bath solution. ( K ) Summary of Na + vs. K + /Ca 2+ selectivity of WT TPC2 and TPC2 N653G channels. Individual data and Mean ± S.E.M are presented (also see ). N.S., no significance. Figure 5—source data 1. Ionic selectivity of WT TPC2 and N653G mutant channels.

Article Snippet: Chemical compound, drug , PI(3,5)P 2 , Echelon Biosciences , Cat. #: P-3508 , .

Techniques: Transferring, Mutagenesis

Journal: bioRxiv

Article Title: Lipid Interactions of a Ciliary Membrane TRP Channel: Simulation and Structural Studies of Polycystin-2 (PC2)

doi: 10.1101/589515

Figure Lengend Snippet: Lipid binding sites suggested by cryoEM maps of PC2 obtained A in the presence of PI(4,5)P 2 (3.0 Å resolution) and B in the presence of PI(3,5)P 2 (3.4 Å resolution). Protein density (contoured at 3.2 sigma) is in grey, detergent density in yellow, lipid density in orange, and cholesterol density in cyan. C Expanded view around the proposed lipid binding site between S3, S4 and S5 showing density from the 3.0 Å map (pink; see SI Fig. S9) and from the 3.4 Å map (grey).

Article Snippet: 18:0-20:4 PI(4,5)P 2 and 18:1 PI(3,5)P 2 lipid extract (Avanti) dissolved in chloroform was dried under an argon stream.

Techniques: Binding Assay

Journal: bioRxiv

Article Title: Lipid Interactions of a Ciliary Membrane TRP Channel: Simulation and Structural Studies of Polycystin-2 (PC2)

doi: 10.1101/589515

Figure Lengend Snippet: Comparison of PI lipids bound to PC2 and to TRPV1 with cryoEM density. A PIP 2 bound to PC2 (as revealed by the current simulation study); B Lipid-like density in the cryoEM maps of PC2 obtained in the presence of PI(3,5)P 2 (3.4 Å resolution; see ); C PI bound to TRPV1 (as revealed by cryoelectron microscopy, PDB ID 5IRZ). In each case the lipid molecule or density is located between the S3, S4 and S5 helices of the VSLD. D A sequence alignment coloured on contacts with PIP 2 in the mixed lipid simulations. Residues of the region around the binding pocket between the S3, S4 and S5 helices are coloured (on a white to red scale) according to the frequency of the interactions of PIP 2 headgroups with each residue. The five basic residues of PC2 which form interactions with the headgroup of PIP 2 at the binding site (i.e. R504, K572, K575, R592 and K595) are boxed.

Article Snippet: 18:0-20:4 PI(4,5)P 2 and 18:1 PI(3,5)P 2 lipid extract (Avanti) dissolved in chloroform was dried under an argon stream.

Techniques: Microscopy, Sequencing, Binding Assay

Journal: Molecular Microbiology

Article Title: Fluconazole‐induced actin cytoskeleton remodeling requires phosphatidylinositol 3‐phosphate 5‐kinase in the pathogenic yeast Candida glabrata

doi: 10.1111/mmi.14110

Figure Lengend Snippet: CgCof1 is a PI(3,5)P2‐binding protein. A. Western blot image showing CgCof1‐SFB‐PI(3,5)P2 interaction. B. Western blot image showing lack of specific interaction between CgCap2‐SFB and PI(3,5)P2. CgCap2 signal was of similar intensity in both lipid‐coated and control beads. C. A western blot image showing the interaction of the recombinant 6xHis‐FLAG‐CgCof1 with PI(3,5)P2. Input indicates the 6xHis‐FLAG‐CgCof1 purified from E. coli . D. Protein‐lipid overlay assay showing binding of 6xHis‐FLAG‐CgCof1 with phosphatidylinositols and phosphatidic acid lipids. Blank spot contains no lipid. [Colour figure can be viewed at http://www.wileyonlinelibrary.com ]

Article Snippet: For lipid‐binding assays, PI(3,5)P2‐coated (Echelon Biosciences; #P‐B035a) and control (No lipid Echelon Biosciences; #P‐B000) beads were used.

Techniques: Binding Assay, Western Blot, Recombinant, Purification, Protein-lipid Overlay Assay (PLOA)