Limits...
Both RyRs and TPCs are required for NAADP-induced intracellular Ca²⁺ release.

Gerasimenko JV, Charlesworth RM, Sherwood MW, Ferdek PE, Mikoshiba K, Parrington J, Petersen OH, Gerasimenko OV - Cell Calcium (2015)

Bottom Line: Antibodies against RyR2 had practically no effect on NAADP-evoked Ca(2+) release, but reduced release in response to cADPR by 55%.We conclude that full NAADP-mediated Ca(2+) release requires both TPCs and RyRs.Our results indicate that the primary, but very small, NAADP-elicited Ca(2+) release via TPCs from endosomes/lysosomes triggers the detectable Ca(2+)-induced Ca(2+) release via RyR1 and RyR3 occurring from the granules and the ER.

View Article: PubMed Central - PubMed

Affiliation: Medical Research Council Group, Cardiff School of Biosciences, Cardiff University, Cardiff, Wales, UK.

No MeSH data available.


Involvement of TPC2 channels in NAADP-elicited Ca2+ release. (A) Trace represents an application of 100 nM NAADP to permeabilized cell isolated from wt mouse (n = 13). (B) Representative trace of application of 100 nM NAADP to permeabilized cell isolated from TPC2-KO mouse (n = 5). (C) Permeabilized cells from wt mice were pre-incubated with TPC2 antibody (20 min, 1:100) followed by addition of 100 nM NAADP (n = 8). (D) Representative trace shows typical response to 100 nM NAADP in permeabilized cells from TPC2-KO mice treated with TPC1 antibody (20 min, 1:100) (n = 11). (E) Comparison of the amplitudes of responses to 100 nM NAADP (n = 13 control; n = 8 with TPC2 antibody treatment), 10 μM IP3 (n = 9 control; n = 8 with TPC2 antibody treatment) or 10 μM cADPR (n = 9 control; n = 5 with TPC2 antibody treatment) in pre-treated or non-treated cells with antibody against TPC2. (F) Summary of NAADP-elicited Ca2+ release from the intracellular stores in permeabilized control cells or pre-treated with antibodies against TPC1 (20.6 ± 0.7%, n = 11), or TPC2 (7.6 ± 1%, SEM, n = 8), or mixture of both (5.3 ± 0.2%, n = 4), in wt mice compared to responses in permeabilized cells isolated from TPC2 KO mice and treated (3.9 ± 0.3%, n = 7) or non-treated with TPC1 antibody (9.8 ± 1.0%, n = 5). (G) Summary of results obtained from permeabilized cells treated with 10 μM thapsigargin to deplete ER followed by subsequent application of NAADP (100 nM) in the presence of antibodies against TPC1 (9.8 ± 0.5%, n = 5, p > 0.08), or TPC2 (1.63 ± 0.3%, n = 6), or a mixture of both (0.83 ± 0.1%, n = 6), or a mixture of antibodies against RyR1 and RyR3 (0.75 ± 0.1%, n = 5) as compared to control NAADP responses (12.4 ± 1.1%, n = 5). Error bars represent ± SEM. Cells were loaded with Fluo-5N in AM form.
© Copyright Policy - CC BY
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC4539342&req=5

fig0015: Involvement of TPC2 channels in NAADP-elicited Ca2+ release. (A) Trace represents an application of 100 nM NAADP to permeabilized cell isolated from wt mouse (n = 13). (B) Representative trace of application of 100 nM NAADP to permeabilized cell isolated from TPC2-KO mouse (n = 5). (C) Permeabilized cells from wt mice were pre-incubated with TPC2 antibody (20 min, 1:100) followed by addition of 100 nM NAADP (n = 8). (D) Representative trace shows typical response to 100 nM NAADP in permeabilized cells from TPC2-KO mice treated with TPC1 antibody (20 min, 1:100) (n = 11). (E) Comparison of the amplitudes of responses to 100 nM NAADP (n = 13 control; n = 8 with TPC2 antibody treatment), 10 μM IP3 (n = 9 control; n = 8 with TPC2 antibody treatment) or 10 μM cADPR (n = 9 control; n = 5 with TPC2 antibody treatment) in pre-treated or non-treated cells with antibody against TPC2. (F) Summary of NAADP-elicited Ca2+ release from the intracellular stores in permeabilized control cells or pre-treated with antibodies against TPC1 (20.6 ± 0.7%, n = 11), or TPC2 (7.6 ± 1%, SEM, n = 8), or mixture of both (5.3 ± 0.2%, n = 4), in wt mice compared to responses in permeabilized cells isolated from TPC2 KO mice and treated (3.9 ± 0.3%, n = 7) or non-treated with TPC1 antibody (9.8 ± 1.0%, n = 5). (G) Summary of results obtained from permeabilized cells treated with 10 μM thapsigargin to deplete ER followed by subsequent application of NAADP (100 nM) in the presence of antibodies against TPC1 (9.8 ± 0.5%, n = 5, p > 0.08), or TPC2 (1.63 ± 0.3%, n = 6), or a mixture of both (0.83 ± 0.1%, n = 6), or a mixture of antibodies against RyR1 and RyR3 (0.75 ± 0.1%, n = 5) as compared to control NAADP responses (12.4 ± 1.1%, n = 5). Error bars represent ± SEM. Cells were loaded with Fluo-5N in AM form.

Mentions: Involvement of TPCs in NAADP-elicited Ca2+ signalling has been shown by many groups [26,34]. To further investigate this issue, we have performed experiments using pancreatic acinar cells isolated from TPC2 KO mice [26]. NAADP (100 nM) induced Ca2+ release from stores was reduced by 64% in cells from TPC2 KO as compared to the release in cells from wt mice (Fig. 3A, B, and F). A similar (72%) level of inhibition was achieved by treatment of permeabilized cells from wt mice with a TPC2 antibody (Fig. 3C and F). In contrast, an antibody against TPC1 was only able to reduce the response to a minor degree (by 24.5%) (Fig. 3F). Responses to cADPR and IP3 were not affected by the TPC2 antibody (Fig. 3E). Pre-incubation of permeabilized cells from TPC2 KO with the TPC1 specific antibody resulted in significant (p < 0.0002) inhibition of the NAADP-elicited Ca2+ release by 85.7% (Fig. 3D and F). Application of a mixture of both antibodies against TPC1 and TPC2 reduced NAADP-induced Ca2+ response by 80.6% (Fig. 3F).


Both RyRs and TPCs are required for NAADP-induced intracellular Ca²⁺ release.

Gerasimenko JV, Charlesworth RM, Sherwood MW, Ferdek PE, Mikoshiba K, Parrington J, Petersen OH, Gerasimenko OV - Cell Calcium (2015)

Involvement of TPC2 channels in NAADP-elicited Ca2+ release. (A) Trace represents an application of 100 nM NAADP to permeabilized cell isolated from wt mouse (n = 13). (B) Representative trace of application of 100 nM NAADP to permeabilized cell isolated from TPC2-KO mouse (n = 5). (C) Permeabilized cells from wt mice were pre-incubated with TPC2 antibody (20 min, 1:100) followed by addition of 100 nM NAADP (n = 8). (D) Representative trace shows typical response to 100 nM NAADP in permeabilized cells from TPC2-KO mice treated with TPC1 antibody (20 min, 1:100) (n = 11). (E) Comparison of the amplitudes of responses to 100 nM NAADP (n = 13 control; n = 8 with TPC2 antibody treatment), 10 μM IP3 (n = 9 control; n = 8 with TPC2 antibody treatment) or 10 μM cADPR (n = 9 control; n = 5 with TPC2 antibody treatment) in pre-treated or non-treated cells with antibody against TPC2. (F) Summary of NAADP-elicited Ca2+ release from the intracellular stores in permeabilized control cells or pre-treated with antibodies against TPC1 (20.6 ± 0.7%, n = 11), or TPC2 (7.6 ± 1%, SEM, n = 8), or mixture of both (5.3 ± 0.2%, n = 4), in wt mice compared to responses in permeabilized cells isolated from TPC2 KO mice and treated (3.9 ± 0.3%, n = 7) or non-treated with TPC1 antibody (9.8 ± 1.0%, n = 5). (G) Summary of results obtained from permeabilized cells treated with 10 μM thapsigargin to deplete ER followed by subsequent application of NAADP (100 nM) in the presence of antibodies against TPC1 (9.8 ± 0.5%, n = 5, p > 0.08), or TPC2 (1.63 ± 0.3%, n = 6), or a mixture of both (0.83 ± 0.1%, n = 6), or a mixture of antibodies against RyR1 and RyR3 (0.75 ± 0.1%, n = 5) as compared to control NAADP responses (12.4 ± 1.1%, n = 5). Error bars represent ± SEM. Cells were loaded with Fluo-5N in AM form.
© Copyright Policy - CC BY
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4539342&req=5

fig0015: Involvement of TPC2 channels in NAADP-elicited Ca2+ release. (A) Trace represents an application of 100 nM NAADP to permeabilized cell isolated from wt mouse (n = 13). (B) Representative trace of application of 100 nM NAADP to permeabilized cell isolated from TPC2-KO mouse (n = 5). (C) Permeabilized cells from wt mice were pre-incubated with TPC2 antibody (20 min, 1:100) followed by addition of 100 nM NAADP (n = 8). (D) Representative trace shows typical response to 100 nM NAADP in permeabilized cells from TPC2-KO mice treated with TPC1 antibody (20 min, 1:100) (n = 11). (E) Comparison of the amplitudes of responses to 100 nM NAADP (n = 13 control; n = 8 with TPC2 antibody treatment), 10 μM IP3 (n = 9 control; n = 8 with TPC2 antibody treatment) or 10 μM cADPR (n = 9 control; n = 5 with TPC2 antibody treatment) in pre-treated or non-treated cells with antibody against TPC2. (F) Summary of NAADP-elicited Ca2+ release from the intracellular stores in permeabilized control cells or pre-treated with antibodies against TPC1 (20.6 ± 0.7%, n = 11), or TPC2 (7.6 ± 1%, SEM, n = 8), or mixture of both (5.3 ± 0.2%, n = 4), in wt mice compared to responses in permeabilized cells isolated from TPC2 KO mice and treated (3.9 ± 0.3%, n = 7) or non-treated with TPC1 antibody (9.8 ± 1.0%, n = 5). (G) Summary of results obtained from permeabilized cells treated with 10 μM thapsigargin to deplete ER followed by subsequent application of NAADP (100 nM) in the presence of antibodies against TPC1 (9.8 ± 0.5%, n = 5, p > 0.08), or TPC2 (1.63 ± 0.3%, n = 6), or a mixture of both (0.83 ± 0.1%, n = 6), or a mixture of antibodies against RyR1 and RyR3 (0.75 ± 0.1%, n = 5) as compared to control NAADP responses (12.4 ± 1.1%, n = 5). Error bars represent ± SEM. Cells were loaded with Fluo-5N in AM form.
Mentions: Involvement of TPCs in NAADP-elicited Ca2+ signalling has been shown by many groups [26,34]. To further investigate this issue, we have performed experiments using pancreatic acinar cells isolated from TPC2 KO mice [26]. NAADP (100 nM) induced Ca2+ release from stores was reduced by 64% in cells from TPC2 KO as compared to the release in cells from wt mice (Fig. 3A, B, and F). A similar (72%) level of inhibition was achieved by treatment of permeabilized cells from wt mice with a TPC2 antibody (Fig. 3C and F). In contrast, an antibody against TPC1 was only able to reduce the response to a minor degree (by 24.5%) (Fig. 3F). Responses to cADPR and IP3 were not affected by the TPC2 antibody (Fig. 3E). Pre-incubation of permeabilized cells from TPC2 KO with the TPC1 specific antibody resulted in significant (p < 0.0002) inhibition of the NAADP-elicited Ca2+ release by 85.7% (Fig. 3D and F). Application of a mixture of both antibodies against TPC1 and TPC2 reduced NAADP-induced Ca2+ response by 80.6% (Fig. 3F).

Bottom Line: Antibodies against RyR2 had practically no effect on NAADP-evoked Ca(2+) release, but reduced release in response to cADPR by 55%.We conclude that full NAADP-mediated Ca(2+) release requires both TPCs and RyRs.Our results indicate that the primary, but very small, NAADP-elicited Ca(2+) release via TPCs from endosomes/lysosomes triggers the detectable Ca(2+)-induced Ca(2+) release via RyR1 and RyR3 occurring from the granules and the ER.

View Article: PubMed Central - PubMed

Affiliation: Medical Research Council Group, Cardiff School of Biosciences, Cardiff University, Cardiff, Wales, UK.

No MeSH data available.