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Transient receptor potential 1 regulates capacitative Ca(2+) entry and Ca(2+) release from endoplasmic reticulum in B lymphocytes.

Mori Y, Wakamori M, Miyakawa T, Hermosura M, Hara Y, Nishida M, Hirose K, Mizushima A, Kurosaki M, Mori E, Gotoh K, Okada T, Fleig A, Penner R, Iino M, Kurosaki T - J. Exp. Med. (2002)

Bottom Line: Here we demonstrate that genetic disruption of transient receptor potential (TRP)1 significantly attenuates both Ca(2+) release-activated Ca(2+) currents and inositol 1,4,5-trisphosphate (IP(3))-mediated Ca(2+) release from endoplasmic reticulum (ER) in DT40 B cells.As a consequence, B cell antigen receptor-mediated Ca(2+) oscillations and NF-AT activation are reduced in TRP1-deficient cells.Thus, our results suggest that CCE channels, whose formation involves TRP1 as an important component, modulate IP(3) receptor function, thereby enhancing functional coupling between the ER and plasma membrane in transduction of intracellular Ca(2+) signaling in B lymphocytes.

View Article: PubMed Central - PubMed

Affiliation: Center for Integrative Bioscience, Department of Information Physiology, National Institute for Physiological Sciences, Myodaiji-cho, Okazaki 444-8585, Japan. moriy@nips.ac.jp

ABSTRACT
Capacitative Ca(2+) entry (CCE) activated by release/depletion of Ca(2+) from internal stores represents a major Ca(2+) influx mechanism in lymphocytes and other nonexcitable cells. Despite the importance of CCE in antigen-mediated lymphocyte activation, molecular components constituting this mechanism remain elusive. Here we demonstrate that genetic disruption of transient receptor potential (TRP)1 significantly attenuates both Ca(2+) release-activated Ca(2+) currents and inositol 1,4,5-trisphosphate (IP(3))-mediated Ca(2+) release from endoplasmic reticulum (ER) in DT40 B cells. As a consequence, B cell antigen receptor-mediated Ca(2+) oscillations and NF-AT activation are reduced in TRP1-deficient cells. Thus, our results suggest that CCE channels, whose formation involves TRP1 as an important component, modulate IP(3) receptor function, thereby enhancing functional coupling between the ER and plasma membrane in transduction of intracellular Ca(2+) signaling in B lymphocytes.

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IP3-induced Ca2+ release is suppressed in TRP1-deficient cells. (A) Intact BCR-induced IP3 production in TRP1-deficient cells. Cells were stimulated with anti-BCR antibody M4 (1 μg/ml) for the indicated time. Data points are the mean ±SE from four experiments. (B) Western blot analysis demonstrating the IP3R-1 or IP3R-2 expression indistinguishable in WT and mutant cells using a polyclonal antibody against either IP3R-1 or IP3R-2. (C) The ER luminal Ca2+ concentration increased with activation of the Ca2+ pump, and declined upon application of IP3. (D) The level of activation of the IP3R can be quantitatively compared by the initial rate of Ca2+ release, which we estimated by fitting an exponential curve (continuous line) to the initial part of the Ca2+ decay signal (black circles). (E) IP3-concentration dependence of Ca2+ release. Release rates were obtained by fitting a single exponential to the initial part of Ca2+ decay signal measured in luminal Ca2+ monitoring (reference 27). The continuous curve and dotted curve represent the best fit hyperbolic equations, rmax/(1 + EC50/[IP3]), where rmax is the extrapolated values of the maximal rate of Ca2+ release, for Ca2+ release rates in WT and TRP1-deficient cells, respectively. rmax and EC50 were 0.153 s-1 and 0.66 μM in WT cells, and 0.118 s−1 and 0.83 μM in mutant cells. Data obtained from TRP1−-14 and TRP1−-16 were combined. Data points are the mean ±SE from six to seven experiments. **P < 0.01.
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fig3: IP3-induced Ca2+ release is suppressed in TRP1-deficient cells. (A) Intact BCR-induced IP3 production in TRP1-deficient cells. Cells were stimulated with anti-BCR antibody M4 (1 μg/ml) for the indicated time. Data points are the mean ±SE from four experiments. (B) Western blot analysis demonstrating the IP3R-1 or IP3R-2 expression indistinguishable in WT and mutant cells using a polyclonal antibody against either IP3R-1 or IP3R-2. (C) The ER luminal Ca2+ concentration increased with activation of the Ca2+ pump, and declined upon application of IP3. (D) The level of activation of the IP3R can be quantitatively compared by the initial rate of Ca2+ release, which we estimated by fitting an exponential curve (continuous line) to the initial part of the Ca2+ decay signal (black circles). (E) IP3-concentration dependence of Ca2+ release. Release rates were obtained by fitting a single exponential to the initial part of Ca2+ decay signal measured in luminal Ca2+ monitoring (reference 27). The continuous curve and dotted curve represent the best fit hyperbolic equations, rmax/(1 + EC50/[IP3]), where rmax is the extrapolated values of the maximal rate of Ca2+ release, for Ca2+ release rates in WT and TRP1-deficient cells, respectively. rmax and EC50 were 0.153 s-1 and 0.66 μM in WT cells, and 0.118 s−1 and 0.83 μM in mutant cells. Data obtained from TRP1−-14 and TRP1−-16 were combined. Data points are the mean ±SE from six to seven experiments. **P < 0.01.

Mentions: Given the evidence that BCR utilizes IP3 to release Ca2+ from the ER stores through its binding to P3Rs (31), the compromised Ca2+ release in TRP1-deficient cells could be explained by the defect in IP3 generation and/or by that in IP3R function. The former possibility is unlikely, because both levels and kinetics of IP3 generation after BCR cross-linking were comparable in mutant and WT DT40 B cells (Fig. 3 A). Then, to examine the latter possibility, we directly assessed the function of IP3Rs by monitoring the Ca2+ concentration in the lumen of the Ca2+ stores (luminal Ca2+ monitoring system) (Fig. 3 C–E; reference 27) and studying the dose–response behavior of IP3-mediated Ca2+ release in WT and TRP1-deficient cells. As shown in Fig. 3 E, TRP1-deficient cells manifested significantly lower IP3 sensitivity compared with WT DT40 cells, suggesting that the diminished activity of IP3Rs per se could at least partly account for the observed attenuation of Ca2+ release in TRP1-deficient B cells. Furthermore, since the expression levels of IP3R-1 and IP3R-2 were indistinguishable in WT and mutant cells (Fig. 3 B), it is likely that reduced activity of a normal number of IP3Rs is responsible for the observed decrease in Ca2+ release rates.


Transient receptor potential 1 regulates capacitative Ca(2+) entry and Ca(2+) release from endoplasmic reticulum in B lymphocytes.

Mori Y, Wakamori M, Miyakawa T, Hermosura M, Hara Y, Nishida M, Hirose K, Mizushima A, Kurosaki M, Mori E, Gotoh K, Okada T, Fleig A, Penner R, Iino M, Kurosaki T - J. Exp. Med. (2002)

IP3-induced Ca2+ release is suppressed in TRP1-deficient cells. (A) Intact BCR-induced IP3 production in TRP1-deficient cells. Cells were stimulated with anti-BCR antibody M4 (1 μg/ml) for the indicated time. Data points are the mean ±SE from four experiments. (B) Western blot analysis demonstrating the IP3R-1 or IP3R-2 expression indistinguishable in WT and mutant cells using a polyclonal antibody against either IP3R-1 or IP3R-2. (C) The ER luminal Ca2+ concentration increased with activation of the Ca2+ pump, and declined upon application of IP3. (D) The level of activation of the IP3R can be quantitatively compared by the initial rate of Ca2+ release, which we estimated by fitting an exponential curve (continuous line) to the initial part of the Ca2+ decay signal (black circles). (E) IP3-concentration dependence of Ca2+ release. Release rates were obtained by fitting a single exponential to the initial part of Ca2+ decay signal measured in luminal Ca2+ monitoring (reference 27). The continuous curve and dotted curve represent the best fit hyperbolic equations, rmax/(1 + EC50/[IP3]), where rmax is the extrapolated values of the maximal rate of Ca2+ release, for Ca2+ release rates in WT and TRP1-deficient cells, respectively. rmax and EC50 were 0.153 s-1 and 0.66 μM in WT cells, and 0.118 s−1 and 0.83 μM in mutant cells. Data obtained from TRP1−-14 and TRP1−-16 were combined. Data points are the mean ±SE from six to seven experiments. **P < 0.01.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2193746&req=5

fig3: IP3-induced Ca2+ release is suppressed in TRP1-deficient cells. (A) Intact BCR-induced IP3 production in TRP1-deficient cells. Cells were stimulated with anti-BCR antibody M4 (1 μg/ml) for the indicated time. Data points are the mean ±SE from four experiments. (B) Western blot analysis demonstrating the IP3R-1 or IP3R-2 expression indistinguishable in WT and mutant cells using a polyclonal antibody against either IP3R-1 or IP3R-2. (C) The ER luminal Ca2+ concentration increased with activation of the Ca2+ pump, and declined upon application of IP3. (D) The level of activation of the IP3R can be quantitatively compared by the initial rate of Ca2+ release, which we estimated by fitting an exponential curve (continuous line) to the initial part of the Ca2+ decay signal (black circles). (E) IP3-concentration dependence of Ca2+ release. Release rates were obtained by fitting a single exponential to the initial part of Ca2+ decay signal measured in luminal Ca2+ monitoring (reference 27). The continuous curve and dotted curve represent the best fit hyperbolic equations, rmax/(1 + EC50/[IP3]), where rmax is the extrapolated values of the maximal rate of Ca2+ release, for Ca2+ release rates in WT and TRP1-deficient cells, respectively. rmax and EC50 were 0.153 s-1 and 0.66 μM in WT cells, and 0.118 s−1 and 0.83 μM in mutant cells. Data obtained from TRP1−-14 and TRP1−-16 were combined. Data points are the mean ±SE from six to seven experiments. **P < 0.01.
Mentions: Given the evidence that BCR utilizes IP3 to release Ca2+ from the ER stores through its binding to P3Rs (31), the compromised Ca2+ release in TRP1-deficient cells could be explained by the defect in IP3 generation and/or by that in IP3R function. The former possibility is unlikely, because both levels and kinetics of IP3 generation after BCR cross-linking were comparable in mutant and WT DT40 B cells (Fig. 3 A). Then, to examine the latter possibility, we directly assessed the function of IP3Rs by monitoring the Ca2+ concentration in the lumen of the Ca2+ stores (luminal Ca2+ monitoring system) (Fig. 3 C–E; reference 27) and studying the dose–response behavior of IP3-mediated Ca2+ release in WT and TRP1-deficient cells. As shown in Fig. 3 E, TRP1-deficient cells manifested significantly lower IP3 sensitivity compared with WT DT40 cells, suggesting that the diminished activity of IP3Rs per se could at least partly account for the observed attenuation of Ca2+ release in TRP1-deficient B cells. Furthermore, since the expression levels of IP3R-1 and IP3R-2 were indistinguishable in WT and mutant cells (Fig. 3 B), it is likely that reduced activity of a normal number of IP3Rs is responsible for the observed decrease in Ca2+ release rates.

Bottom Line: Here we demonstrate that genetic disruption of transient receptor potential (TRP)1 significantly attenuates both Ca(2+) release-activated Ca(2+) currents and inositol 1,4,5-trisphosphate (IP(3))-mediated Ca(2+) release from endoplasmic reticulum (ER) in DT40 B cells.As a consequence, B cell antigen receptor-mediated Ca(2+) oscillations and NF-AT activation are reduced in TRP1-deficient cells.Thus, our results suggest that CCE channels, whose formation involves TRP1 as an important component, modulate IP(3) receptor function, thereby enhancing functional coupling between the ER and plasma membrane in transduction of intracellular Ca(2+) signaling in B lymphocytes.

View Article: PubMed Central - PubMed

Affiliation: Center for Integrative Bioscience, Department of Information Physiology, National Institute for Physiological Sciences, Myodaiji-cho, Okazaki 444-8585, Japan. moriy@nips.ac.jp

ABSTRACT
Capacitative Ca(2+) entry (CCE) activated by release/depletion of Ca(2+) from internal stores represents a major Ca(2+) influx mechanism in lymphocytes and other nonexcitable cells. Despite the importance of CCE in antigen-mediated lymphocyte activation, molecular components constituting this mechanism remain elusive. Here we demonstrate that genetic disruption of transient receptor potential (TRP)1 significantly attenuates both Ca(2+) release-activated Ca(2+) currents and inositol 1,4,5-trisphosphate (IP(3))-mediated Ca(2+) release from endoplasmic reticulum (ER) in DT40 B cells. As a consequence, B cell antigen receptor-mediated Ca(2+) oscillations and NF-AT activation are reduced in TRP1-deficient cells. Thus, our results suggest that CCE channels, whose formation involves TRP1 as an important component, modulate IP(3) receptor function, thereby enhancing functional coupling between the ER and plasma membrane in transduction of intracellular Ca(2+) signaling in B lymphocytes.

Show MeSH
Related in: MedlinePlus