An inositol 1,4,5-trisphosphate receptor-dependent cation entry pathway in DT40 B lymphocytes

Abstract

We examined the roles of inositol 1,4,5-trisphosphate (IP3) receptors (IP3R) in calcium signaling using DT40 B lymphocytes, and a variant lacking the three IP3R isoforms (IP3R-KO). In wild-type cells, B cell receptor (BCR) stimulation activates a cation entry route that exhibits significantly greater permeability to Ba2+ than does capacitative calcium entry. This cation entry is absent in IP3R-KO cells. Expression of the type-3 IP3R (IP3R-3) in the IP3R-KO cells rescued not only agonist-dependent release of intracellular Ca2+, but also Ba2+ influx following receptor stimulation. Similar results were obtained with an IP3R-3 mutant carrying a conservative point mutation in the selectivity filter region of the channel (D2477E); however, an IP3R-3 mutant in which this same aspartate was replaced by alanine (D2477A) failed to restore either BCR-induced Ca2+ release or receptor-dependent Ba2+ entry. These results suggest that in DT40 B lymphocytes, BCR stimulation activates a novel cation entry across the plasma membrane that depends upon, or is mediated by, fully functional IP3R.

Fig. 1. The IP₃R inhibitor xestospongin C effectively suppresses both BCR-induced Ca²⁺ release and calcium entry in DT40 cells B lymphocytes, but does not affect thapsigargin-induced Ca²⁺ release or entry. DT40 cells were incubated for 45 min in the presence of 25 µM xestospongin C. Cells were then bathed in nominally Ca²⁺-free medium, exposed to 5 µg/ml anti-IgM antibody (top) or 2 µM thapsigargin (bottom), as indicated. Ca²⁺ (1.5 mM) was added where indicated. In this experiment, relatively higher ratio values were obtained as compared with other experiments due to the use of a different Ca²⁺ measuring system; in this series (and for experiments in Figure 3), a photon counting system was used, while in the other series, an imaging system was used. Black trace: control (no xestospongin C); gray trace, 25 µM xestospongin C. Shown are representative traces from three independent experiments.

Fig. 2. BCR activation, but not store depletion, induces Ba²⁺ entry in DT40 B lymphocytes. (A) Fura-2-loaded WTDT40 cells were incubated in nominally Ca²⁺-free medium and then exposed to 2 µM thapsigargin in order to deplete intracellular Ca²⁺ stores. After cytosolic Ca²⁺ returned to basal levels, Ba²⁺ (10 mM) was added to the medium. A representative trace from at least five independent experiments is shown. (B) Wild-type (WTDT40) or IP₃R-KO (broken line) DT40 cells were maintained in nominally Ca²⁺-free medium, exposed to 5 µg/ml anti-IgM antibody, and then Ba²⁺ (10 mM) was added where indicated. Representative traces from at least five independent experiments are shown.

Fig. 3. Activation of the G-protein-coupled M5 muscarinic receptor results in stimulation of Ba²⁺ entry in wild-type but not in IP₃R-KO DT40 cells. Ba²⁺ influx was measured in single Fura-2-loaded wild-type (WTDT40) or IP₃R-KO (dotted line) DT40 cells transfected with the human M5 muscarinic receptor. The cells were maintained in nominally Ca²⁺-free medium, exposed to 100 µM carbachol, and then Ba²⁺ (10 mM) was added where indicated. The relatively higher ratio values in this experiment are due to the use of a different Ca²⁺ measuring system; in this series, a photon counting system was used, while in the other series, an imaging system was used. Representative traces from three independent experiments are shown.

Fig. 4. BCR-dependent activation of Ba²⁺ entry in wild-type but not in IP₃R-KO DT40 cells. Fura-2-loaded wild-type (WTDT40) and IP₃R-KO DT40 cells were incubated in nominally Ca²⁺-free medium and then exposed to 2 µM thapsigargin. After complete store depletion, Ba²⁺ (10 mM) was added to the medium where indicated, and 2 min later anti-IgM (5 µg/ml) was added. Representative traces from three independent experiments are shown.

Fig. 5. Transient expression of an IP₃R restores agonist-induced Ba²⁺ entry in IP₃R-KO DT40 cells. Ba²⁺ influx was measured in Fura-2-loaded IP₃R-KO DT40 cells transfected with either the rat IP₃R-3 or its vector (Mock). The cells were maintained in nominally Ca²⁺-free medium, exposed to 5 µg/ml anti-IgM antibody, and then Ba²⁺ (10 mM) was added where indicated. Representative traces from three independent experiments are shown.

Fig. 6. Effect of single point mutations within the putative pore forming region of IP₃R-3 on restoration of agonist-induced Ba²⁺ entry in IP₃R-KO DT40 cells. The top of the figure shows an alignment of a region including the predicted pore of rat IP₃R-1–3 (the putative selectivity filter sequence is underlined). D2477 of rat IP₃R-3, which was mutated into glutamate (D2477E) or alanine (D2477A), corresponds to D2550 of rat IP₃R-1. In the experiment shown below, Ba²⁺ influx was measured in Fura-2-loaded IP₃R-KO DT40 cells transfected with either the D2477E (solid line) or the D2477A (broken line) mutants of the rat IP₃R-3 (see Materials and methods for details on the mutations). The cells were maintained in nominally Ca²⁺-free medium, exposed to 5 µg/ml anti-IgM antibody, and then Ba²⁺ (10 mM) was added where indicated. Representative traces from three independent experiments are shown. At the bottom, immunolocalization of transiently expressed rat IP₃R-3 (pCB6⁺ vector) in DT40 B cells is shown. IP₃R-KO cells untransfected, or transfected with the wild-type rat IP₃R-3 (WT-IP₃R), or the rat IP₃R-3 mutant D2477A (D2477A-IP₃R; see text for details) were incubated with a mouse anti-IP₃R-3 monoclonal antibody and then with the secondary Alexa Fluor 488-labeled anti-mouse IgG antibody. The fluorescence images were acquired with a Zeiss 410 confocal microscope.

Fig. 7. The D2477A mutant of the rat IP₃R-3 does not restore BCR-dependent activation of Ba²⁺ entry in Ca²⁺-depleted, IP₃R-KO DT40 cells. Fura-2-loaded IP₃R-KO DT40 cells transfected with either the wild type or the D2477A mutant of the rat IP₃R-3 were incubated in nominally Ca²⁺-free medium and then exposed to 2 µM thapsigargin. After complete store depletion, Ba²⁺ (10 mM) was added to the medium where indicated, and 2 min later anti-IgM (5 µg/ml) was added. Solid line, wild-type IP₃R; dashed line, D2477A IP₃R. Representative traces from three independent experiments are shown.