Human Trp3 forms both inositol trisphosphate receptor-dependent and receptor-independent store-operated cation channels in DT40 avian B lymphocytes

Abstract

Mammalian Trp proteins are candidates for plasma membrane calcium channels regulated by receptor activation or by intracellular calcium store depletion [capacitative calcium entry (CCE)]. One extensively investigated member of the Trp family, the human Trp3 (hTrp3), behaves as a receptor-activated, calcium-permeable, nonselective cation channel when expressed in cell lines and does not appear to be activated by store depletion. Nonetheless, there is good evidence that Trp3 can be regulated by interacting with inositol trisphosphate receptors (IP(3)Rs), reminiscent of the conformational coupling mode of CCE. To investigate the role of Trp3 in CCE, and its regulation by IP(3)R, we transiently expressed hTrp3 in the wild-type DT40 chicken B lymphocyte cell line and its variant lacking IP(3)R. Expression of hTrp3 in either wild-type or IP(3)R-knockout cells did not increase basal membrane permeability, but resulted in a substantially greater divalent cation entry after thapsigargin-induced store depletion. This hTrp3-dependent divalent cation entry was significantly greater in the wild type than in IP(3)R-knockout cells. Thus, it appears that in this cell line, hTrp3 forms channels that are store-operated by both IP(3)R-dependent and IP(3)R-independent mechanisms. Trp3, or one of its structural relatives, is a candidate for the store-operated, nonselective cation channels observed in smooth muscle cells and other cell types.

Figure 1

Store depletion induces Ca²⁺ entry in hTrp3-transfected wild-type DT40 cells. Fura-2-loaded wild-type DT40 cells transfected with either hTrp3 or its vector (Mock), along with a construct encoding eGFP as transfection marker (see Materials and Methods), were incubated in a nominally Ca²⁺-free medium and then exposed to 2 μM thapsigargin to deplete intracellular Ca²⁺ stores. After cytosolic Ca²⁺ returned to basal levels, Ca²⁺ (1.5 mM) was re-added to the medium. Shown are average curves ± SEM (in gray) from three independent experiments, each of them performed on at least four single cells. Measurements were performed on positively transfected cells, which were selected by their green fluorescence when excited at 488 nm (eGFP-positive cells).

Figure 2

Store depletion induces Ca²⁺ entry in hTrp3-transfected IP₃R-KO DT40 cells. The procedure was the same as for Fig. 1, except that hTrp3 was transfected into the IP₃R-KO line of DT40. Shown are average curves ± SEM (in gray) from three independent experiments, each of them performed on at least four single cells.

Figure 3

Store depletion induces Ba²⁺ entry in hTrp3-transfected wild-type and IP₃R-KO DT40 cells. Ba²⁺ influx was measured in Fura-2-loaded DT40 cells transfected with either hTrp3 (wild type, IP₃R-KO) or its vector (pcDNA3, Mock, wild type only) as described in Fig. 1 legend. The cells were maintained in a nominally Ca²⁺-free medium, exposed to 2 μM thapsigargin, and then Ba²⁺ (10 mM) was added where indicated. Shown are average curves ± SEM (in gray) from four independent experiments, each of them performed on at least four single cells.

Figure 4

Agonist activation of the B cell receptor induces Ba²⁺ entry in hTrp3-transfected wild-type but not IP₃R-KO DT40 cells. Ba²⁺ influx was measured in Fura-2-loaded, hTrp3-transfected wild-type or hTrp3-transfected IP₃R-KO cells as described in Fig. 1 legend. The cells were maintained in a nominally Ca²⁺-free medium, exposed to 2 μg/ml anti-IgM, and then Ba²⁺ (10 mM) was added where indicated. Shown are average curves ± SEM (in gray) from experiments performed on six single cells. The data are representative of three independent experiments (nine cells).

Figure 5

OAG stimulation of Ba²⁺ entry in hTrp3-transfected DT40 cells. Ba²⁺ influx was measured in Fura-2-loaded wild type or IP₃R-KO cells transfected with hTrp3. The cells were maintained in a nominally Ca²⁺-free medium and then exposed to 100 μM of the membrane-permeant diacylglycerol OAG. When indicated, Ba²⁺ (10 mM) was added to the medium. Shown are average curves ± SEM (in gray) from experiments performed on six single cells. The data are representative of four independent experiments (12 cells).