Characterization of G proteins involved in activation of nonselective cation channels by endothelin(B) receptor

Abstract

1: We recently demonstrated that endothelin-1 (ET-1) activates two types of Ca(2+)-permeable nonselective cation channels (NSCC-1 and NSCC-2) in Chinese hamster ovary cells expressing endothelin(B) receptors (CHO-ET(B)R) that couple with G(q) and G(i). The purpose of the present study was to identify the G proteins involved in the activation of these Ca(2+) channels by ET-1. For this purpose, we constructed CHO cells expressing an unpalmitoylated (Cys(402)Cys(403) Cys(405)-->Ser(402)Ser(403)Ser(405)) ET(B)R (CHO-SerET(B)R) and ET(B)R truncated at the cytoplasmic tail downstream of Cys(403) (CHO-ET(B)RDelta403). 2: Based on the data obtained from actin stress fibre formation, CHO-ET(B)R couple with G(13). Therefore, CHO-ET(B)R couple with G(q), G(i) and G(13). CHO-SerET(B)R and CHO-ET(B)RDelta403 couple with G(13) and G(q), respectively. 3: ET-1 activated NSCC-1 in CHO-ET(B)R preincubated with phospholipase C (PLC) inhibitor, U73122, and in CHO-SerET(B)R. On the other hand, ET-1 failed to activate Ca(2+) channels in CHO-ET(B)RDelta403. Microinjection of dominant negative mutants of G(13) (G(13)G225A) abolished activation of NSCC-1 and NSCC-2 in CHO-ET(B)R and that of NSCC-1 in CHO-SerET(B)R. 4: Y-27632, a specific Rho-associated kinase (ROCK) inhibitor, did not affect the ET-1-induced transient and sustained increase in [Ca(2+)](i) in CHO-ET(B)R. 5: These results indicate that (1) the cytoplasmic tail downstream of the palmitoylation sites of ET(B)R, but not the palmitoylation site itself, is essential for coupling with G(13), (2) the activation mechanism of each Ca(2+) channel by ET-1 is different in CHO-ET(B)R. NSCC-1 activation depends on G(13)-dependent cascade, and NSCC-2 activation depends on both G(q)/PLC- and G(13)-dependent cascades. Moreover, ROCK-dependent cascade is not involved in the activation of these channels.

Figure 1

Effects of Y-27632, U73122, G₁₂G228A and G₁₃G225A on the ET-1-induced actin stress fibre formation in CHO-ET_BR. Cells were stimulated with (B∼E) or without (A) 10 nM ET-1. The effects of preincubation with 10 μM Y-27632 (C), combination of G₁₃G225A microinjection and 5 μM U73122 preincubation (D) and combination of G₁₃G228A microinjection, PTX (50 ng ml⁻¹) preincubation and 5 μM U73122 preincubation (E) on ET-1-induced stress fibre formation are shown. Y-27632, U73122 and PTX were added 15 min before stimulation with ET-1. Expression plasmids encoding for G₁₂G228A and G₁₃G225A were microinjected into the cell nuclei 24 h before stimulation with ET-1. Actin stress fibers were visualized as described in Methods. Representative examples of stress fibres in individual cells are shown.

Figure 2

Effects of Y27632, G₁₂G228A and G₁₃G225A on ET-1-induced actin stress fiber formation in CHO-SerET_BR. Cells were stimulated with (B∼E) or without (A) 10 nM ET-1. (C) Y-27632 at 10 μM was added 15 min before stimulation with ET-1. Expression plasmids encoding for G₁₂G228A (D) and G₁₃G225A (E) were microinjected into cell nuclei 24 h before stimulation with ET-1. Actin stress fibres were visualized as described in Methods. Representative examples of stress fibres in individual cells are shown.

Figure 3

Effects of Y27632, U73122, G₁₂G228A and G₁₃G225A on ET-1-induced actin stress fiber formation in CHO-ET_BRΔ403. Cells were stimulated with (B∼F) or without (A) 10 nM ET-1. Y-27632 at 10 μM (C) and U73122 at 5 μM (D) were added 15 min before stimulation with ET-1. Expression plasmids encoding G₁₂G228A (E) and G₁₃G225A (F) were microinjected into the cell nuclei 24 h before stimulation with ET-1. Actin stress fibres were visualized as described in Methods. Representative examples of stress fibres in individual cells are shown.

Figure 4

Original tracings illustrating the effects of ET-1 on the increase in [Ca²⁺]_i in CHO-ET_BR (A), CHO-ET_BRΔ403 (B) and CHO-SerET_BR (C). The cells were loaded with fluo-3 and stimulated with 10 nM ET-1 at the time indicated by arrows. Effects of various concentrations of ET-1 on the transient increase in [Ca²⁺]_i (D) and the sustained increase in [Ca²⁺]_i (E) in CHO-ET_BR, CHO-ET_BRΔ403 and CHO-SerET_BR. The values for CHO-ET_BR, CHO-ET_BRΔ403 and CHO-SerET_BR were represented by circles, squares and triangles, respectively. Each point represents the mean±s.e.m. of five experiments.

Figure 5

Original tracings illustrating the effects of various concentrations of LOE 908 and SK&F 96365 on the ET-1-induced sustained increase in [Ca²⁺]_i in CHO-ET_BR (A,B) and CHO-SerET_BR (C,D). The cells were loaded with fluo-3 and stimulated with 10 nM ET-1 at the time indicated by arrows. After [Ca²⁺]_i reached a steady-state, various concentrations of LOE 908 or SK&F 96365 was added as indicated by arrows. Effects of maximally effective concentration of LOE 908, SK&F 96365 and their combination on the ET-1-induced sustained increase in [Ca²⁺]_i in CHO-ET_BR (E) and CHO-SerET_BR (F). The experimental protocols were described in Methods, and the values of [Ca²⁺]_i following addition of 10 μM LOE 908 and/or 10 μM SK&F 96365 were determined. Each point represents the mean±s.e.m. of five experiments.

Figure 6

Original tracings illustrating the effects of maximally effective concentration of LOE 908 (A) and SK&F 96365 (B) on the ET-1-induced sustained increase in [Ca²⁺]_i in CHO-ET_BR pretreated with U73122. The cells loaded with fluo-3 were incubated with 5 μM U73122 for 10 min before 10 nM ET-1 stimulation. After [Ca²⁺]_i reached a steady-state, 10 μM LOE 908 or 10 μM SK&F 96365 was added at the time indicated by horizontal bars. (C) Effects of maximally effective concentration of LOE 908 and SK&F 96365 on the ET-1-induced sustained increase in [Ca²⁺]_i in CHO-ET_BR pretreated with U73122. The experimental protocols were described in Methods, and the values of [Ca²⁺]_i following addition of 10 μM LOE 908 or 10 μM SK&F 96365 were determined. Each point represents the mean±s.e.m. of five experiments.

Figure 7

Effects of G₁₃G225A on ET-1-induced sustained increase in [Ca²⁺]_i in CHO-SerET_BR (A) and CHO-ET_BR (B). The cells loaded with fluo-3 were stimulated by 10 nM ET-1. The sustained increase in [Ca²⁺]_i in the presence of G₁₃G225A is represented as a percentage of values in its absence. Data are presented as mean±s.e.m. of three experiments.

Figure 8

(A) Original tracing illustrating the effects of Y-27632 on the ET-1-induced increase in [Ca²⁺]_i in CHO-ET_BR. The cells loaded with fluo-3 were incubated with 10 μM Y-27632 for 15 min before 10 nM ET-1 stimulation. (B, C) Effects of 10 μM Y-27632 on 10 nM ET-1-induced transient (B) and sustained (C) increase in [Ca²⁺]_i in CHO-ET_BR. The experimental protocols were described in Methods. Data are presented as mean±s.e.m. of three experiments.