Differential effects of the Gβ5-RGS7 complex on muscarinic M3 receptor-induced Ca2+ influx and release

Abstract

The G protein β subunit Gβ5 uniquely forms heterodimers with R7 family regulators of G protein signaling (RGS) proteins (RGS6, RGS7, RGS9, and RGS11) instead of Gγ. Although the Gβ5-RGS7 complex attenuates Ca(2+) signaling mediated by the muscarinic M3 receptor (M3R), the route of Ca(2+) entry (i.e., release from intracellular stores and/or influx across the plasma membrane) is unknown. Here, we show that, in addition to suppressing carbachol-stimulated Ca(2+) release, Gβ5-RGS7 enhanced Ca(2+) influx. This novel effect of Gβ5-RGS7 was blocked by nifedipine and 2-aminoethoxydiphenyl borate. Experiments with pertussis toxin, an RGS domain-deficient mutant of RGS7, and UBO-QIC {L-threonine,(3R)-N-acetyl-3-hydroxy-L-leucyl-(aR)-a-hydroxybenzenepropanoyl-2,3-idehydro-N-methylalanyl-L-alanyl-N-methyl-L-alanyl-(3R)-3-[[(2S,3R)-3-hydroxy-4- methyl-1-oxo-2-[(1-oxopropyl)amino]pentyl]oxy]-L-leucyl-N,O-dimethyl-,(7→1)-lactone (9CI)}, a novel inhibitor of Gq, showed that Gβ5-RGS7 modulated a Gq-mediated pathway. These studies indicate that Gβ5-RGS7, independent of RGS7 GTPase-accelerating protein activity, couples M3R to a nifedipine-sensitive Ca(2+) channel. We also compared the action of Gβ5-RGS7 on M3R-induced Ca(2+) influx and release elicited by different muscarinic agonists. Responses to Oxo-M [oxotremorine methiodide N,N,N,-trimethyl-4-(2-oxo-1-pyrrolidinyl)-2-butyn-1-ammonium iodide] were insensitive to Gβ5-RGS7. Pilocarpine responses consisted of a large release and modest influx components, of which the former was strongly inhibited whereas the latter was insensitive to Gβ5-RGS7. McN-A-343 [(4-hydroxy-2-butynyl)-1-trimethylammonium-3-chlorocarbanilate chloride] was the only compound whose total Ca(2+) response was enhanced by Gβ5-RGS7, attributed to, in part, by the relatively small Ca(2+) release this partial agonist stimulated. Together, these results show that distinct agonists not only have differential M3R functional selectivity, but also confer specific sensitivity to the Gβ5-RGS7 complex.

Fig. 1.

Gβ5-RGS7 attenuation of M3R-stimulated Ca²⁺ release. CHO-K1 cells on glass coverslips were transiently transfected with M3R or M3R + Gβ5-RGS7 and loaded with fura-2 AM. Fluorescence images were recorded in real time using MetaFluor software as described in Materials and Methods. The entire visual field, containing 20–40 cells responding to muscarinic agonists, was used as a region of interest. The traces represent a mean of the 340/380 ratios recorded in at least two independent experiments, with three to six replicates per experiment. (A) CCh (1 μM) was applied for 30 seconds as indicated by the horizontal bar above the traces in the presence or absence of cotransfected Gβ5-RGS7 in complete HBSS buffer containing 1.26 mM CaCl₂. (B) Cells were stimulated with CCh in Ca²⁺-free HBSS. (C) The mean amplitude ± S.D. of the maximal responses (n = 9) with and without extracellular Ca²⁺. White bars represent cells transfected with M3R and pcDNA3 plasmid. Gray bars represent M3R cotransfected with Gβ5 and RGS7.

Fig. 2.

Gβ5-RGS7 augments M3R-stimulated Ca²⁺ influx. Transfected CHO-K1 were cells loaded with fura-2 and superfused with 10 μM ATP in Ca²⁺-free buffer to deplete intracellular Ca²⁺ stores. (A) Subsequent stimulation with CCh (time 260 seconds) in Ca²⁺-free buffer resulted in no measurable rise in [Ca²⁺]_i. (B) Following ATP-stimulated Ca²⁺ depletion as in A, cells were treated with Ca²⁺-containing buffer (gray box) in the absence of CCh. (C) Cells were treated with ATP as in A and B, but 1 μM CCh was delivered in Ca²⁺-containing HBSS buffer. The traces in A–C represent an average of three independent experiments with three (A and B) and five (C) replicates per experiment. (D) The mean amplitude ± S.D. of the maximal responses for total Ca²⁺ response (shown in Fig. 1A), Ca²⁺ release (Fig. 1B), and Ca²⁺ influx (Fig. 2C). *P ≤ 0.05.

Fig. 3.

Effects of 2-APB, nifedipine, and PMA on M3R-stimulated Ca²⁺ signaling. Cells were transfected and treated as in Fig. 2. The panels show only the Ca²⁺ transients after depletion of intracellular stores. (A) Cells activated with 1 μM CCh in the presence of Ca²⁺. (B) Cells were treated with 100 µM 2-APB added prior to and during stimulation with 1 μM CCh. (C) Cells were treated with 10 µM nifedipine and stimulated with CCh as in B. (D) Cells expressing M3R or M3R + Gβ5-RGS7 were treated with 100 nM PMA. Each trace represents an average of three replicates in two (B and D) or three (C) independent experiments.

Fig. 4.

Effect of Gβ5-RGS7 on M3R-stimulated changes in membrane potential. CHO cells transfected with pcDNA, M3R, or M3R + Gβ5-RGS7 were incubated with blue FLIPR Membrane Potential Reagent, as described in Materials and Methods. Fluorescence intensity was recorded using the FLIPR Tetra. An increase in fluorescence intensity indicates membrane depolarization. At the time of 10 seconds, CCh was added to final concentrations from 100 nM to 1 mM as indicated in A–E. The traces represent the mean changes in fluorescence intensity collected from quadruplicate wells on the 384-well plate. (F) Concentration dependencies of the maximal change in fluorescence at the last time point recorded in A–E.

Fig. 5.

M3R Ca²⁺ signaling in transfected CHO cells only involves Gq. (A) Transfected cells were incubated with or without PTX, as described in Materials and Methods, and stimulated with 10 μM 5-hydroxytryptamine (5-HT). Traces show Ca²⁺ increase after store depletion as in Figs. 2 and 3. (B) Cells with or without PTX pretreatment were stimulated with CCh. (C) Cells were transfected with M3R or M3R together with Gβ5 plus full-length RGS7 or the mutant lacking the RGS domain (RGS7^ΔRGS). (D) Cells were treated with UBO-QIC, as described in Materials and Methods, and the total Ca²⁺ response to 1 μM CCh was recorded as in Fig. 1A. WT, wild-type.

Fig. 6.

Influence of Gβ5-RGS7 on acetylcholine-stimulated Ca²⁺ responses. We used 10 μM acetylcholine ACh [acetylcholine (2-acetyloxyethyl-trimethylazanium)] to elicit M3R-mediated Ca²⁺ release and influx as described in Figs. 1 and 2, respectively. (A) Total ACh response in complete HBSS buffer. (B) ACh-stimulated release was measured using Ca²⁺-free HBSS buffer. (C) ACh in complete HBSS buffer stimulates Ca²⁺ influx response after ATP-induced Ca²⁺ depletion. (D) The bar graphs represent the mean amplitude ± S.D. of the Ca²⁺ responses from two independent experiments with three replicates in each. **P ≤ 0.01.

Fig. 7.

Gβ5-RGS7 does not influence M3R stimulated with Oxo-M. We used 0.1 μM Oxo-M to elicit M3R-mediated Ca²⁺ release and influx as described in Figs. 1 and 2. (A) Total Oxo-M response in complete HBSS buffer. (B) Oxo-M–stimulated release was measured using Ca²⁺-free HBSS buffer. (C) Oxo-M in complete HBSS buffer stimulates Ca²⁺ influx response after ATP-induced Ca²⁺ depletion. (D) The bar graphs show the mean amplitude ± S.D. of the Ca²⁺ responses from two independent experiments with three replicates in each.

Fig. 8.

Stimulation of M3R with pilocarpine renders it more sensitive to Gβ5-RGS7 regulation. Concentration dependencies were obtained for M3R and M3R + Gβ5-RGS7 stimulated with CCh (A) and pilocarpine (B) using the same method as described in Fig. 1. Nonlinear regression curves were fit using GraphPad Prism sigmoidal dose-response (variable slope) equation. Each data point represents the mean amplitude of the maximal response ± S.D. (n = 4).

Fig. 9.

Gβ5-RGS7 selectively inhibits pilocarpine-stimulated Ca²⁺ release. (A) Total Ca²⁺ response to 10 μM pilocarpine in complete HBSS buffer. (B) Pilocarpine-stimulated Ca²⁺ release in Ca²⁺-free buffer. (C) Ca²⁺ influx response to pilocarpine in complete HBSS buffer following ATP-induced Ca²⁺ depletion. (D) The mean amplitude ± S.D. of the Ca²⁺ responses (n = 3; three replicates in each experiment). **P ≤ 0.01.

Fig. 10.

Effects of Gβ5-RGS7 on McN-A-343–stimulated Ca²⁺ influx and release. (A) Total response of M3R to 100 μM McN-A-343 in complete HBSS buffer, in the absence and presence of Gβ5-RGS7. (B) McN-A-343–stimulated Ca²⁺ release. (C) Ca²⁺ response to McN-A-343 in complete HBSS following ATP depletion in Ca²⁺-free buffer. (D) The mean amplitude ± S.D. of the Ca²⁺ responses from two independent experiments with three replicates in each. *P ≤ 0.05; **P ≤ 0.01.