Membrane cholesterol content influences binding properties of muscarinic M2 receptors and differentially impacts activation of second messenger pathways

Abstract

We investigated the influence of membrane cholesterol content on preferential and non-preferential signaling through the M(2) muscarinic acetylcholine receptor expressed in CHO cells. Cholesterol depletion by 39% significantly decreased the affinity of M(2) receptors for [(3)H]-N-methylscopolamine ([(3)H]-NMS) binding and increased B(max) in intact cells and membranes. Membranes displayed two-affinity agonist binding sites for carbachol and cholesterol depletion doubled the fraction of high-affinity binding sites. In intact cells it also reduced the rate of agonist-induced receptor internalization and changed the profile of agonist binding from a single site to two affinity states. Cholesterol enrichment by 137% had no effects on carbachol E(max) of cAMP synthesis inhibition and on cAMP synthesis stimulation and inositolphosphates (IP) accumulation at higher agonist concentrations (non-preferred pathways). On the other hand, cholesterol depletion significantly increased E(max) of cAMP synthesis inhibition or stimulation without change in potency, and decreased E(max) of IP accumulation. Noteworthy, modifications of membrane cholesterol had no effect on membrane permeability, oxidative activity, protein content, or relative expression of G(s), G(i/o), and G(q/11) alpha subunits. These results demonstrate distinct changes of M(2) receptor signaling through both preferential and non-preferential G-proteins consequent to membrane cholesterol depletion that occur at the level of receptor/G-protein/effector protein interactions in the cell membrane. The significant decrease of IP accumulation by cholesterol depletion was also observed in cells expressing M(3) receptors and by both cholesterol depletion and enrichment in cells expressing M(1) receptors indicating relevance of reduced G(q/11) signaling for the pathogenesis of Alzheimer's disease.

Fig. 1

Binding of [³H]-NMS and carbachol in cells and membranes after cholesterol depletion or cholesterol enrichment. A and B: Specific [³H]-NMS binding in intact cells (A) and membranes (B) is fitted as a saturation isotherm (abscissa, concentration of [³H]-NMS; ordinate, specific binding in pmol/mg protein). Shown is a representative experiment. Each point is the mean±S.E.M. of triplicate values. When S.E.M. is not depicted it was smaller than the symbol. Parameters of non-linear regression fits are shown in Table 2. C and D: Displacement of 0.6 nM [³H]-NMS by carbachol (abscissa, log M) is expressed as percent of control (ordinate) in intact cells (C) and membranes (D) without or with added GTP as indicated. Each point is the mean±S.E.M. of six values from two independent experiments. One- or two-site competition equation is fitted to data as appropriate. Parameters of fits are shown in Table 3. Control, cells or membranes derived from untreated cells; MBCD, cholesterol-depleted cells or membranes prepared from these cells; Cholesterol-MBCD, cholesterol-enriched cells or membranes derived from these cells. Dashed lines in (D) denote competition curves in membranes incubated in the presence of 10 mM GTP.

Fig. 2

Influence of changes in membrane cholesterol content on carbachol-induced modulation of cAMP synthesis in intact cells. A: Raw data of concentration-response relationship of carbachol effects (abscissa, log M) on forskolin-stimulated cAMP synthesis are expressed as percent of incorporated radioactivity (ordinate). Bell-shaped concentration-response equation is fitted to data obtained in native cells (full lines) and Sigmoidal concentration-response equation with slope of unity to data obtained in pertussis toxin-pretreated cells (dashed lines). Statistical evaluation of the effect of treatments on resting values is shown in Table 4. B: For better clarity, effects of cholesterol depletion or enrichment on carbachol-induced changes of forskolin-stimulated cAMP synthesis is expressed as a net change (ordinate; in percent of incorporated radioactivity). The net inhibition of cAMP synthesis was obtained by subtracting basal values from values measured in the presence of carbachol up to a concentration of 3 μM shown in A. The net stimulation of cAMP synthesis in native cells by carbachol from a concentration of 10 μM up was obtained by subtracting calculated maximal inhibition values. The net stimulation of cAMP synthesis in pertussis toxin-treated cells (dashed lines) was obtained by subtracting measured basal values from those in the presence of carbachol. Sigmoidal concentration-response equation with slope of unity was fitted to the data. Parameters of fits are shown in Table 5. Symbols are as described in Fig. 1.

Fig. 3

Influence of changes in membrane cholesterol content on carbachol-induced inositol phosphates (IP) accumulation in intact cells expressing M₂, M₁, and M₃ receptors. A and B: Shown are raw data (ordinate, percent of incorporated radioactivity; abscissa, log M concentration of carbachol) in cells expressing M₂ receptor (A) or M₁ and M₃ receptors (B). C and D: For easier comparison, concentration-responses of carbachol-induced (abscissa, log M) IP accumulation are shown as a net increase above basal in percent of incorporated radioactivity (ordinate). The net stimulation of IP accumulation was calculated by subtracting resting values. A sigmoidal concentration-response equation was fitted to data. Statistical evaluation of the effect of treatments on resting values is shown in Table 4 and parameters of fits are shown in Table 5. Symbols are the same as in Figure 1. CHO-M₁-M₃, CHO cells expressing M₁-M₃ receptors individually.

Fig. 4

Effects of changes in membrane cholesterol content on internalization of M₂ receptors induced by carbachol stimulation. A: Concentration-response of carbachol during 20 min incubations (abscissa log M) on the number of plasma membrane M₂ receptors in intact cells. B and C: Time-course (abscissa, time in min) of 10 μM (B) and 1 mM (C) carbachol effects on the number of plasma membrane M₂ receptors in intact cells is expressed as percent of control values (ordinates). Points are mean±S.E.M. when bigger than symbols, of six values from two independent experiments. Symbols and descriptions are the same as in Figure 1. Sigmoidal concentration-response equation with slope of unity (A) or single phase exponential decay equation (B and C) was fitted to data. Parameters of fits are shown in Table 6.

Fig. 5

Influence of treatments on protein content and cell integrity. A: Protein content in control cells and in cells treated with MBCD or Ch-MBCD. Results are expressed as percent (ordinate) of protein content in control cells in six independent experiments. B: Relative content in percent of controls (ordinate) of G_s, G_i/o, and G_q/11 G-protein α-subunits in membranes prepared from MBCD and Ch-MBCD treated cells. Data are shown as mean±S.E.M. of three independent experiments. Representative blots are shown above the columns. C: Rate of calcein leakage during one hour incubation in MBCD and cholesterol-MBCD treated cells expressed in percent (ordinate) of leakage in control cells. Columns represent mean±S.E.M. of two independent experiments in octaplicates. D: Oxidative activity of MBCD and cholesterol-MBCD treated cells is expressed in percent (ordinate) of that in control cells. Columns represent mean±S.E.M. of two independent experiments in octaplicates.