Cellular cholesterol controls TRPC3 function: evidence from a novel dominant-negative knockdown strategy

Abstract

TRPC3 (canonical transient receptor potential protein 3) has been suggested to be a component of cation channel complexes that are targeted to cholesterol-rich lipid membrane microdomains. In the present study, we investigated the potential role of membrane cholesterol as a regulator of cellular TRPC3 conductances. Functional experiments demonstrated that cholesterol loading activates a non-selective cation conductance and a Ca2+ entry pathway in TRPC3-overexpressing cells but not in wild-type HEK-293 (human embryonic kidney 293) cells. The cholesterol-induced membrane conductance exhibited a current-to-voltage relationship similar to that observed upon PLC (phospholipase C)-dependent activation of TRPC3 channels. Nonetheless, the cholesterol-activated conductance lacked negative modulation by extracellular Ca2+, a typical feature of agonist-activated TRPC3 currents. Involvement of TRPC3 in the cholesterol-dependent membrane conductance was further corroborated by a novel dominant-negative strategy for selective blockade of TRPC3 channel activity. Expression of a TRPC3 mutant, which contained a haemagglutinin epitope tag in the second extracellular loop, conferred antibody sensitivity to both the classical PLC-activated as well as the cholesterol-activated conductance in TRPC3-expressing cells. Moreover, cholesterol loading as well as PLC stimulation was found to increase surface expression of TRPC3. Promotion of TRPC3 membrane expression by cholesterol was persistent over 30 min, while PLC-mediated enhancement of plasma membrane expression of TRPC3 was transient in nature. We suggest the cholesterol content of the plasma membrane as a determinant of cellular TRPC3 activity and provide evidence for cholesterol dependence of TRPC3 surface expression.

Figure 1. Acute administration of MβCD-saturated cholesterol induces a cation current in TRPC3-overexpressing HEK-293 cells

(A) Representative time courses of membrane currents recorded at −70 mV showing acute stimulation of TRPC3-overexpressing HEK-293 cells (T3-9) by either cholesterol-saturated MβCD (left panel) or 200 μM carbachol (CCH; right panel) as indicated. (B) Representative current responses recorded using ramp-like voltage protocols in T3-9 cells after challenge with cholesterol (1) and carbachol (2). Experiments were performed at room temperature. (C) Mean values for peak current density recorded in T3-9 cells are shown for cells exposed to cholesterol or carbachol in the absence or presence of extracellular calcium. Values represent the means±S.E.M. (n=5–17). Asterisks denote significant difference versus non-stimulated T3-9 cells. (D) Pairs of T3-9 whole-cell current values measured at −70 mV before and after administration of cholesterol or carbachol in the presence and absence of extracellular calcium.

Figure 2. Combined stimulation of TRPC3-expressing HEK-293 cells with cholesterol and carbachol

(A) Time course of whole-cell current recorded at −70 mV from a TRPC3-overexpressing HEK-293 cell (T3-9) during repetitive stimulation with 200 μM carbachol (CCh), as indicated, after incubation with cholesterol-saturated MβCD for 45 min. (B) Representative current responses recorded using ramp-like voltage protocols in T3-9 cells pre-incubated with cholesterol and challenged with carbachol as indicated.

Figure 3. Membrane loading with cholesterol promotes basal Ca²⁺ entry into T3-9 cells

Representative traces of Ca²⁺-sensitive fura 2/AM fluorescence ratios (F_340/380) recorded in HEK-293 wild-type (wt) cells (A) and TRPC3-overexpressing HEK-293 cells (T3-9) (B) incubated in serumfree medium in the absence (control; ●) or presence (△) of cholesterol-saturated MβCD (cholesterol; 10 mM, 45 min). During the experiments, cells were initially kept in Ca²⁺-free solution, and Ca²⁺-containing solution was added to initiate calcium influx at the time point indicated. (C) Average of maximal Ca²⁺ or Ba²⁺ net influx signal derived from fura 2/AM imaging in wild-type and T3-9 cells under control conditions (open bars) and loaded with cholesterol-saturated MβCD (filled bars). Values represent means±S.E.M. (n=40–70).

Figure 4. ExoHA-tagged TRPC3 cation channels are sensitive to anti-HA antibody

(A) Representative time course of carbachol (CCh; 200 μM)-induced current recorded at −70 mV in an HEK-TSA cell transfected to express exoHA–TRPC3. Experiments were performed with cells pre-incubated for 30 min in the presence or absence of anti-HA antibody (1:200). (B) Comparison of carbachol-induced current densities measured at −70 mV in exoHA–TRPC3-expressing cells after pre-incubation in the absence and presence of anti-HA antibody. Values represent the means±S.E.M. (n=6–8). The asterisk denotes significant difference versus control.

Figure 5. Cholesterol-induced TRPC3-mediated currents in exoHA-tagged TRPC3-expressing cells are sensitive to anti-HA antibody

Shown are mean carbachol- and cholesterol-induced current densities recorded in T3-9 cells transiently transfected with exoHA-tagged TRPC3. Responses in T3-9 wild-type cells are shown for comparison. The cells were kept in Ca²⁺-free solution when stimulated with carbachol, and in Ca²⁺-containing solution when challenged by acute administration of cholesterol-saturated MβCD. Carbachol- and cholesterol-mediated conductances in T3-9 cells transiently transfected with exoHA-tagged TRPC3 were significantly inhibited by pre-incubation with anti-HA antibody (1:200, 30 min), while carbachol-induced currents in T3-9 cells were not affected by anti-HA antibody. Values represent the means±S.E.M. (n=7–10). Asterisks denote significant differences versus carbachol- and cholesterol-induced conductance in the absence of antibody.

Figure 6. Effects of carbachol and cholesterol on plasma membrane presentation of TRPC3 and analysis of the effects of BFA on membrane presentation and function of TRPC3

(A) T3-9 cells were either stimulated with carbachol (CCh; 200 μM, 1 min) in Ca²⁺-free buffer or incubated with FCS-free DMEM containing cholesterol–MβCD (10 mM, 45 min), or with FCS-free DMEM containing cholesterol–MβCD subsequent to a pre-incubation with BFA (10 μg/ml, 60 min). HA–TRPC3 abundance in total lysates (150 μg; input) and biotinylated fractions (PM, plasma membrane fraction) was measured by immunoblotting with anti-HA antibody. Results are representative of three individual experiments. (B) Statistical analysis of the TRPC3 immunoreactivity detected in biotinylated fractions of controls, in cells after stimulation with carbachol or cholesterol and in BFA-treated cells stimulated with cholesterol (BFA+cholesterol). * Indicate values that are significantly different from control; ** indicates significant difference versus cholesterol stimulation. (C) Inhibition of cholesterol-induced Ca²⁺ signalling by BFA. Representative traces of Ca²⁺-sensitive fura 2/AM fluorescence ratios (F_340/380) recorded in TRPC3-overexpressing HEK-293 cells pre-incubated in FCS-free medium in the absence (control; ●) or presence (△) of cholesterol–MβCD (10 mM, 45 min) subsequent to pre-incubation with BFA (10 μg/ml, 60 min) (BFA, filled triangles).