Modulation of Transient Receptor Potential C Channel Activity by Cholesterol

Abstract

Changes of cholesterol level in the plasma membrane of cells have been shown to modulate ion channel function. The proposed mechanisms underlying these modulations include association of cholesterol to a single binding site at a single channel conformation, association to a highly flexible cholesterol binding site adopting multiple poses, and perturbation of lipid rafts. These perturbations have been shown to induce reversible targeting of mammalian transient receptor potential C (TRPC) channels to the cholesterol-rich membrane environment of lipid rafts. Thus, the observed inhibition of TRPC channels by methyl-β-cyclodextrin (MβCD), which induces cholesterol efflux from the plasma membrane, may result from disruption of lipid rafts. This perturbation was also shown to disrupt multimolecular signaling complexes containing TRPC channels. The Drosophila TRP and TRP-like (TRPL) channels belong to the TRPC channel subfamily. When the Drosophila TRPL channel was expressed in S2 or HEK293 cells and perfused with MβCD, the TRPL current was abolished in less than 100 s, fitting well the fast kinetic phase of cholesterol sequestration experiments in cells. It was thus suggested that the fast kinetics of TRPL channel suppression by MβCD arise from disruption of lipid rafts. Accordingly, lipid raft perturbation by cholesterol sequestration could give clues to the function of lipid environment in TRPC channel activity and its mechanism.

Keywords: TRP-like (TRPL) channel; caveolae; cholesterol recognition amino acid consensus sequence (CRAC); lipid rafts; methyl-β-cyclodextrin.

Figure 1

A sterol binding pocket in the TRPC4_DR structure. Electron cryo-microscopy structure of zebra fish TRPC4 (TRPC4_DR) channel in its unliganded closed state, at an overall resolution of 3.6 Å. The transmembrane S1–S6 helices structure revealed that in the pre-S1 elbow domain inside the membrane, a cavity is formed with helices S1 and S4, in which a density corresponding to a sterol is formed. (Reproduced from Vinayagam et al. (2018) with permission from eLife.)

Figure 2

(A) The phosphoinosite (PI) cycle. In the phototransduction cascade, light triggers the activation of phospholipase Cβ (PLCβ, encoded by norpA). This catalyzes hydrolysis of the membrane phospholipid PI(4,5)P₂ (PIP₂) into IP₃ and diacylglycerol (DAG). DAG is transported by endocytosis to the endoplasmic reticulum and inactivated by phosphorylation converting it into phosphatidic acid (PA) via DAG kinase (DGK, encoded by rdgA) and to CDP-DAG via CDP-DAG synthase. Subsequently, CDP-DAG is converted into phosphatidyl inositol (PI), which is transferred back to the microvillar membrane, by the PI transfer protein (encoded by rdgB). PIP and PIP₂ are produced at the microvillar membrane by PI kinase and PIP kinase, respectively. PA can also be converted back to DAG by lipid phosphate phosphohydrolase (Lpp, encoded by laza). PA is also produced from phosphatidyl choline (PC) by phospholipase D (PLD). DAG is also converted in two enzymatic stages, one of them is by DAG lipase (encoded by inaE), into polyunsaturated fatty acids (PUFAs). (B–D) MβCD blocks constitutive TRPL channels activity. (B) Current–voltage relationships (I–V curves) measuring TRPL-dependent currents. I–V curves obtained in response to voltage ramp (of 1 s duration) from S2 cells expressing TRPL and showing basal channel activity with strong outward rectification, typical for TRPL-dependent current (1). The TRPL channel activity was highly reduced after perfusion with 10 mM methyl-β-cyclodextrin (MβCD) (2) and the effect was irreversible, even after washout of MβCD (3) (n > 10). (C) Time course of the MβCD effects on TRPL currents in S2 cells. Current densities are shown as a function of time. Series of I–V curves were derived from repeatedly applied voltage ramps every 5 s, and currents were measured at ±120 mV holding potentials as a function of time under the various experimental conditions as indicated. The numbers correspond to the numbers on the I–V curves in (B). (D) Statistics of the cholesterol depletion experiments in S2 cells. (A) Cholesterol depletion by MβCD had a significant effect on the positive TRPL currents at 120 mV (n = 5, values are average ± SEM, paired Student t-test, *p ≤ 0.05). Reproduced from Katz and Minke, 2009 with permission from Frontiers. (B–D) Reproduced from Peters et al., 2017 with permission from Elsevier, license number 4676401165468.)

Figure 3

(A–B) Cholesterol depletion suppressed receptor-activated TRPL-dependent current. (A) TRP-like–green fluorescent protein (TRPL–GFP) did not show any spontaneous activity in HEK293 cells, but it could be readily activated via PLC and blocked by MβCD: current–voltage relationships measured from HEK293 cells expressing TRPL–GFP, showing no basal channel activity (1). However, coexpression of the hM1 muscarinic receptor and application of carbachol (CCH) activated the expressed TRPL–GFP channels via endogenous PLC-mediated cascade (2) and the TRPL-dependent current in the presence of CCH was suppressed by application of MβCD (3), while subsequent application of LA, a strong activator of TRPL channels, did not activate the channels after the application of MβCD (4). (B) Time course of the receptor-activated TRPL-dependent current and the effect of cholesterol depletion on the receptor-activated TRPL currents in HEK293 cells. Current densities are shown as a function of time. Series of i–V curves were derived from repeatedly applied voltage ramps every 5 s, and currents were measured at ±120 mV holding potentials as a function of time under the various experimental conditions as indicated (C–H) Cholesterol depletion did not affect receptor-induced PLC activity. No effects of cholesterol depletion on PLC activity as monitored by translocation of the PIP₂ sensor PH–GFP: representative series of multiphoton images of HEK293 cells coexpressing eGFP-tagged PH domain and hM1 receptor. Application of CCH to the bathing solution, in a concentration that activated the TRPL channels (10 μM CCH), induced similar translocation of the eGFP-tagged PH domain to the cell body, with and without MβCD, indicating the PLC-mediated hydrolysis of PIP₂ is not affected by MβCD. (C) The time course of fluorescence changes measured in the cytosol before application of MβCD: graph plotting the relative mean pixels’ intensity (red curve) as a function of time measured from multiphoton images of HEK293 cells expressing PH–GFP and hM1 receptors. Before PLC stimulation by CCH application (white background), the GFP–PH is associated with the plasma membrane where most PIP₂ is located and the cell body fluorescence is low (for quantification, see G, H). Once PLC is activated and PIP₂ is hydrolyzed (green background), the PH–GFP translocates to the cytosol and there is a marked increase in fluorescence intensity at the cytosol. The individual single-cell measurements are shown by noisy dim gray traces. (D) The time course of fluorescence changes measured in the cytosol after application of MβCD: similar graph as in (C), but measured following application of MβCD. (E) Multiphoton images of HEK293 cells expressing PH–GFP and hM1 receptor without application of MβCD: Left: GFP fluorescence of cells before application of CCH, little PH–GFP translocation was observed. Right: GFP fluorescence of cells after perfusion with the M1 agonist CCH. Translocation of PH–GFP is observed. MβCD was not applied (n > 50). (F) Multiphoton images of HEK293 cells expressing PH–GFP and hM1 receptor after application of MβCD: Similar images of HEK293 cells expressing TRPL PH–GFP and hM1 receptor before (left) and after application of CCH (right). MβCD was applied, but it did not affect PH–GFP translocation (n > 50). (G, H) Graphs plotting the PH–GFP fluorescence intensity as a function of cell position: fluorescence intensity of images showing cross sections of two representative cells along the red line, before application of CCH (red curve), and after application of CCH (black curve) in the absence of MβCD (G) and after application of MβCD (H). (Reproduced from Peters et al., 2017 with permission from Elsevier, license number 4676401165468.)