Diacylglycerol Activates the Drosophila Light Sensitive Channel TRPL Expressed in HEK Cells

Abstract

Physiological activation by light of the Drosophila TRP and TRP-like (TRPL) channels requires the activation of phospholipase Cβ (PLC). The hydrolysis of phosphatidylinositol 4,5, bisphosphate (PIP₂) by PLC is a crucial step in the still-unclear light activation, while the generation of Diacylglycerol (DAG) by PLC seems to be involved. In this study, we re-examined the ability of a DAG analogue 1-oleoyl-2-acetyl-sn-glycerol (OAG) to activate the TRPL channels expressed in HEK cells. Unlike previous studies, we added OAG into the cytosol via a patch-clamp pipette and observed robust activation of the expressed TRPL channels. However, TRPL channel activation was much slower than the physiologically activated TRPL by light. Therefore, we used a picosecond-fast optically activated DAG analogue, OptoDArG. Inactive OptoDArG was added into the intracellular solution with the patch-clamp pipette, and it slowly accumulated on the surface membrane of the recorded HEK cell in the dark. A fast application of intense UV light to the recorded cell resulted in a robust and relatively fast TRPL-dependent current that was greatly accelerated by the constitutively active TRPL^F557I pore-region mutation. However, this current of the mutant channel was still considerably slower than the native light-induced TRPL current, suggesting that DAG alone is not sufficient for TRPL channel activation under physiological conditions.

Keywords: (OptoDArG); 1-oleoyl-2-acetyl-sn-glycerol (OAG); 3-hydroxypropane-1,2-diylbis(4-(4-((E)-(4-butylphenyl) diazenyl) phenyl; Diacylglycerol (DAG); Drosophila TRP/TRPL channels; phospholipase C (PLC).

Figure 1

Heterologous expression, localization, and activation of the TRPL channel in HEK cells. (A) Western-blot analysis of T-REx-293 cells expressing dTRPL^WT (third lane), using α-dTRPL antibody. The Drosophila trpl³⁰² mutant fly heads lacking the TRPL channel (first lane) and un-transfected T-REx cells (second lane) were used as negative controls. The Drosophila trp^P343 mutant fly heads expressing only the TRPL channel (fourth lane), and the Drosophila WT fly heads (fifth lane) were used as positive controls. α-Actin antibody was used as protein loading control (n = 4). Molecular mass marker (in kDa) is indicated to the left of the gel. (B) Representative confocal fluorescence images of Naïve T-REx-293 cells co-transfected with hTRPC3^WT-mCherry (red, left) and with dTRPL-GFP (green, middle). The right image shows the merged localization of hTRPC3^WT-mCherry together with dTRPL^WT-GFP. Scale bar 10 μm (applicable for all three images). The three lines crossing the cell (1, 2, 3) indicate the location of the analysis performed in section C. The total number of analyzed cells is 12. (C) Line profile graphs of the fluorescent intensity along the three white lines crossing the plasma membrane in the right merged image (1, 2, 3). The red graph represents hTRPC3^WT-mCherry expression, and the green graph represents dTRPL^WT-GFP expression. (D) Representative current–voltage relationship (i-V curves) obtained from patch clamp whole cell current measurements from a T-REx-293 cell heterologously expressing dTRPL^WT, GFP and the human Muscarinic Receptor 1 (hM1R) in response to voltage ramps from −150 mV to +150 mV (in 1 s), before bath application of carbachol (CCh, red, 1), after application of 100 μM CCh (black, 2) and after application of Standard External Solution (SES, E1) to which 10 mM Gd³⁺ (TRPL channels blocker) was added (blue, 3). The total number of analyzed cells is 6. (E) Corresponding currents measured at +140 mV and −140 mV. Numbers indicate the time of the selected i-V curves.

Figure 2

Activation of expressed TRPL channels by intracellular OAG. (A) Representative i-V curves obtained from patch clamp whole cell current measurements from a T-REx-293 cell expressing dTRPL^WT and GFP, in response to voltage ramps from −150 mV to +150 mV, during continuous intracellular application of 30 μM OAG, a short time (red, 1) and long time (black, 2) after whole cell formation and after application of E1, SES with 10 mM Gd³⁺ at the end of the experiment (blue, 3). (B) Corresponding currents at +140 mV and −140 mV. Numbers indicate the time of the selected i-V curves. The top red dot circled in black represents 13 superimposed dots, each at a time point of 5 s after the previous dot. Inset, an enlarged segment of the corresponding currents, from 1150 s to 1200 s, indicating no significant change in current density. (C) A bar chart showing the mean maximal current density measured at +140 mV after the bath application of 100 μM CCh in T-REx-293 cells expressing dTRPL^WT, GFP and hM1R (red, n = 6, see Figure 1D,E). The bar chart also shows mean maximal current density measured at +140 mV after intracellular application of 30 μM OAG via the patch clamp pipette in cells expressing dTRPL^WT and GFP (black, n = 5, see Figure 3B). Error bars show SEM. p values were calculated using a two-tailed Mann–Whitney U-test and are indicated below the bars. Values from individual experiments are shown for each of the columns (circles). (D) A bar chart showing the mean time to response-onset measured after bath application of 100 μM CCh in T-REx-293 cells expressing dTRPL^WT, GFP and hM1R (red, n = 6, see Figure 2B) or after whole cell formation with a pipette containing 30 μM OAG (intracellular application) in cells expressing dTRPL^WT and GFP (black, n = 5, see Figure 4B). Values from individual experiments are shown for each of the columns (circles). (E) A bar chart showing the time from the response onset to the maximal measured current (when no significant change in the i-V curve amplitude was observed during 50 s) in the same cell types as in the bar chart described in D. Error bars show SEM. p value was calculated using a two-tailed Mann–Whitney U-test and is indicated below the bar. Values from individual experiments are shown for each of the columns (circles).

Figure 3

Activation of TRPL channels expressed in HEK cells by an optically controlled DAG analogue in comparison to light activation of Drosophila TRPL. (A) A representative current as a function of time showing an outward current induced by an intense UV (380 nm) light applied to an HEK cell expressing the TRPL channel, in which 30 μM OptoDArG was included in the intracellular solution of the patch clamp pipette. The UV light was applied 16 min after whole cell formation, while the cell was kept in the dark during that time to keep the OptoDArG in its inactive trans configuration. Then, the membrane voltage was increased in the dark from 0 mV to +100 mV holding potential and the UV light was turned on, resulting in a robust outward current (red trace). The upper black trace depicts the onset of the UV (380 nm) light measured by a light detector. The yellow arrow and the dashed line indicate the time of UV light onset. Left inset, the accurate open time of the electromechanical shutter, which allowed the accurate application of UV light as measured by the light detector. Right inset, a bar chart showing the mean time to response onset as measured from the turn-on time of the UV light to the initiation of the outward current (n = 5). Error bar shows SEM. Values from individual experiments are shown (circles). (B) A cluster of representative i-V curves obtained from patch clamp whole cell current measurements of a T-REx cell expressing TRPL, in response to voltage ramps from −150 mV to +150 mV, following intracellular application of 30 μM OptoDArG for 6 min (from whole cell formation) in the dark, followed by application of the intense UV light. The first trace (designated 0 s) is in black and indicates the UV 380 nm light onset. The time between each i-V curve is 5 s and the brightening of the trace colors indicates the progression of time during the recordings. The gradual increase in i-V curve amplitude presumably reflects the accumulation of active OptoDArG in the surface membrane. (C) A representative current as a function of time showing that negligible outward current was induced by the intense UV (380 nm) light applied to an HEK cell expressing the TRPL channel, in which 30 μM OptoDArG was included in the intracellular solution of the patch clamp pipette. The UV light was applied 2.5 min after whole cell formation, and was allowed 30 s to develop a response before recording was terminated. This time span was deemed the cutoff time (bottom inset). The upper black trace depicts the UV 380 nm light measured by the light detector. The yellow arrow and the dashed line indicate the time of UV light onset. Inset, a bar chart showing the upper limit of the cutoff time from UV light onset to the termination of the recording. During all 5 measurements, no response was evident within the cutoff time; therefore, there is no SEM. Values from individual experiments are shown (circles). (D) A representative trace showing whole cell current induced by orange light with intensity of 3 × 10⁶ EP/s (EF, effective photons) of the Drosophila trp^P343 mutant photoreceptor (expressing only TRPL channels) in a patch clamp whole cell recording at a holding potential of −70 mV (red). The upper black trace depicts the light measured by the light detector. The yellow arrow and the dashed line indicate the time of orange light onset. Top inset, magnified waveform of the light stimulus as measured by the light detector and the initial light induced current. Bottom inset, a bar chart showing the mean time to response onset, from the time of light turn-on to the initial TRPL current (n = 12). Error bar shows SEM. Values from individual experiments are shown for each of the columns (circles).

Figure 4

Intracellular OAG enhanced and accelerated the initial basal current of the constitutively active TRPL^F557I mutant channel. (A) A representative control i-V curve of the initial current obtained from patch clamp whole cell measurements from a T-REx-293 cell expressing TRPL^WT and GFP, in response to voltage ramps from −150 mV to +150 mV. (B) Representative i-V curve of the initial current obtained from patch clamp whole cell measurements from a T-REx-293 cell expressing TRPL^F557I mutant channel and GFP, in response to voltage ramps from −150 mV to +150 mV. (C) Representative i-V curve of the initial current obtained from patch clamp whole cell measurements from a T-REx-293 cell expressing TRPL^F557I and GFP, in response to voltage ramps from −150 mV to +150 mV applied every 5 s. The i-V curves were measured 18 ± 1.2 s after whole cell formation while the pipette solution included 30 μM OAG. (D) A representative current as a function of time showing an outward current induced by an intense UV (380 nm) light applied to an HEK cell expressing the TRPL^F557I mutant channel, in which 30 μM OptoDArG was included in the intracellular solution of the patch clamp pipette. The UV light was applied ~6 min after whole cell formation, while the cell was kept in the dark during that time to keep the OptoDArG in its inactive trans configuration. Then, the membrane voltage was increased in the dark from 0 mV to +100 mV holding potential and the UV light was turned on, resulting in a robust outward current (red trace). The upper black trace depicts the onset of the UV (380 nm) light measured by a light detector. The yellow arrow and the dashed line indicate the time of UV light onset. (E) A bar chart showing the mean initial current density measured at +140 mV from T-REx-293 cells expressing TRPL^WT and GFP (red, n = 6) and cells expressing mutant TRPL^F557I and GFP with (blue, n = 5) and without the intracellular application of 30 μM OAG (black, n = 7). Error bars show SEM. p values were calculated using a two-tailed Mann–Whitney U-test and are indicated below the bars. Values from individual experiments are shown for each of the columns (circles). (F) A bar chart comparing the mean time to response onset from the time of UV light switched on in HEK cells expressing the TRPL^WT channel (red, n = 5) and the TRPL^F557I mutant channel (black, n = 5), when 30 μM of OptoDArG was included in the intracellular solution of the patch pipette. Error bars show SEM. p values were calculated using a two-tailed Mann–Whitney U-test and are indicated below the bars. Values from individual experiments are shown for each of the columns (circles).