The history of the Drosophila TRP channel: the birth of a new channel superfamily

Abstract

Transient receptor potential (TRP) channels are polymodal cellular sensors involved in a wide variety of cellular processes, mainly by changing membrane voltage and increasing cellular Ca(2+). This review outlines in detail the history of the founding member of the TRP family, the Drosophila TRP channel. The field began with a spontaneous mutation in the trp gene that led to a blind mutant during prolonged intense light. It was this mutant that allowed for the discovery of the first TRP channels. A combination of electrophysiological, biochemical, Ca(2+) measurements, and genetic studies in flies and in other invertebrates pointed to TRP as a novel phosphoinositide-regulated and Ca(2+)-permeable channel. The cloning and sequencing of the trp gene provided its molecular identity. These seminal findings led to the isolation of the first mammalian homologues of the Drosophila TRP channels. We now know that TRP channel proteins are conserved through evolution and are found in most organisms, tissues, and cell-types. The TRP channel superfamily is classified into seven related subfamilies: TRPC, TRPM, TRPV, TRPA, TRPP, TRPML, and TRPN. A great deal is known today about participation of TRP channels in many biological processes, including initiation of pain, thermoregulation, salivary fluid secretion, inflammation, cardiovascular regulation, smooth muscle tone, pressure regulation, Ca(2+) and Mg(2+) homeostasis, and lysosomal function. The native Drosophila photoreceptor cells, where the founding member of the TRP channels superfamily was found, is still a useful preparation to study basic features of this remarkable channel.

Figure 1. The phylogenetic tree of TRP channels superfamily

Depicted are the 7 subfamilies that constitute the TRP family. The 4 different species are indicated by different colors. Only some of the Drosophila and C. elegans (worm) members are included. For more details see (Venkatachalam & Montell, 2007). (From Nilius and Mahieu, (Nilius & Mahieu, 2006)).

Figure 2

The original electroretinogram (ERG) comparing the response to a light pulse between wild type Drosophila (wt) and a spontaneously formed mutant (m) that was designated later trp by Minke and colleagues. The traces were photographed from an oscilloscope and the different characters indicate various phases in the ERG response. Note that phases c,d,e,f are missing in the mutant. The upper traces represent the light monitor. The * at the upper panel indicates 2.5 faster timescale. Also note that all the traces are presented in an unconventional manner so that negative voltage (corneal negative) is presented upward (From (Cosens, 1971)).

Figure 3. The waveforms and light-induced bump noise of wild type and trp mutant during intense lights

A. The effect of lowering external Ca²⁺ on light responses of Limulus ventral photoreceptor in response to dim (−log I/I₀=5) and intense (−log I/I₀=3) lights, recorded by two electrode voltage clamp. Lowering external Ca²⁺ concentration increased the bump noise (left) and the steady state noise of the macroscopic response to light (right, from (Wong et al., 1982)). B, top. Intracellular recordings of the receptor potential and the following Prolonged Depolarizing Afterpotential (PDA) in response to intense blue lights in the trp^CM mutant raised at 19°C (top) and in WT Drosophila (B bottom). In the trp^CM mutant a large increase of bump noise is observed during the steady state phase of the light response and during the PDA of the mutant (top), but not in WT flies (bottom). Suppression of the PDA by red light resulted it a prompt suppression of the bump noise (B, top, red light) (from (Minke et al., 1975). C. The decay of the response to light of the trp^CM mutant raised at 24°C is accompanied by a conductance decrease. The figure shows intracellular bridge measurement made in the trp mutant before, during and after green light stimulus. The bridge, which was balanced in the dark, shows during light an initial conductance increase that then decreases in parallele with the decrease in voltage, showing that the decay of the response is accompanied by closure of channels (from (Minke, 1982). D. The decay time of the intracellularly recorded response to light of the trp^CM mutant raised at 24°C depends on light intensity. At very dim light the response does not decay to baseline (from (Minke, 1982).

Figure 4

The phosphoinositide cascade of vision. Cloned genes (for all of which mutants are available) are shown in italics, alongside their corresponding proteins. Upon absorption of light, rhodopsin (ninaE gene) is converted to the active metarhodopsin state, which activates a heterotrimeric G protein (dGq). This leads to activation of phospholipase C (PLCβ, norpA gene) and subsequent opening of two classes of light-sensitive channels encoded at by trp and trpl genes. PLC catalyzes the hydrolysis of phosphatidylinositol 4,5-bisphosphate (PIP₂) into the soluble inositol 1,4,5-trisphosphate (IP₃) and the membrane-bound diacylglycerol (DAG). DAG is recycled to PIP₂ by the phosphatidylinositol (PI) cycle shown in an extension of the smooth endoplasmic reticulum called submicrovillar cisternae (SMC, shown on the bottom). DAG is converted to phosphatidic acid (PA) via DAG kinase (DGK, rdgA gene). After conversion to PI, PI is presumed to be transported back to the microvillar membrane by the PI transfer protein (PITP encoded by the rdgB gene). The InsP₃ receptor (IP₃R), which is an internal Ca²⁺ channel that opens and releases Ca²⁺ upon binding of InsP₃. (From (Minke & Cook, 2002)).

Figure 5. Biochemical measurements of light-induced and G-protein-dependent hydrolysis of PIP₂ and its absence by the norpA mutant which abolishes reversibly the response to light

A. Light-induced hydrolysis of phosphoinositides in Musca eye membrane preparation. Equivalents of 100 eyes were cut into halves and were incubated in the dark for 4 h at 30°C with [³H]inositol. Preparation of eye membranes and measurements of light dependent phosphoinositide hydrolysis were carried out as described previously (Devary et al., 1987). The upper panels show production of InsP₂ and the lower panels show production of InsP3, which is a precursor of InsP₂. The right column (top and bottom) shows the effect of the InsP₃ phosphatase inhibitor (2,3 DPG) that causes an increase in InsP₃ (right bottom) and concurrent reduction in InsP₂ right top. The figure shows light dependent PLC activation leading to production of InsP₃ and subsequent production of InsP₂. B. Light-induced PIP₂ hydrolysis is G-protein dependent. As in A, the figure shows enhancement of InsP₂ production by GTPγS (10μM) and suppression of InsP₂ production by GDPβS (100μM, right top as compared to left top) on inositol phosphate production. Due to the fast conversion of InsP₃ to InsP₂ only a small increase in InsP₃ could be measured without 2,3 DPG. C. Light-induced PIP₂ hydrolysis in eye membrane preparation of wild type (W.T., left panels) and its absence at elevated temperatures in the norp ^H52 temperature sensitive mutant of Drosophila. The experimental system was similar to that of panel A except that Drosophila heads were used. Systems depicted by dotted lines were pre-incubated for 4 min at the indicated temperatures in medium lacking ATP and ATP regenerating system which does not allow the biochemical reaction. The latter components were subsequently added (arrow) to initiate the reaction.

Figure 6. The trp^CM mutant is not a null allele and expresses reduced amount of TRP

Western blot analysis of the homozygote trp^CM mutant, raised at 24°C and at 19°C (as indicated) using momoclonal antibody against the TRP protein (Pollock et al., 1995). The TRP protein of WT and the mutant appears on the gel. (from (Yoon et al., 2000)).

Figure 7. Lanthanum (La³⁺) mimics the trp phenotype in wild type fly but has no effect on a trp homologue mutant

Intracellular recordings from single photoreptor cell of white-eyed Musca domestica (A) and from white-eyed nss mutant of Lucilia cuprina (Howard, 1984), which is a mutant homologue of the Drosophila trp (B). Responses to increasing intensities of orange lights are shown. The left columns show responses before application of La³⁺ (CONTROL). La³⁺ was applied by pressure injection into the extracellular space. Partial recovery of WT phenotype was observed 20 min after injection (A, right). Injection of La³⁺ to the extracellular space of the nss mutant had no effect on the rate of decline, but induced a small reduction in the amplitude of the initial peak response (from (Suss Toby et al., 1991)).

Figure 8. Model scheme that summarizes fly phototransduction according to the “conformational coupling” hypothesis

According to this model, TRP (trp) is a new type of channel/transporter, which is activated by light-induced depletion of the InsP₃-sensitive Ca²⁺ stores (SMC), following production of InsP₃ by G-protein (G) activated PLC and binding of InsP₃ to the InsP₃ Receptor. According to this model depletion of Ca²⁺ from the stores, couples TRP to the InsP₃ receptor and opens the TRP Ca²⁺ channel (from (Minke & Selinger, 1991)).

Figure 9. Calcium indicator fluorescence reveals light-induced large elevation in intracellular Ca²⁺, which is smaller in the trp mutant relative to WT and it is totally abolished in a PLC null mutant

A and B: Calcium green-5N fluorescence (Kd< 30 μM) measured in WT at negative (−50 mV, A) and positive (+50 mV, B) membrane potentials. The reduced Ca²⁺ signal at positive membrane potential is due to reduced driving force for Ca²⁺ influx. The dotted lines indicate resting Ca²⁺ level in all panels. C and D: Measurements similar to A and B performed in the trp^CM mutant (raised at 24°C) showing a large reduction of Ca²⁺ influx in the mutant. E: Both the light-induced current (lower trace in each pair) and increase in cellular Ca²⁺ are absent in the norpA^P24, a virtually null PLC mutant. Fluo 3 (Kd< 1μM) fluorescence revealed only the resting Ca²⁺ level that was largely reduced by prolonged exposure to EGTA. (modified from (Peretz et al., 1994))

Figure 10. Current Voltage relationships of the native TRPL and TRP in WT and in the trp and trpl mutants

A. Current Voltage (I–V) relationships determined from voltage ramps in photoreceptors of wild type (WT), trp^P301 and trpl³⁰² mutants during whole cell recordings from ommatidia bathed in physiological Ringer’s solution containing nominal 0 Ca²⁺ and 4mM Mg²⁺. The current in trp shows simple exponential outward rectification (similar to that of panel A), however, in both WT and trpl flies there is a conspicuous S shape inward and outward rectification, which are markedly different than the I–V curve of TRP in pannel A (from (Reuss et al., 1997). B. Comparison of the I–V curves recorded by whole cell measurements from a photoreceptor of the trp mutant (expressing only TRPL) and the S2 cell expressing heterologously the TRPL channel. The top panel shows I–V curves derived from voltage ramps, while the bottom traces show voltage clamped currents in response to voltage steps between −100 mV and +80 mV in 20 mV increments (bottom traces). A striking similarity is observed between the data obtained from native and the heterologously expressed TRPL channels (from (Hardie et al., 1997).