A transient receptor potential ion channel in Chlamydomonas shares key features with sensory transduction-associated TRP channels in mammals

Abstract

Sensory modalities are essential for navigating through an ever-changing environment. From insects to mammals, transient receptor potential (TRP) channels are known mediators for cellular sensing. Chlamydomonas reinhardtii is a motile single-celled freshwater green alga that is guided by photosensory, mechanosensory, and chemosensory cues. In this type of alga, sensory input is first detected by membrane receptors located in the cell body and then transduced to the beating cilia by membrane depolarization. Although TRP channels seem to be absent in plants, C. reinhardtii possesses genomic sequences encoding TRP proteins. Here, we describe the cloning and characterization of a C. reinhardtii version of a TRP channel sharing key features present in mammalian TRP channels associated with sensory transduction. In silico sequence-structure analysis unveiled the modular design of TRP channels, and electrophysiological experiments conducted on Human Embryonic Kidney-293T cells expressing the Cr-TRP1 clone showed that many of the core functional features of metazoan TRP channels are present in Cr-TRP1, suggesting that basic TRP channel gating characteristics evolved early in the history of eukaryotes.

Figure 1.

Cr-TRP1 within the SSN of the TRP Family. Nodes represent protein sequences, and edges represent the lowest reciprocal BLASTP E-values that exceed a given threshold. Colored nodes correspond to functionally characterized TRP channels, and different shapes are used to identify channels from C. reinhardtii, V. carteri, and other algae or unicellular organisms. (A) SSN of 2841 representative sequences with edges filtered to E-value < 1e⁻¹⁸, median alignment length of 651 residues, and median identity of 35.8%. (B) SSN of 7126 sequences with edges filtered to E-value < 1e⁻⁸⁷, median alignment length of 812 residues, and median identity of 48%.

Figure 2.

Phylogenetic Inference for Selected Members of the TRP Family. Branch confidence values are shown next to nodes. Taxa in color correspond to the 68 functionally characterized TRP subgroup members (same color scheme as in Figure 1). Taxa in black (including Cr-TRP1 in yellow with black border) are the 33 TRP channels identified here in algae and unicellular organisms. Proteomes obtained from Phytozome are as follows: 1, C. reinhardtii (Cr-TRP1, Cr-TRP2, Cr-TRP6, Cr-TRP11, Cr-TRP13, Cr-TRP16, Cr-TRPP2, CrTRP21, CrTRP22, Cr-TRP23); 2, V. carteri (Vc-TRP1, Vc-TRP2, Vc-TRP3, Vc-TRP4); 3, Coccomyxa subellipsoidea C-169 (Cs-TRP1, Cs-TRP2); 4, Micromonas pusilla CCMP1545 (Mp_ccmp-TRP1); 5, Micromonas pusilla RCC299 (Mp_rcc-TRP2). Proteomes obtained from UniProt are as follows: 1, Dictyostelium discoideum (Dd-TRP1, Dd-TRP2); 2, Dictyostelium purpureum (Dp-TRP1, Dp-TRP2, Dp-TRP3); 3, Leishmania infantum (Li-TRP1); 4, Leishmania major (Lm-TRP1); 5, Leishmania mexicana (strain MHOM/GT/2001/U1103) (Lmex-TRP1, Lmex-TRP2); 6, Paramecium tetraurelia (Pt-TRP1, Pt-TRP2, Pt-TRP3); 7, Trypanosoma cruzi (strain CL Brener) (TcCL-TRP1, TcCL-TRP2); 8, Trypanosoma cruzi (Tc-TRP1).

Figure 3.

Structural Domains in Cr-TRP1. (A) Protein illustration for Cr-TRP1 including the identified domains. (B) Multiple sequence alignment depicting the TD in Cr-TRP1 and other TRPN, TRPC, TRPV, and TRPM family representatives. Note the conservation of the positively charged residues in Cr-TRP1 responsible for PIP2 binding in mammalian TRPs (box). Conservation values range between 0 and 9; the larger the number, the larger the conservation. Only conservation values above 5 are shown. Consensus_aa values are as follows: conserved amino acid residues are boldface and uppercase; h, hydrophobic; s, small; p, polar; o, alcohol; l, aliphatic; +, positively charged. Consensus_ss values are as follows: h, α-helix; e, β-strand.

Figure 4.

Functional Characterization of TRP1 Expressed in HEK-293T Cells. (A) Transfection of the TRP1-GFP clone in HEK-293T cells. The images present one cell in epifluorescence mode (left) and one cell in TIRF mode (right). Bar = 10 μm. (B) Current-voltage relations determined from voltage ramps from −100 to +140 mV obtained from HEK-293T cells transfected with TRP1 in pTracer vector (untagged). The curves were obtained under symmetrical Na⁺ conditions (box; concentrations in mM), and each represents the average of four independent experiments. Statistics are available in Supplemental Figure 4. (C) Dose-response curve showing the blocking effect of BCTC on the TRP1 conductance (IC₅₀ = 1.03 μM; n = 5). Error bars correspond to se. (D) Representative curves showing changes in the V_rev (E_rev) we used to determine the permeability ratio P_X/P_Na when the external solution was replaced. The table above the plot contains the calculated permeability ratios and the corresponding E_rev (n = 4).

Figure 5.

Voltage and Temperature Dependence of TRP1 Expressed in HEK-293T Cells. (A) Instantaneous current protocol for TRP1 (top left). The cell was exposed to a +160-mV depolarizing potential pulse and then pulsed to voltages between −160 and +160 mV in 20-mV increments (bottom left). Macroscopic tail currents were fitted with a single exponential function (dotted line) to obtain the instantaneous currents at zero time (bottom right). The box depicts the ionic concentrations (in mM) in the whole-cell configuration used. The blue and red arrows here and in (B) indicate the steady state and instantaneous currents, respectively. (B) The steady state curve (closed gray circles, blue arrow) rectifies while the instantaneous current-voltage curve (open orange circles, red arrow) follows an ohmic behavior. Closed black circles correspond to the uncorrected raw data, showing a discrete rectification of the ohmic behavior. (C) Representative traces obtained in transfected cells subjected to voltage steps from −160 to +200 mV in 20-mV increments. The holding potential was 0 mV, and the tail current was taken at −100 mV. Ionic concentrations (in mM) used in the whole-cell configuration are depicted in the box. (D) G-V curves were obtained by plotting peak tail currents at −100 mV in response to voltage-activated steady state currents as in Figure 5C (wild type, z = 1.09, V_0.5 = −39.3 mV [n = 9]; R759K, z = 0.8, V_0.5 = +148 mV [n = 5]). (E) Representative traces obtained from transiently transfected HEK-293T cells subjected to voltage steps from −100 to +160 mV in 20-mV increments at three different temperatures. (F) Current-voltage (I-V) relations were obtained by plotting steady state currents depicted in Figure 5E. Each represents the average of five independent experiments. (G) Current density at different temperatures taken from two fixed voltages (−100 and +100 mV). Error bars correspond to se.