Abstract
In order to identify channels involved in membrane depolarization, Ulva compressa was incubated with agonists of TRP channels C5, A1 and V1, and the level of intracellular calcium was detected. Agonists of TRPC5, A1 and V1 induced increases in intracellular calcium at 4, 9, and 11 min of exposure, respectively, and antagonists of TRPC5, A1, and V1 corresponding to SKF-96365 (SKF), HC-030031 (HC), and capsazepin (CPZ), respectively, inhibited calcium increases indicating that functional TRPs exist in U. compressa. In addition, copper excess induced increases in intracellular calcium at 4, 9, and 12 min which were inhibited by SKF, HC, and CPZ, respectively, indicating that copper activate TRPC5, A1, and V1 channels. Moreover, copper-induced calcium increases were inhibited by EGTA, a non-permeable calcium chelating agent, but not by thapsigargin, an inhibitor of endoplasmic reticulum (ER) calcium ATPase, indicating that activation of TRPs leads to extracellular calcium entry. Furthermore, copper-induced calcium increases were not inhibited by W-7, an inhibitor of CaMs, and staurosporine, an inhibitor of CDPKs, indicating that extracellular calcium entry did not require activation of CaMs and CDPKs. In addition, copper induced membrane depolarization events at 4, 8, and 11 min and these events were inhibited by SKF, HC, CPZ, and bathocuproine, a specific copper chelating agent, indicating that copper entry through TRP channels leads to membrane depolarization. Moreover, membrane depolarization events were inhibited by W-7 and staurosporine, indicating that activation of CaMs and CDPKs is required to allow copper entry through TRPs. Interestingly, copper-induced calcium increases and depolarization events were light-dependent and were inhibited by DCMU, an inhibitor of photosystem II, and ATP-γ-S, a non-hydrolizable analog of ATP, suggesting that ATP derived from photosynthesis is required to activate TRPs. Thus, light-dependent copper-induced activation TRPC5, A1 and V1 promotes extracellular calcium entry leading to activation of CaMs and CDPKs which, in turn, promotes copper entry through TRP channels and membrane depolarization.
Highlights
Transient Receptor Potential (TRP) channels are ionotropic cation channels involved in the perception of local environment, present in organisms such as humans, mammals, flies, nematodes, and yeast (Nilius and Owsianik, 2011)
Compressa In order to analyze the existence of functional TRP channels in U. compressa, the alga was loaded with the fluorophor Fluo-3AM and cultivated in control condition or with 1 m M of the specific agonists of TRPC5, La+3, Gd+3, and sphingosine-1-P, for 15 min and the increase of intracellular calcium was detected
Functional TRPC5 exists in the plasma membrane of U. compressa and its activation leads to intracellular calcium increase at 4 min of exposure
Summary
Transient Receptor Potential (TRP) channels are ionotropic cation channels involved in the perception of local environment, present in organisms such as humans, mammals, flies, nematodes, and yeast (Nilius and Owsianik, 2011). TRPs are activated by stimuli such as: injury, temperature, pH, osmolarity, and pressure; by pungent compounds extracted from plants such as vanillin, capsaicin, cinnamaldehyde, allylisothiocyanate, camphor, menthol, cannabinoids, among others; and by inflammatory and signaling mediators such as prostaglandines, cytokines, inositol phosphates, diacylglycerol, etc. The structure of TRPs is reminiscent to Voltage-Dependent Calcium Channels (VDCC), comprising six putative trans-membrane domains (S1 to S6), free intracellular N and C terminal domains, and the pore-forming loop located between S5 and S6. TRPC and TRPV family members contain domain 3 to 4 ankyrin repeats in the N terminal whereas TRPA1 contain 14 ankyrin motifs and this sequence is absent in other TRP family members (Owsianik et al, 2006). It has been shown that the ankyrin repeats allow protein-protein interactions and binding of ligands such as ATP and calmodulins (CaMs) (Gaudet, 2008)
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