Regulation of Drosophila transient receptor potential-like (TrpL) channels by phospholipase C-dependent mechanisms

Stimulation of isolated Drosophila photoreceptor cells by light causes an increase in membrane conductance that requires phospholipase C (PLC) (Bloomquist et al. 1988) and reflects the activity of both the transient receptor potential channel (Trp) and the Trp-like channel (TrpL) (Niemeyer et al. 1996). It was originally thought that light-activated channels were localized in the photoreceptor cell to the base of the microvilli of the rhabdomere (Pollock et al. 1995). The close proximity of the Trp channels to the submicrovilli cisternae, i.e. the internal Ca2+ storage compartment, led to the suggestion that an inositol-1,4,5-trisphosphate (Ins(1,4,5)P3)-induced Ca2+ release activated Trp and TrpL via a capacitative Ca2+ entry (CCE) mechanism analogous to receptor-mediated activation of Ca2+ entry in mammalian non-excitable cells (Hardie & Minke, 1993). Subsequent studies, however, have shown that the microvilli, which are 1-2 μm long and 50 nm wide, have Trp, TrpL and PLC located along their entire length (Niemeyer et al. 1996; Chevesich et al. 1997; Tsunoda et al. 1997), placing most of the ion channels at a considerable distance from the nearest internal Ca2+ store. Furthermore, recent studies have shown that the Ins(1,4,5)P3 receptor is not required for activation of Trp or TrpL by light (Acharya et al. 1997; Raghu et al. 2000). Heterologous expression of TrpL gives rise to Ca2+-permeable, non-selective cation currents that are unaffected by depletion of the internal Ca2+ stores by thapsigargin, but are activated following stimulation of membrane receptors linked to phosphoinositide turnover (Vaca et al. 1994; Hu & Schilling, 1995; Harteneck et al. 1995; Dong et al. 1995; Obukhov et al. 1996; Kunze et al. 1997). Trp and TrpL were the first cloned members of what now appears to be a large family of ion channels found in a variety of mammalian non-excitable and excitable tissues. To date, seven mammalian Trp homologues have been identified (Wes et al. 1995; Zhu et al. 1995, 1996; Philipp et al. 1996; Boulay et al. 1997; Okada et al. 1998, 1999; Liman et al. 1999). Heterologous expression studies have shown that receptor-mediated changes in [Ca2+]i and whole-cell membrane currents are increased in cells expressing mammalian Trp3, Trp4, Trp5, Trp6 and Trp7 (Zitt et al. 1997, 1998; Boulay et al. 1997; Okada et al. 1998, 1999; Hurst et al. 1998; Hofmann et al. 1999; Schaefer et al. 2000). As with the Drosophila homologues, regulation of the mammalian Trp channels by receptor stimulation appears to require phosphoinositide hydrolysis since this regulation can be blocked by {"type":"entrez-nucleotide","attrs":{"text":"U73122","term_id":"4098075","term_text":"U73122"}}U73122, a specific inhibitor of PLC (Zhu et al. 1998; Okada et al. 1999; Kamouchi et al. 1999; Schaefer et al. 2000). The link between PLC and channel activation, however, remains obscure. Although several studies demonstrate that mammalian Trp channels are not activated by depletion of internal Ca2+ stores, at least when heterologously expressed (Zitt et al. 1997; Boulay et al. 1997; Zhu et al. 1998; Sinkins et al. 1998; Okada et al. 1998, 1999; Hurst et al. 1998; Hofmann et al. 1999; Kamouchi et al. 1999; Schaefer et al. 2000), other studies have shown that Trp4 and Trp5 may function as store-operated channels (Philipp et al. 1996, 1998; Warnat et al. 1999). Furthermore, studies using antisense oligonucleotides directed against trp1 and/or trp3 suggest they may play a role in CCE in HEK cells (Wu et al. 2000) and rat submandibular epithelial cells (Liu et al. 2000). Trp3, Trp5 and Trp7 may also be activated by a rise in [Ca2+]iper se (Zitt et al. 1997; Okada et al. 1998, 1999; but see Kamouchi et al. 1999). One consistent result is that Drosophila TrpL and three mammalian Trp homologues, Trp3, Trp6 and Trp7, appear to be activated by exogenous application of diacylglycerols (DAGs) or poly-unsaturated fatty acids (PUFAs) (Okada et al. 1999; Hofmann et al. 1999; Chyb et al. 1999). However, it is not clear whether the effects of DAGs or PUFAs are through direct interaction with the channels or are indirect via some other membrane-associated mechanism(s). Furthermore, the physiological concentration of DAG or PUFA generated in the membrane following receptor stimulation and its relationship to pharmacological doses experienced by the channels during exogenous application of these agents remains unclear. The consequences of PLC activation may not be limited to the downstream effects of Ins(1,4,5)P3, DAG or Ca2+. Studies have shown that the activity of the cardiac Na+-Ca2+ exchanger and several K+ channels (Hilgemann & Ball, 1996; Zheng & Makielski, 1997; Huang et al. 1998; Sui et al. 1998) can be regulated in excised membrane patches by phosphatidylinositol-4,5-bisphosphate (PIP2), suggesting an important role for this minor membrane lipid in the direct regulation of membrane-associated ion transporters. Thus, the effect of PLC on the Trp family of channels may reflect, at least in part, alterations in PIP2 levels following activation of membrane receptors. Because of the complexity of the PLC/Ins(1,4,5)P3 signalling pathway, it is difficult to distinguish between channel regulatory or modulatory mechanisms and the initial PLC-dependent activation step. To address this issue, the experiments of the present study compare and contrast receptor/PLC-dependent regulation of TrpL channels measured using fura-2 fluorescence in intact cells with results obtained using patch-clamp techniques. With regard to the latter, TrpL channels were recorded in the cell-attached mode (intact cells), the whole-cell configuration (dialysed intact cell), and at the single channel level in excised inside-out membrane patches. For these studies, TrpL was expressed in Sf9 insect cells using baculovirus expression vectors. The Sf9 cell has several advantages over other expression systems for the study of ion channels involved in Ca2+ signalling. Importantly, this method of expression involves viral infection (rather than transfection). Host cell protein synthesis decreases immediately after infection and is essentially zero after 24 h (O’Reilly et al. 1992). Thus, problems associated with up-regulation or co-expression of endogenous proteins is minimized, whereas the probability of measuring homomeric channels is enhanced. Furthermore, Sf9 cells have the molecular machinery necessary for receptor-mediated Ca2+ signalling; responses essentially identical to those observed in mammalian cells are seen in Sf9 cells stimulated with either receptor agonists or thapsigargin (Hu et al. 1994). Lastly, Sf9 cells lack endogenous voltage-gated and Ca2+-activated channels, which potentially may complicate data analysis in other expression systems such as the Xenopus oocyte, CHO cells or HEK cells. The results of the present study demonstrate that receptor-mediated activation of TrpL (1) is dependent upon the activity of PLC, (2) is influenced by DAG and/or PUFAs, at least in part through a PLC-dependent mechanism, and (3) may reflect hydrolysis of PIP2 in addition to the generation of DAG. However, neither hydrolysis of PIP2 nor the release of DAG alone can fully account for receptor-induced activation of TrpL.