TheDrosophilaTRP and TRPL are assembled as homomultimeric channels in vivo

Abstract

SummaryFamily members of the cationic transient receptor potential (TRP) channels serve as sensors and transducers of environmental stimuli. The ability of different TRP channel isoforms of specific subfamilies to form heteromultimers and the structural requirements for channel assembly are still unresolved. Although heteromultimerization of different mammalian TRP channels within single subfamilies has been described, even within a subfamily (such as TRPC) not all members co-assemble with each other. In Drosophila photoreceptors two TRPC channels, TRP and TRP-like protein (TRPL) are expressed together in photoreceptors where they generate the light-induced current. The formation of functional TRP-TRPL heteromultimers in cell culture and in vitro has been reported. However, functional in vivo assays have shown that each channel functions independently of the other. Therefore, the issue of whether TRP and TRPL form heteromultimers in vivo is still unclear. In the present study we investigated the ability of TRP and TRPL to form heteromultimers, and the structural requirements for channel assembly, by studying assembly of GFP-tagged TRP and TRPL channels and chimeric TRP and TRPL channels, in vivo. Interaction studies of tagged and native channels as well as native and chimeric TRP-TRPL channels using coimmunoprecipitation, immunocytochemistry and electrophysiology, critically tested the ability of TRP and TRPL to interact. We found that TRP and TRPL assemble exclusively as homomultimeric channels in their native environment. The above analyses revealed that the transmembrane regions of TRP and TRPL do not determine assemble specificity of these channels. However, the C-terminal regions of both TRP and TRPL predominantly specify the assembly of homomeric TRP and TRPL channels.

In vivo identification of Drosophila rhodopsin interaction partners by biotin proximity labeling

“…TRPL was also suggested to be part of the signalplex via heteromultimerization with TRP 28 . However, while the assembly into a signaling complex of TRP, PLCβ and ePKC by INAD is generally accepted, the binding of significant amounts of Rh1 to the INAD signaling complex as well as formation of TRP/TRPL heteromultimers is not supported by other studies 29 – 31 . The scaffolding protein INAD and the protein kinase C (ePKC), were also detected in the Rh1::TbID wt sample, albeit at a relatively low abundance.…”

Immunocytochemical Labeling of Rhabdomeric Proteins in Drosophila Photoreceptor Cells Is Compromised by a Light-dependent Technical Artifact

“…3C). 33 We performed electroretinogram recordings of eyes from TRPL::SNAP expressing flies in various genetic backgrounds using a combination of orange and blue stimuli. According to our data, TRPL::SNAP forms functional TRPL channels and the tag has no obvious effect on amplitude, on-/off-transients, inactivation, deactivation, or prolonged depolarization afterpotential when compared to wild type flies (Fig.…”

The latency of the light response is modulated by the phosphorylation state of Drosophila TRP at a specific site

“…To rule out possible effects of the transgenic expression of TRP per se, we performed several control experiments. In principle, an abnormal light response may be associated with impaired function of TRP channels due to effects on channel gating, large reduction in channel protein levels, 3 failure to interact with the INAD scaffold protein, 4 failure to assemble and form homotetramers, 5 or mislocalization of the TRP channel outside the signaling compartment, the rhabdomere. 5 Accordingly, in the previous study, we performed the above controls for the transgenic flies on a single null trp P343 background.…”

“…In principle, an abnormal light response may be associated with impaired function of TRP channels due to effects on channel gating, large reduction in channel protein levels, 3 failure to interact with the INAD scaffold protein, 4 failure to assemble and form homotetramers, 5 or mislocalization of the TRP channel outside the signaling compartment, the rhabdomere. 5 Accordingly, in the previous study, we performed the above controls for the transgenic flies on a single null trp P343 background. In the present study, we repeated the necessary controls on transgenes expressed on double null trpl 302 ;trp P343 background.…”