Role of the Transient Receptor Potential Vanilloid 5 (TRPV5) Protein N Terminus in Channel Activity, Tetramerization, and Trafficking

Abstract

The epithelial Ca 2؉ channel transient receptor potential vanilloid 5 (TRPV5) constitutes the apical entry site for active Ca 2؉ reabsorption in the kidney. The TRPV5 channel is a member of the TRP family of cation channels, which are composed of four subunits together forming a central pore. Regulation of channel activity is tightly controlled by the intracellular N and C termini. The TRPV5 C terminus regulates channel activity by various mechanisms, but knowledge regarding the role of the N terminus remains scarce. To study the role of the N terminus in TRPV5 regulation, we generated different N-terminal deletion constructs. We found that deletion of the first 32 residues did not affect TRPV5-mediated 45 Ca 2؉ uptake, whereas deletion up to residue 34 and 75 abolished channel function. Immunocytochemistry demonstrated that these mutant channels were retained in the endoplasmic reticulum and in contrast to wildtype TRPV5 did not reach the Golgi apparatus, explaining the lack of complex glycosylation of the mutants. A limited amount of mutant channels escaped the endoplasmic reticulum and reached the plasma membrane, as shown by cell surface biotinylation. These channels did not internalize, explaining the reduced but significant amount of these mutant channels at the plasma membrane. Wild-type TRPV5 channels, despite significant plasma membrane internalization, showed higher plasma membrane levels compared with the mutant channels. The assembly into tetramers was not affected by the N-terminal deletions. Thus, the N-terminal residues 34 -75 are critical in the formation of a functional TRPV5 channel because the deletion mutants were present at the plasma membrane as tetramers, but lacked channel activity.The epithelial Ca 2ϩ channel TRPV5, 3 the gatekeeper of active Ca 2ϩ reabsorption in the kidney, is a member of the TRP superfamily. TRP channels are involved in many different biological processes ranging from sensory physiology, contributing to taste, olfaction, and vision to muscle proliferation and Ca 2ϩ and Mg 2ϩ homeostasis of the body (1, 2). Based on their sequence and structural homology, the mammalian TRP channel superfamily is divided into six subfamilies: TRPA (ankyrin), TRPC (canonical), TRPM (melastatin), TRPP (polycystin), TRPML (mucolipin), and TRPV (vanilloid). All TRP proteins have a common topology, including six transmembrane segments flanked by large cytoplasmic N-and C-terminal domains. They are suggested to assemble into tetramers, the hydrophobic loop between transmembrane 5 and 6 forming a putative core domain, ultimately leading to the formation of a cation-selective channel (1, 3).Despite this common topology, the structure and function of the cytosolic N-and C-terminal domains between the different TRP subfamilies are quite diverse (4). The N terminus of the TRPM subfamily holds four TRPM homology regions, which are essential for proper functioning of these channels (5-8).Highly conserved ankyrin repeats are present in the N termini of TRPC, TRPV, and TRPA channels. Ankyrin repeats a...

A TRPV4 Channel C-terminal Folding Recognition Domain Critical for Trafficking and Function

“…We propose that mutational effects are probably results of disruption of the conformation of this region required for the normal protein maturation process. Rescue of TRPV4 G849A/P851A channel function by lowering incubation temperature is fully consistent with this hypothesis, indicating that misfolding is probably the cause for ER retention and loss of function, as previously documented in other misfolding proteins (30,48,49). Given that segment deletions within a broad neighboring region of the C terminus were tolerable, results from the present study are best explained by a working model assuming that the region around residues of Gly 849 and Pro 851 is essential for correct folding of the TRPV4 channel.…”

Bidirectional Modulation of Thermal and Chemical Sensitivity of TRPM8 Channels by the Initial Region of the N-terminal Domain

“…The fully functional mM8-myc appears to be largely distributed in vesicles (Fig. 1C), with a pattern similar to that displayed by other TRP channels when they are overexpressed in HEK293 cells (51). In contrast, the ⌬41-57 deletion displays a substantial overlap with an ER marker (Fig.…”

“…Numerous studies indicate that ER export is limited primarily by quality control (47). For example, studies of several splice variants or TRP channel mutants reported their accumulation in the endoplasmic reticulum due to misfolding or compromised tetramerization rather than trafficking regulation mechanisms (22,43,51). In addition, it is possible that truncations that affect the quaternary structure also impair channel assembly and function.…”

“…Moreover, retention in the ER can occur even if the tetramerization of the channel is not prevented. For example, the ⌬1-38 TRPV5 mutant produces a nonfunctional channel that lacks mature glycosylation and is mainly located in the ER (51). In the study, the authors investigated the oligomerization state of the deletion using the perfluorooctanoic acid-PAGE technique and concluded that although formation of the tetramer occurs, the ⌬1-38 subunits were probably not folded correctly and were therefore retained in the ER (51).…”

New TRPM6 missense mutations linked to hypomagnesemia with secondary hypocalcemia

“…Subsequently, proteins present at the cell surface were biotinylated with 0.5 mg/ml sulfo-NHS-LC-LC-Biotin (Pierce, Rockford, IL, USA) in PBS-CM for 30 min at 4 1C as previously described. 25 Immunoblots were incubated with mouse anti-HA (Cell Signalling Technology, Danvers, MA, USA). Blots were incubated with sheep horseradish peroxidaseconjugated anti-mouse (Jackson ImmunoResearch, West Grove, PA, USA) and then visualized using the enhanced chemiluminescence system.…”