Influence of membrane cholesterol in the molecular evolution and functional regulation of TRPV4

https://doi.org/10.1016/j.bbrc.2014.11.077Get rights and content

Highlights

  • A single copy of TRPV4 is maintained in all vertebrates except frogs.

  • Molecular evolution of TRPV4 correlates with vertebrate evolution and cholesterol biosynthesis pathway.

  • TRPV4 contains highly conserved cholesterol-binding motif sequences.

  • TRPV4 interacts with cholesterol, its metabolic precursors and derivatives.

  • TRPV4 mobility in membrane is cholesterol dependent.

Abstract

TRPV4 is involved in several physiological and sensory functions as well as with several diseases and genetic disorders, though the molecular mechanisms for these are unclear. In this work we have analyzed molecular evolution and structure–function relationship of TRPV4 using sequences from different species. TRPV4 has evolved during early vertebrate origin (450 million years). Synteny analysis confirms that TRPV4 has coevolved with two enzymes involved in sterol biosynthesis, namely MVK and GLTP. Cholesterol-recognizing motifs are present within highly conserved TM4–Loop4–TM5 region of TRPV4. TRPV4 is present in lipid raft where it co-localizes with Caveolin1 and Filipin. TM4–Loop4–TM5 region as well as Loop4 alone can physically interact with cholesterol, its precursor mevalonate and derivatives such as stigmasterol and aldosterone. Mobility of TRPV4-GFP depends on membrane cholesterol level. Molecular evolution of TRPV4 shared striking parallelism with the cholesterol bio-synthesis pathways at the genetic, molecular and metabolic levels. We conclude that interaction with sterols and cholesterol-dependent membrane dynamics have influence on TRPV4 function. These results may have importance on TRPV4-medaited cellular functions and pathophysiology.

Introduction

TRPs represent a group of non-selective channels that are permeable to different cations. Among all, TRPV4 is unique as it is activated by several physical and chemical stimuli such as temperature, mechanical pressure, osmolarity, infrared, and compounds like vanilloids, 4αPDD, Apigenin, dimethylallyl pyrophosphate and PUFAs [1], [2], [3], [4]. A common aspect of TRPV4-specific agonists is their high hydrophobicity, suggesting that these compounds primarily act on the transmembrane regions. Membrane deformation by stretch too causes rapid activation of TRPV4 [5]. All these suggest that TRPV4 function is dependent on the biochemical composition, structure and by the biophysical nature of the membrane. TRPV4 is specifically present in the cholesterol-enriched detergent-resistant membrane fraction (lipid raft) [6]. At the molecular level, TRPV4 forms signaling complex, which includes membranous components and sub-membranous cytoskeleton [7]. Such complexes are critical for proper function and regulation.

TRPV4 is present in several animals and is involved in detection of different physical and chemical stimuli. The primary function of TRPV4 remains conserved across different species. For example, hTRPV4 (but not hTRPV1) can rescue the defects in transduction of osmotic and mechanical stimuli in osm-9 (but not ocr-2) mutants in Caenorhabditis elegans and low sequence similarities between hTRPV4 with osm-9 suggests that smaller regions are sufficient to perform key tasks [8], [9]. TRPV4-mediated sensory functions significantly contribute to the natural selection in specific habitats which favor organism’s survival as fittest. Therefore, TRPV4 might have influence on the adaptation, speciation and evolution of different species, especially in response to certain selection pressure where the above mentioned sensory processes are involved. Indeed, TRPV4 can regulate certain behaviors that are linked with adaptation [8]. In this work we tested the molecular evolution of TRPV4 per se. TRPV4 physically interacts with sterols and some of its functions are dependent on availability of the cholesterol. We demonstrate that molecular evolution of TRPV4 has been influenced by the cholesterol-biosynthesis pathway, an unexpected finding that may also explain the molecular mechanism of TRPV4.

Section snippets

Sequence retrieval and protein sequence analysis

All TRPV4 sequences were retrieved either from Ensembl or NCBI database (provided in Tab:S1). These protein sequences were aligned by using MUSCLE alignment tool with its default parameters within MEGA5 software suite. Histone H4 and Cytochrome C sequences were retrieved from ENSEMBL and NCBI databases as described previously [10].

Fragmentation of TRPV4 into different domains and motifs

Conservation of different domains and motifs of TRPV4 were analyzed separately (Table 1). In all cases, we used the hTRPV4 sequence (ENSP00000261740) as template.

TRPV4 has evolved during Silurian era

We reconstructed the phylogenetic history of vertebrate TRPV4 using Bayesian phylogenetic method (Fig. 1A). TRPV4-mediated functions in C. elegans can be rescued by hTRPV4, suggesting that certain functional features of TRPV4 are conserved throughout the evolution [16]. However, hTRPV4 protein shares less identity (∼20%) and homology (∼36%) with Osm9 (TRPV4 homologue in C. elegans). Similarly, NAN (homologue from Drosophila) also shares less homology with hTRPV4. Invertebrate homologues show

Discussion

We have combined protein sequence, genomic data, structural information, biochemical and cell-biological experimental results, which demonstrate that TRPV4 is a highly conserved protein and has evolved ∼450 MY before when vertebrate evolution started. TRPV4 physically interacts with cholesterol, its precursor molecule mevalonate and some of its derivatives through the Loop4 and nearby helices, a region of TRPV4 which is 100% identical in all vertebrates. In agreement with this physical

Acknowledgments

We acknowledge Dr. P.V. Alone for GST vector. Funding from NISER and DBT – India (BT-BRB-TF-2-2011) are acknowledged.

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