TRP: Cell Lines Expressing a DNA Construct Expressing Mutant TRPV4 (UCLA Case No. 2021-361)

UCLA researchers from the Department of Molecular, Cell, and Developmental Biology have developed stable cell lines expressing wild-type or disease-associated mutant TRPV4 channels, providing a powerful platform for mechanistic and drug discovery studies.

BACKGROUND: TRPV4 is a non-selective cation channel that regulates calcium influx in response to diverse stimuli, including mechanical, osmotic, and thermal fluctuations. TRPV4 is expressed in multiple tissues and cell types, and mutations in TRPV4 have been implicated in a broad spectrum of human diseases, ranging from skeletal dysplasias and arthropathies to peripheral neuropathies. However, in vitro models for studying TRPV4 function, regulation, and disease-causing mutations remain limited. Thus, cell-based systems that stably express both wild-type (WT) and clinically relevant mutant TRPV4 variants are critical for investigating pathogenic mechanisms and screening for therapeutic modulators.

INNOVATION: Researchers at UCLA led by Dr. Daniel Cohn have developed four stable cell lines for investigation of TPV4 function and pathophysiology. One cell line expresses two copies of wild-type (WT) TRPV4, while the other three express one WT copy and one distinct mutant copy of TRPV4, each corresponding to a disease-associated variant. In all constructs, the paired TRPV4 genes are expressed as a single transcript from a doxycycline-inducible promoter, allowing temporal control over expression. Each TRPV4 protein is tagged with FLAG and V5 tags to enable versatile detection and purification. Additionally, a P2A peptide sequence separates the two TRPV4 proteins post-translation, ensuring proper individual folding and function. This unique design enables precise mechanistic studies that compare WT and mutant TRPV4 activity, as well as therapeutic screening.

POTENTIAL APPLICATIONS:

• Functional studies of WT and mutant TRPV4 physiology and pathophysiology
• Mechanistic studies of disease-associated TRPV4 variants
• Screening and validation of TRPV4-targeting therapies
• Development of mutation-specific therapies

ADVANTAGES:

• Cell lines express clinically relevant mutations
• Cell lines with matched wild-type and mutant controls enable direct comparisons
• Doxycycline-inducible expression allows temporal regulation
• Dual epitope tagging facilitates detection/purification

DEVELOPMENT-TO-DATE: UCLA researchers have developed four wild-type and mutant TRPV4 expressing cell lines.

Related Papers (from the inventors only):

1. Weinstein MM, Kang T, Lachman RS, Bamshad M, Nickerson DA, Krakow D, Cohn DH. Somatic mosaicism for a lethal TRPV4 mutation results in non-lethal metatropic dysplasia. Am J Med Genet A. 2016 Dec;170(12):3298-3302. doi: 10.1002/ajmg.a.37942. Epub 2016 Aug 17. PMID: 27530454; PMCID: PMC5115972.
2. Rock MJ, Prenen J, Funari VA, Funari TL, Merriman B, Nelson SF, Lachman RS, Wilcox WR, Reyno S, Quadrelli R, Vaglio A, Owsianik G, Janssens A, Voets T, Ikegawa S, Nagai T, Rimoin DL, Nilius B, Cohn DH. Gain-of-function mutations in TRPV4 cause autosomal dominant brachyolmia. Nat Genet. 2008 Aug;40(8):999-1003. doi: 10.1038/ng.166. Epub 2008 Jun 29. PMID: 18587396; PMCID: PMC3525077.
3. Krakow D, Vriens J, Camacho N, Luong P, Deixler H, Funari TL, Bacino CA, Irons MB, Holm IA, Sadler L, Okenfuss EB, Janssens A, Voets T, Rimoin DL, Lachman RS, Nilius B, Cohn DH. Mutations in the gene encoding the calcium-permeable ion channel TRPV4 produce spondylometaphyseal dysplasia, Kozlowski type and metatropic dysplasia. Am J Hum Genet. 2009 Mar;84(3):307-15. doi: 10.1016/j.ajhg.2009.01.021. Epub 2009 Feb 19. PMID: 19232556; PMCID: PMC2667978.

Keywords: TRPV4, cell lines, skeletal dysplasias, doxycycline-inducible, ion channel, DNA constructs, cation channel, P2A, FLAG tag, V5 tag