Theses Doctoral

Functional Characterization of the Mammalian TRPV4 Channel: Yeast Screen Reveals Gain-of-Function Mutations

Doyle, Christina

Transient receptor potential (TRP) channels are a class of six-transmembrane (6-TM) cation-permeable channels that mediate flux of calcium and sodium into cells, leading to depolarization as well as activation of calcium-mediated second-messenger signaling pathways. The TRP channel family is large and diverse in terms of tissue expression, mechanism, and function; therefore, sub-classification is primarily through amino acid homology. A general role has emerged for TRP channels, though, in the processing of sensory stimuli at both the cellular and organismal level. The goal of this study was to perform mutagenesis screens of mammalian TRP channels to reveal key structural determinants of channel activity (such as gating, permeation, and selectivity). We screened for gain-of-function alleles of TRP channels by their ability to rescue growth deficiency of a strain of the yeast Saccharomyces cerevisiae caused by lack of ion efflux. Channels were further characterized through electrophysiological analysis of their activity when heterologously expressed in Xenopus laevis oocytes. Of the subset of mammalian TRP channels tested, only wild type TRPV4 rescued the ability of the yeast strain trk1Δ trk2Δ to grow on low potassium media. The TRPV4 channel is important in thermosensitive, osmosensitive, and mechanosensitive processes; recently, mutations of TRPV4 have been linked to human skeletal and neurodegenerative disorders. We obtained a loss-of-function variant of TRPV4 containing the substitutions K70E (N-terminal tail) and M605T (intracellular linker between transmembrane helices S4 and S5) that failed to rescue low potassium growth of trk1Δ trk2Δ. Therefore, we screened for compensatory mutations that would restore the ability of the V4-K70E/M605T channel to rescue the yeast growth phenotype. Five gain-of-function clones were isolated, containing a total of seven mutations: three substitutions in the N-terminal tail (R151W, P152S, L154F), one substitution in the pore-lining S5 transmembrane helix (M625I), one substitution in the C-terminal tail (H787Y), and two truncations of the C-terminal tail (N789Δ and Q790Δ). Each of these mutations was assayed, in both the variant V4-K70E/M605T and the wild type TRPV4 background, for effect on rescue of trk1Δ trk2Δ yeast low-potassium growth, as well as degree of salt sensitivity conferred on wild type yeast. We also performed two-electrode voltage-clamp (TEVC) recordings of the mutant channels expressed in Xenopus oocytes, obtaining preliminary data on the ability of the mutations to restore a calcium-activated sodium current to V4-K70E/M605T that was present in wild type TRPV4. Given the known importance of the S5 helix in gating, the mutation M625I most likely has an effect on gating of the intracellular pore. This mutation showed strong rescue of low potassium growth and salt sensitivity in yeast, and preliminary data showed strong rescue of calcium-activated current in oocytes. An autoinhibitory channel structure is formed by binding of the C-terminal calmodulin-binding domain to a portion of the N-terminus, which is disrupted by the binding of calcium-calmodulin to the C-terminal domain. The point mutations we isolated in the N- and C-termini lie just outside these respective regions, leading us to believe that the gain-of-function phenotype could be due to disruption of this autoinhibitory structure. Although the C-terminal truncations were isolated with a gain-of-function phenotype in V4-K70E/M605T (rescue of low-potassium yeast growth), introduction of the truncations into wild type TRPV4 led to a loss-of-function phenotype: truncated channels no longer induced yeast salt sensitivity and exhibited no calcium-activated current in oocytes. This phenotype could be due to the loss of the calmodulin-binding domain, suggesting that the potentiation of channel activity by calcium involves mechanisms other than simply the disruption of the autoinhibitory domain. However, it is also possible that the phenotype is merely the result of reduced membrane expression: some studies indicate that truncation of the C-terminal tail leads to ER retention of the protein. Taken together, the results of our three assays provide insight into the mechanisms of TRP channel function. Combined with what is already known about these channel regions, we are able to draw conclusions as to the potential contribution of these residues on channel activity and add to the body of evidence regarding mechanisms of function of the TRPV4 channel.



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More About This Work

Academic Units
Biological Sciences
Thesis Advisors
Yang, Jian
Ph.D., Columbia University
Published Here
March 23, 2012