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A novel ion channel formed by interaction of TRPML3 with TRPV5.

Guo Z, Grimm C, Becker L, Ricci AJ, Heller S - PLoS ONE (2013)

Bottom Line: It has pharmacological similarity with TRPML3 and requires functional TRPML3 as well as functional TRPV5.Single channel analyses revealed that TRPML3 and TRPV5 heteromers have different features than the respective homomers, and furthermore, that they occur in potentially distinct stoichiometric configurations.Based on overlapping expression of TRPML3 and TRPV5 in the kidney and the inner ear, we propose that TRPML3 and TRPV5 heteromers could have a biological function in these organs.

View Article: PubMed Central - PubMed

Affiliation: Department of Otolaryngology-HNS, Stanford University School of Medicine, Palo Alto, California, United States of America.

ABSTRACT
TRPML3 and TRPV5 are members of the mucolipin (TRPML) and TRPV subfamilies of transient receptor potential (TRP) cation channels. Based on sequence similarities of the pore forming regions and on structure-function evidence, we hypothesized that the pore forming domains of TRPML and TRPV5/TRPV6 channels have similarities that indicate possible functional interactions between these TRP channel subfamilies. Here we show that TRPML3 and TRPV5 associate to form a novel heteromeric ion channel. This novel conductance is detectable under conditions that do not activate either TRPML3 or TRPV5. It has pharmacological similarity with TRPML3 and requires functional TRPML3 as well as functional TRPV5. Single channel analyses revealed that TRPML3 and TRPV5 heteromers have different features than the respective homomers, and furthermore, that they occur in potentially distinct stoichiometric configurations. Based on overlapping expression of TRPML3 and TRPV5 in the kidney and the inner ear, we propose that TRPML3 and TRPV5 heteromers could have a biological function in these organs.

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Interaction between TRPML channels and TRPV5/6.(A) Phylogenetic tree, based on full length sequence comparisons of human TRP channel proteins. (B) Phylogenetic tree, based on sequence comparisons of the pore forming domains of human TRP channel proteins. (C) Fluorescence energy resonance transfer (FRET) experiments showing representative FRET efficiencies among TRPML3, TRPV5, and TRPV6 channels. FRET efficiencies were determined by measuring the recovery of CFP fluorescence during YFP photobleaching. (D) Average FRET efficiencies reported as mean values± SEM (n = parenthesized). Shown are efficiencies for TRPML, TRPV5, and TRPV6 channel homo- and heteromers, as well as PKD2, TRPC6, TRPV2, and TRPA1 as controls. YFP and CFP indicate the fluorescent tag, which was carboxyl-terminal in all cases. Control indicates a pcDNA3.1-based expression vector for the corresponding fluorescent protein (YFP in the example shown). ***p<0.0001 and **p<0.001, Student’s t-test, unpaired, comparison with TRPC6/TRPML3 coexpression as negative control.
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pone-0058174-g001: Interaction between TRPML channels and TRPV5/6.(A) Phylogenetic tree, based on full length sequence comparisons of human TRP channel proteins. (B) Phylogenetic tree, based on sequence comparisons of the pore forming domains of human TRP channel proteins. (C) Fluorescence energy resonance transfer (FRET) experiments showing representative FRET efficiencies among TRPML3, TRPV5, and TRPV6 channels. FRET efficiencies were determined by measuring the recovery of CFP fluorescence during YFP photobleaching. (D) Average FRET efficiencies reported as mean values± SEM (n = parenthesized). Shown are efficiencies for TRPML, TRPV5, and TRPV6 channel homo- and heteromers, as well as PKD2, TRPC6, TRPV2, and TRPA1 as controls. YFP and CFP indicate the fluorescent tag, which was carboxyl-terminal in all cases. Control indicates a pcDNA3.1-based expression vector for the corresponding fluorescent protein (YFP in the example shown). ***p<0.0001 and **p<0.001, Student’s t-test, unpaired, comparison with TRPC6/TRPML3 coexpression as negative control.

Mentions: Functional analyses of the Va mutant isoform of TRPML3 revealed susceptibility for helix-breaking mutations in the fifth transmembrane-spanning domain (TM5), which results in constitutive channel activity [1], [8]–[10]. The propensity for pore property alteration by helix-breaking mutations is a common feature of TRPML channels and surprisingly also for the two TRPV family members TRPV5 and TRPV6 [8], [17]. Based on these previous findings, we became intrigued by a potential similitude of TRPML with TRPV5 and TRPV6 channels, particularly with respect to the pore region. At first glance, based on overall protein sequence homology, TRPML and TRPV channels are quite dissimilar and form discrete subfamilies (Fig. 1A). When we conducted a sequence comparison that was restricted to the pore regions including sequences from the start of TM5 to the end of TM6, however, we found that TRPV5 and TRPV6 formed a distinct subfamily that was phylogenetically closer to the TRPML channel subfamily than to the TRPV1-4 subfamily cluster (Fig. 1B). A closer look at the individual sequences revealed that the similarity of TRPML channels with TRPV5 and TRPV6 is greatest for the stretch of amino acids comprising TM5 (Fig. S1). This sequence similarity combined with the previous observations of susceptibility for helix-breaking mutations raised our curiosity whether TRPML and TRPV5/6 channels might be able to interact physically. We investigated whether co-expressed TRPML and TRPV5/6 channels are in close enough association to display Förster/fluorescence resonance energy transfer (FRET). Homomeric pairs of cyan fluorescent protein (CFP) and yellow fluorescent protein (YFP)-tagged TRPML channels as well as TRPV5 and TRPV6 showed FRET efficiencies between 14–22% (Fig. 1C,D), indicative of close proximity of the two fluorophores. Previously known heteromeric channel pairs such as TRPML1/TRPML3, TRPML2/TRPML3, and TRPV5/TRPV6, as well as homomeric TRPML1, TRPML3, TRPV5, TRPV6, PKD2, TRPC6, TRPV2, and TRPA1 channels displayed equally pronounced FRET efficiencies ranging from 10–20%. All combinations of TRPML1 and TRPML3 with TRPV5 and TRPV6 also exhibited FRET ranging from 12–17% efficiency. Negative controls including pairs of TRPML3 with PKD2, TRPC6, TRPV2, and TRPA1 or YFP alone showed no FRET, indicated by thresholds below 5%. These results suggested that TRPML3 is able to closely associate with TRPV5 and TRPV6.


A novel ion channel formed by interaction of TRPML3 with TRPV5.

Guo Z, Grimm C, Becker L, Ricci AJ, Heller S - PLoS ONE (2013)

Interaction between TRPML channels and TRPV5/6.(A) Phylogenetic tree, based on full length sequence comparisons of human TRP channel proteins. (B) Phylogenetic tree, based on sequence comparisons of the pore forming domains of human TRP channel proteins. (C) Fluorescence energy resonance transfer (FRET) experiments showing representative FRET efficiencies among TRPML3, TRPV5, and TRPV6 channels. FRET efficiencies were determined by measuring the recovery of CFP fluorescence during YFP photobleaching. (D) Average FRET efficiencies reported as mean values± SEM (n = parenthesized). Shown are efficiencies for TRPML, TRPV5, and TRPV6 channel homo- and heteromers, as well as PKD2, TRPC6, TRPV2, and TRPA1 as controls. YFP and CFP indicate the fluorescent tag, which was carboxyl-terminal in all cases. Control indicates a pcDNA3.1-based expression vector for the corresponding fluorescent protein (YFP in the example shown). ***p<0.0001 and **p<0.001, Student’s t-test, unpaired, comparison with TRPC6/TRPML3 coexpression as negative control.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3585263&req=5

pone-0058174-g001: Interaction between TRPML channels and TRPV5/6.(A) Phylogenetic tree, based on full length sequence comparisons of human TRP channel proteins. (B) Phylogenetic tree, based on sequence comparisons of the pore forming domains of human TRP channel proteins. (C) Fluorescence energy resonance transfer (FRET) experiments showing representative FRET efficiencies among TRPML3, TRPV5, and TRPV6 channels. FRET efficiencies were determined by measuring the recovery of CFP fluorescence during YFP photobleaching. (D) Average FRET efficiencies reported as mean values± SEM (n = parenthesized). Shown are efficiencies for TRPML, TRPV5, and TRPV6 channel homo- and heteromers, as well as PKD2, TRPC6, TRPV2, and TRPA1 as controls. YFP and CFP indicate the fluorescent tag, which was carboxyl-terminal in all cases. Control indicates a pcDNA3.1-based expression vector for the corresponding fluorescent protein (YFP in the example shown). ***p<0.0001 and **p<0.001, Student’s t-test, unpaired, comparison with TRPC6/TRPML3 coexpression as negative control.
Mentions: Functional analyses of the Va mutant isoform of TRPML3 revealed susceptibility for helix-breaking mutations in the fifth transmembrane-spanning domain (TM5), which results in constitutive channel activity [1], [8]–[10]. The propensity for pore property alteration by helix-breaking mutations is a common feature of TRPML channels and surprisingly also for the two TRPV family members TRPV5 and TRPV6 [8], [17]. Based on these previous findings, we became intrigued by a potential similitude of TRPML with TRPV5 and TRPV6 channels, particularly with respect to the pore region. At first glance, based on overall protein sequence homology, TRPML and TRPV channels are quite dissimilar and form discrete subfamilies (Fig. 1A). When we conducted a sequence comparison that was restricted to the pore regions including sequences from the start of TM5 to the end of TM6, however, we found that TRPV5 and TRPV6 formed a distinct subfamily that was phylogenetically closer to the TRPML channel subfamily than to the TRPV1-4 subfamily cluster (Fig. 1B). A closer look at the individual sequences revealed that the similarity of TRPML channels with TRPV5 and TRPV6 is greatest for the stretch of amino acids comprising TM5 (Fig. S1). This sequence similarity combined with the previous observations of susceptibility for helix-breaking mutations raised our curiosity whether TRPML and TRPV5/6 channels might be able to interact physically. We investigated whether co-expressed TRPML and TRPV5/6 channels are in close enough association to display Förster/fluorescence resonance energy transfer (FRET). Homomeric pairs of cyan fluorescent protein (CFP) and yellow fluorescent protein (YFP)-tagged TRPML channels as well as TRPV5 and TRPV6 showed FRET efficiencies between 14–22% (Fig. 1C,D), indicative of close proximity of the two fluorophores. Previously known heteromeric channel pairs such as TRPML1/TRPML3, TRPML2/TRPML3, and TRPV5/TRPV6, as well as homomeric TRPML1, TRPML3, TRPV5, TRPV6, PKD2, TRPC6, TRPV2, and TRPA1 channels displayed equally pronounced FRET efficiencies ranging from 10–20%. All combinations of TRPML1 and TRPML3 with TRPV5 and TRPV6 also exhibited FRET ranging from 12–17% efficiency. Negative controls including pairs of TRPML3 with PKD2, TRPC6, TRPV2, and TRPA1 or YFP alone showed no FRET, indicated by thresholds below 5%. These results suggested that TRPML3 is able to closely associate with TRPV5 and TRPV6.

Bottom Line: It has pharmacological similarity with TRPML3 and requires functional TRPML3 as well as functional TRPV5.Single channel analyses revealed that TRPML3 and TRPV5 heteromers have different features than the respective homomers, and furthermore, that they occur in potentially distinct stoichiometric configurations.Based on overlapping expression of TRPML3 and TRPV5 in the kidney and the inner ear, we propose that TRPML3 and TRPV5 heteromers could have a biological function in these organs.

View Article: PubMed Central - PubMed

Affiliation: Department of Otolaryngology-HNS, Stanford University School of Medicine, Palo Alto, California, United States of America.

ABSTRACT
TRPML3 and TRPV5 are members of the mucolipin (TRPML) and TRPV subfamilies of transient receptor potential (TRP) cation channels. Based on sequence similarities of the pore forming regions and on structure-function evidence, we hypothesized that the pore forming domains of TRPML and TRPV5/TRPV6 channels have similarities that indicate possible functional interactions between these TRP channel subfamilies. Here we show that TRPML3 and TRPV5 associate to form a novel heteromeric ion channel. This novel conductance is detectable under conditions that do not activate either TRPML3 or TRPV5. It has pharmacological similarity with TRPML3 and requires functional TRPML3 as well as functional TRPV5. Single channel analyses revealed that TRPML3 and TRPV5 heteromers have different features than the respective homomers, and furthermore, that they occur in potentially distinct stoichiometric configurations. Based on overlapping expression of TRPML3 and TRPV5 in the kidney and the inner ear, we propose that TRPML3 and TRPV5 heteromers could have a biological function in these organs.

Show MeSH
Related in: MedlinePlus