Human TRPV4 Channel Splice Variants Revealed a Key Role of Ankyrin Domains in Multimerization and Trafficking

Abstract

The TRPV4 cation channel exhibits a topology consisting of six predicted transmembrane domains (TM) with a putative pore loop between TM5 and TM6 and intracellular N- and C-tails, the former containing at least three ankyrin domains. Functional transient receptor potential (TRP) channels are supposed to result following the assembly of four subunits. However, the rules governing subunit assembly and protein domains implied in this process are only starting to emerge. The ankyrin, TM, and the C-tail domains have been identified as important determinants of the oligomerization process. We now describe the maturation and oligomerization of five splice variants of the TRPV4 channel. The already known TRPV4-A and TRPV4-B (Δ384–444) variants and the new TRPV4-C (Δ237–284), TRPV4-D (Δ27–61), and TRPV4-E (Δ237–284 and Δ384–444) variants. All alternative spliced variants involved deletions in the cytoplasmic N-terminal region, affecting (except for TRPV4-D) the ankyrin domains. Subcellular localization, fluorescence resonance energy transfer, co-immunoprecipitation, glycosylation profile, and functional analysis of these variants permitted us to group them into two classes: group I (TRPV4-A and TRPV4-D) and group II (TRPV4-B, TRPV4-C, and TRPV4-E). Group I, unlike group II variants, were correctly processed, homo- and heteromultimerized in the endoplasmic reticulum, and were targeted to the plasma membrane where they responded to typical TRPV4 stimuli. Our results suggest that: 1) TRPV4 biogenesis involves core glycosylation and oligomerization in the endoplasmic reticulum followed by transfer to the Golgi apparatus for subsequent maturation; 2) ankyrin domains are necessary for oligomerization of TRPV4; and 3) lack of TRPV4 oligomerization determines its accumulation in the endoplasmic reticulum. The TRPV4 cation channel exhibits a topology consisting of six predicted transmembrane domains (TM) with a putative pore loop between TM5 and TM6 and intracellular N- and C-tails, the former containing at least three ankyrin domains. Functional transient receptor potential (TRP) channels are supposed to result following the assembly of four subunits. However, the rules governing subunit assembly and protein domains implied in this process are only starting to emerge. The ankyrin, TM, and the C-tail domains have been identified as important determinants of the oligomerization process. We now describe the maturation and oligomerization of five splice variants of the TRPV4 channel. The already known TRPV4-A and TRPV4-B (Δ384–444) variants and the new TRPV4-C (Δ237–284), TRPV4-D (Δ27–61), and TRPV4-E (Δ237–284 and Δ384–444) variants. All alternative spliced variants involved deletions in the cytoplasmic N-terminal region, affecting (except for TRPV4-D) the ankyrin domains. Subcellular localization, fluorescence resonance energy transfer, co-immunoprecipitation, glycosylation profile, and functional analysis of these variants permitted us to group them into two classes: group I (TRPV4-A and TRPV4-D) and group II (TRPV4-B, TRPV4-C, and TRPV4-E). Group I, unlike group II variants, were correctly processed, homo- and heteromultimerized in the endoplasmic reticulum, and were targeted to the plasma membrane where they responded to typical TRPV4 stimuli. Our results suggest that: 1) TRPV4 biogenesis involves core glycosylation and oligomerization in the endoplasmic reticulum followed by transfer to the Golgi apparatus for subsequent maturation; 2) ankyrin domains are necessary for oligomerization of TRPV4; and 3) lack of TRPV4 oligomerization determines its accumulation in the endoplasmic reticulum. The non-selective cation channel TRPV4 is a member of the transient receptor potential (TRP) 3The abbreviations used are: TRPtransient receptor potential4α-PDD4α-phorbol 12,13-didecanoateANKankyrin domainERGICendoplasmic reticulum/Golgi intermediate compartmentFRETfluorescence resonance energy transferTMtransmembrane domainsERendoplasmic reticulumYFPyellow fluorescent proteinCYPcyan fluorescent proteinPNGase FN-glycosidase FEndo Hendoglycosidase H. 3The abbreviations used are: TRPtransient receptor potential4α-PDD4α-phorbol 12,13-didecanoateANKankyrin domainERGICendoplasmic reticulum/Golgi intermediate compartmentFRETfluorescence resonance energy transferTMtransmembrane domainsERendoplasmic reticulumYFPyellow fluorescent proteinCYPcyan fluorescent proteinPNGase FN-glycosidase FEndo Hendoglycosidase H. family of channels (1Montell C. Sci. STKE. 2005; 2005: re3PubMed Google Scholar). 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Pharmacol. 2003; 140: 413-421Crossref PubMed Scopus (61) Google Scholar), although this variant was not further investigated because of its unresponsiveness to hypotonic stimuli. transient receptor potential 4α-phorbol 12,13-didecanoate ankyrin domain endoplasmic reticulum/Golgi intermediate compartment fluorescence resonance energy transfer transmembrane domains endoplasmic reticulum yellow fluorescent protein cyan fluorescent protein N-glycosidase F endoglycosidase H. transient receptor potential 4α-phorbol 12,13-didecanoate ankyrin domain endoplasmic reticulum/Golgi intermediate compartment fluorescence resonance energy transfer transmembrane domains endoplasmic reticulum yellow fluorescent protein cyan fluorescent protein N-glycosidase F endoglycosidase H. The general topology of a TRP subunit consists of six predicted transmembrane domains (TM) with a putative pore loop between TM5 and TM6 and intracellular N- and C-terminal regions of variable length, the former containing multiple ankyrin (ANK) repeats in the TRPC, TRPA, TRPN, and TRPV subfamilies (1Montell C. Sci. STKE. 2005; 2005: re3PubMed Google Scholar). ANK repeats are modular protein interaction domains, each composed by ≈33 amino acids with a highly conserved secondary structure of helix turn helix that determines its interaction properties (20Mosavi L.K. Cammett T.J. Desrosiers D.C. Peng Z.Y. Protein Sci. 2004; 13: 1435-1448Crossref PubMed Scopus (655) Google Scholar). Functional TRP channels are supposed to result following the assembly of four TRP subunits. Although multimerization has been already demonstrated for various members of the TRP family (including TRPV4) (21Gillo B. Chorna I. Cohen H. Cook B. Manistersky I. Chorev M. Arnon A. Pollock J.A. Selinger Z. Minke B. 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Chem. 2004; 279: 54062-54068Abstract Full Text Full Text PDF PubMed Scopus (130) Google Scholar). were and into the and the of the C-terminal and of the used the The of each of the four domains and in TRPV4 channels was to the following and the Cell and human epithelial and cells were as (5Arniges M. Vazquez E. Fernandez-Fernandez J.M. Valverde M.A. J. Biol. Chem. 2004; 279: 54062-54068Abstract Full Text Full Text PDF PubMed Scopus (130) Google Scholar, 11Andrade Y.N. Fernandes J. Vazquez E. Fernandez-Fernandez J.M. Arniges M. Sanchez T.M. Villalon M. Valverde M.A. J. Cell Biol. 2005; 168: 869-874Crossref PubMed Scopus (163) Google Scholar). resonance energy transfer was carried out cells with of TRPV4 channel and of subunit cells were in and with of and of and functional were carried out cells with of the TRPV4 and of with the functional at a were to each with were with a TRPV4 (5Arniges M. Vazquez E. Fernandez-Fernandez J.M. Valverde M.A. J. Biol. 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Cell 2003; PubMed Scopus Google Scholar). was expressed as the of a of with were carried out following the N. Albrecht N. Harteneck C. Schultz G. Schaefer M. J. Cell Sci. 2005; 118: 917-928Crossref PubMed Scopus (243) Google for and for in the intracellular were in cells with for at followed by for at The of fluorescence following at and was as J.M. Nobles M. Currid A. Vazquez E. Valverde M.A. Am. J. Physiol. 2002; 283: C1705-C1714Crossref PubMed Scopus (84) Google Scholar). The pH The hypotonic pH was by from the and was with are expressed as of were from cells at from to in the A. E. B. J. Pflugers Arch. PubMed Scopus Google Scholar). The was used for and subsequent and were with a containing and pH The was pH were at and at were at of TRPV4-A and was as the of channels channel from and the of of TRPV4 from process identified variants of the TRPV4 channel in human epithelial of the cloned to the already TRPV4 and TRPV4 exon amino was as lacking exon although subsequent analysis of the structure of the TRPV4 identified a further exon the TRPV4 We also identified three new splice variants, TRPV4-C, and TRPV4-E variants affecting the cytoplasmic N-terminal TRPV4-C exon amino TRPV4-D a exon amino and TRPV4-E and amino is by a alternative lacking and for the TRPV4-D variant are by two and for and sites, All variants cloned the in for these five variants have been also from the human epithelial cell not Functional of the TRPV4 TRPV4-A channel to a of (13Nilius B. Vriens J. Prenen J. Droogmans G. Voets T. Am. J. Physiol. 2004; 286: C195-C205Crossref PubMed Scopus (365) Google Scholar). TRPV4 variants cells and intracellular in to three known of TRPV4 a cells were with the and in for TRPV4-A and TRPV4-D channels was for TRPV4-B TRPV4-C and TRPV4-E cells in the were between TRPV4-A and TRPV4-D variants. analysis of TRPV4-A and TRPV4-D cells 2) channel at was by the of to in TRPV4-A and from to in TRPV4-D in and were also between TRPV4-A and and TRPV4-D and not in cells with in the of and Subcellular of TRPV4 of TRPV4 variants was carried out cells with TRPV4 (5Arniges M. Vazquez E. Fernandez-Fernandez J.M. Valverde M.A. J. Biol. Chem. 2004; 279: 54062-54068Abstract Full Text Full Text PDF PubMed Scopus (130) Google Scholar, 11Andrade Y.N. Fernandes J. Vazquez E. Fernandez-Fernandez J.M. Arniges M. Sanchez T.M. Villalon M. Valverde M.A. J. Cell Biol. 2005; 168: 869-874Crossref PubMed Scopus (163) Google Scholar). shows that TRPV4-A and TRPV4-D a cell TRPV4-C, and TRPV4-E a intracellular of in the and variants were in the glycosylation and carried out with in cell protein was with to high of F to high and glycosylation of TRPV4-A was used as a in three protein with of and were for TRPV4-A results not in The the of the The not the was to and were to F. the the high and the the An glycosylation was for the TRPV4-D not An glycosylation and F was for the TRPV4-D not the and variants to two at and and and and The with F the of a lack of further of TRPV4-A because F a with to the predicted of the the suggest that and variants are in the TRPV4-A and TRPV4-D are and the plasma membrane where they functional of for and variants was by All three variants with the of TRPV4-B variant with the intermediate compartment the Golgi and was results were with TRPV4-C and of TRPV4 TRP channel the tetrameric assembly of M. Pflugers Arch. 2005; PubMed Scopus Google Scholar). to the including and the have been identified as for oligomerization Neurosci. Full Text PDF PubMed Scopus Google Scholar, A. Cell Biol. PubMed Scopus Google Scholar). the multimerization of the TRPV4 variants. this the of and TRPV4 variants, as of subunit assembly by N. Albrecht N. Harteneck C. Schultz G. Schaefer M. J. Cell Sci. 2005; 118: 917-928Crossref PubMed Scopus (243) Google Scholar). The and fluorescence were for cell to of TRPV4 and to the between and subunits. shows in cells with TRPV4 TRPV4-A and TRPV4-D variants a and with TRPV4-C, and TRPV4-D results that only TRPV4-A and TRPV4-D into channel TRPV4 channels in the carried out analysis of cells with and TRPV4-A variants and for with the intracellular from the to the Golgi J.G. J. J. Cell Biol. PubMed Scopus Google Scholar). for TRPV4-A were not between and cells and result that assembly of in the and that of and variants is to to in the the intracellular of channel involves the of are the tetrameric structure in channel intracellular N. B. Y.N. Neuron. 22: Full Text Full Text PDF PubMed Scopus Google Scholar). TRPV4 protein two the N-terminal and and two the C-terminal and intracellular of TRPV4-B variants containing in the of of the four not the of TRPV4-B not a TRPV4-B in four were by was in the and not to the Golgi of TRPV4 assembly of TRP channels a in the intracellular the of the new protein E. S. Bindels R.J. J.G. J. Biol. Chem. 2004; 279: Full Text Full Text PDF PubMed Scopus Google Scholar, C. G. L. J. Biol. Chem. 2003; 278: Full Text Full Text PDF PubMed Scopus Google Scholar, C. J.D. J. Biol. Chem. 2004; 279: Full Text Full Text PDF PubMed Scopus (93) Google Scholar). TRPV4-A TRPV4-C, and TRPV4-E in the and variants in cells and the by in plasma membrane was only in cells TRPV4-A and TRPV4-D in intracellular was for of TRPV4 variants. is also the lack of to the plasma membrane of the intracellular variants and following with the TRPV4-A TRPV4-C, and TRPV4-E to the plasma membrane with of of TRPV4 variants was also by and as the and variants as a high of only demonstrated for the channel between TRPV4-A and TRPV4-D subunits. for TRPV4-A with TRPV4-C, TRPV4-E TRP the of the channel with the a also of in cells channels not from the TRPV4-A and TRPV4-D channels with and the the with the protein with in were to only the TRPV4-D variant was with to the of the variants, and the with the of the are also between and variants was not Our of localization, and channel of human TRPV4 splice variants has us to the N-terminal ANK repeats as determinants of subunit assembly and subsequent of the channel. a of have five TRPV4 variants cloned from human epithelial three of were for the We have them into two classes: group I (including TRPV4-A and TRPV4-D) and group II (including TRPV4-C, and TRPV4-E). Group I variants are correctly and targeted to the plasma membrane where they functional channels with from group are lacking of the ANK domains are to and were in the channel identified and by in the Neurosci. Full Text PDF PubMed Scopus Google Scholar, R. Physiol. Biochem. Pharmacol. 2004; PubMed Scopus Google Scholar). it is important to the domains involved in the oligomerization process. the of TRP of the assembly is starting to emerge M. Pflugers Arch. 2005; PubMed Scopus Google Scholar). The of TRP as it for E. S. Bindels R.J. J.G. J. Biol. Chem. 2004; 279: Full Text Full Text PDF PubMed Scopus Google Scholar), has also been identified as a multimerization domain X.Z. Li H.S. Guggino W.B. Montell C. Cell. 1997; 89: 1155-1164Abstract Full Text Full Text PDF PubMed Scopus (277) Google Scholar, 24Erler I. Hirnet D. Wissenbach U. Flockerzi V. Niemeyer B.A. J. Biol. Chem. 2004; 279: 34456-34463Abstract Full Text Full Text PDF PubMed Scopus (112) Google Scholar, 25Hellwig N. Albrecht N. Harteneck C. Schultz G. Schaefer M. J. Cell Sci. 2005; 118: 917-928Crossref PubMed Scopus (243) Google Scholar, M. B. K. Res. PubMed Scopus Google Scholar, M. O. P. C. U. C. E. O. A. J. FEBS Lett. 2002; PubMed Scopus Google Scholar, E. S. Bindels R.J. J.G. J. Biol. Chem. 2004; 279: Full Text Full Text PDF PubMed Scopus Google Scholar). the of and a oligomerization domain has been to the N-terminal ANK domains I. Hirnet D. Wissenbach U. Flockerzi V. Niemeyer B.A. J. Biol. Chem. 2004; 279: 34456-34463Abstract Full Text Full Text PDF PubMed Scopus (112) Google Scholar, E. S. Bindels R.J. J.G. J. Biol. Chem. 2004; 279: Full Text Full Text PDF PubMed Scopus Google Scholar). and transmembrane domains have also been to in the oligomerization of TRP channels E. S. Bindels R.J. J.G. J. Biol. Chem. 2004; 279: Full Text Full Text PDF PubMed Scopus Google Scholar, N. A. C. R. E. G. A. J. Neurosci. 2004; PubMed Scopus Google Scholar), including TRPV4 N. Albrecht N. Harteneck C. Schultz G. Schaefer M. J. Cell Sci. 2005; 118: 917-928Crossref PubMed Scopus (243) Google Scholar). Our of and splice variants of the ANK by in and has us to for the three important of TRPV4 1) of TRPV4 channel involves to Golgi with the in the from the high of the to the of the Golgi 2) TRPV4-A in the in were in the of of to Golgi 3) subunit assembly of II variants is because of the lack of N-terminal ANK domains and protein in the Although that of and variants is to to that such is because of protein is a of channel of channel also subunit is not Neurosci. Full Text PDF PubMed Scopus Google Scholar, N. D.M. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). for of involves of a is in channels N. B. Y.N. Neuron. 22: Full Text Full Text PDF PubMed Scopus Google Scholar, R. R. Pharmacol. 2001; PubMed Scopus Google Scholar). a in the assembly of and TRPV4 four two in each intracellular However, of not the intracellular of II variants, to have been for membrane F. E. C. F. J. Biol. Chem. 2005; Full Text Full Text PDF PubMed Scopus Google Scholar). The of L. M. A. 286: PubMed Scopus Google Scholar, D. 2002; PubMed Scopus Google in the of II TRPV4 variants is a that to TRPV4 lacking the three ANK domains a to W. Choe Y. Marti-Renom M.A. Bell A.M. Denis C.S. Sali A. Hudspeth A.J. Friedman J.M. Heller S. Cell. 2000; 103: 525-535Abstract Full Text Full Text PDF PubMed Scopus (1036) Google Scholar). these to results not functional the by and W. Choe Y. Marti-Renom M.A. Bell A.M. Denis C.S. Sali A. Hudspeth A.J. Friedman J.M. Heller S. Cell. 2000; 103: 525-535Abstract Full Text Full Text PDF PubMed Scopus (1036) Google in of results and of of TRPV4 H. Vriens J. Benham C.D. Droogmans G. Nilius B. J. Biol. Chem. 2002; 277: Full Text Full Text PDF PubMed Scopus Google and I. Hirnet D. Wissenbach U. Flockerzi V. Niemeyer B.A. J. Biol. Chem. 2004; 279: 34456-34463Abstract Full Text Full Text PDF PubMed Scopus (112) Google channels with ANK domains. channel functional is by the of splice variants and of and regulatory subunits. is a to protein Biochem. 2003; PubMed Scopus Google Scholar). the TRP family of channels splice variants have been of them in lack of to typical stimuli, the pore and C. J.D. J. Biol. Chem. 2004; 279: Full Text Full Text PDF PubMed Scopus (93) Google Scholar, L. F. K. FEBS Lett. 2001; PubMed Scopus Google Scholar, B. Am. J. Physiol. 2001; PubMed Google Scholar, M.A. I. Levine J.D. J. Biol. Chem. 2000; Full Text Full Text PDF PubMed Scopus Google Scholar, J. A. Flockerzi V. J. Biol. Chem. 2005; Full Text Full Text PDF PubMed Scopus Google Scholar). Group II TRPV4 splice variants have been identified in two human epithelial cell and the of TRPV4 channels in epithelial (5Arniges M. Vazquez E. Fernandez-Fernandez J.M. Valverde M.A. J. Biol. Chem. 2004; 279: 54062-54068Abstract Full Text Full Text PDF PubMed Scopus (130) Google Scholar, 11Andrade Y.N. Fernandes J. Vazquez E. Fernandez-Fernandez J.M. Arniges M. Sanchez T.M. Villalon M. Valverde M.A. J. Cell Biol. 2005; 168: 869-874Crossref PubMed Scopus (163) Google Scholar, D.M. Pflugers Arch. 2005; PubMed Scopus Google Scholar), it that a in the expressed of group I to group II variants, the epithelial by S. 2002; PubMed Scopus Google including and of them also of the it to such the expressed of TRPV4 group I to group II variants. We E. Y. and J. Fernandes for Y. for and I. and G. for at of this TRPV4 channel splice variants a of ankyrin domains in multimerization and of PDF