participants were evaluated at stanford university medical center and the university of washington medical school . weakness was graded as mild if the medical research council scale score was 4/5 , moderate if 3 and < 4 , and severe if 2 . sensory loss was determined to be moderate or mild by the examiner based on vibration testing . genomic dna was isolated from blood leukocytes using standard extraction protocols and examined by direct cmt gene testing . the ard is currently the only trpv4 domain for which a high - resolution structure has been determined . in this study , swiss - model was used to generate a 4-fold symmetric tetramer model of human trpv4 ( residues 148755 of 871 ) using the apo rat trpv1 electron cryomicroscopy structure as a template ( pdbid 3j5p ) . rat trpv1-ard and human trpv4-ard have 56% sequence identity , and their core c atoms have a root mean square deviation of 1.6  , allowing us to place the experimentally determined trpv4-ard crystal structure with high confidence within our homology model . therefore , after generation of a full model , the swiss - model  generated ard was removed and replaced with the experimentally determined x - ray crystal structure of the human trpv4-ard ( pdbid 4dx2 , chain b , residues 148389 ) after alignment to residues 350389 ( ankyrin repeat 6 ) in coot . to relieve any resulting clashes , 10 rounds of geometry minimization of residues 390470 and 635666 were performed in phenix.refine with the 4-fold symmetry restrained . the c - terminal -strand ( residues 752762 in the 3j5p trpv1 structure ) was then added to the homology model to illustrate the position of this structural feature . t cells were cultured in dulbecco 's modified eagle 's medium supplemented with 10% ( vol / vol ) fetal calf serum at 37c with 6% co2 . cells were transfected with pcdna3.1-trpv4-flag ( wild - type [ wt ] or mutant ) constructs using lipofectamine ltx ( life technologies , grand island , ny ) and then replated onto poly - l - ornithine  coated coverslips 4.5 hours after transfection in the absence or presence of the trpv4-specific antagonist hc-067047 ( sigma - aldrich , st . 24 hours after transfection , cells were fixed in 4% paraformaldehyde in 0.1 m tris - buffered saline for 15 minutes at room temperature and then processed as described previously . t cells were lysed 24 hours after transfection in radioimmunoprecipitation assay buffer ( sigma - aldrich ) supplemented with protease inhibitors ( sigma - aldrich ) and sonicated . protein lysates were resolved on 4%15% tgx gels ( bio - rad laboratories , inc , hercules , ca ) and transferred to polyvinylidene fluoride membranes . primary antibodies used were rabbit anti - flag ( 1:1,000 ; cell signaling technology , danvers , ma ; # 2368 ) and rabbit anti--actin ( 1:1,000 ; cell signaling technology ; # 4970 ) , followed by a donkey anti - rabbit - hrp  conjugated secondary antibody ( 1:200,000 ; ge healthcare life sciences , pittsburgh , pa ) . membranes were developed using supersignal west femto maximum sensitivity substrate ( thermo scientific , waltham , ma ) . hek293 t and mn-1 ( mouse motor neuron  neuroblastoma fusion ) cells were cotransfected with pcdna3-mcherry and pcdna3.1-trpv4-flag ( wt or mutant ) constructs using lipofectamine ltx . calcium imaging was performed on a zeiss axio observer.z1 inverted microscope equipped with a lambda dg-4 ( sutter instrument company , novato , ca ) wavelength switcher . cells were bath - loaded with fura-2 am ( 8 m ; life technologies ) for 30 minutes at 37c in calcium - imaging buffer ( 150 mm nacl , 5 mm kcl , 1 mm mgcl2 , 2 mm cacl2 , 10 mm glucose , 10 mm hepes , ph 7.4 ) containing 0.04% pluronic f-127 . cell death assays were performed 24 hours after transfection using the cytotoxicity detection kit ( ldh ) kit ( roche diagnostics , indianapolis , in ) according to the manufacturer 's instructions . cytotoxicity in this assay is calculated by subtracting the absorbance of a background control ( normal medium ) from the absorbance of the experimental samples ( supernatant from transfected cells ) . results are expressed as a percentage relative to the absorbance of a high control ( supernatant from cells treated with lysis buffer ) . statistical significance was determined using the mann - whitney u test ( calcium imaging ) or unpaired student t test ( cytotoxicity assays ) . participants were evaluated at stanford university medical center and the university of washington medical school . weakness was graded as mild if the medical research council scale score was 4/5 , moderate if 3 and < 4 , and severe if 2 . sensory loss was determined to be moderate or mild by the examiner based on vibration testing . genomic dna was isolated from blood leukocytes using standard extraction protocols and examined by direct cmt gene testing . the ard is currently the only trpv4 domain for which a high - resolution structure has been determined . in this study , swiss - model was used to generate a 4-fold symmetric tetramer model of human trpv4 ( residues 148755 of 871 ) using the apo rat trpv1 electron cryomicroscopy structure as a template ( pdbid 3j5p ) . rat trpv1-ard and human trpv4-ard have 56% sequence identity , and their core c atoms have a root mean square deviation of 1.6  , allowing us to place the experimentally determined trpv4-ard crystal structure with high confidence within our homology model . therefore , after generation of a full model , the swiss - model  generated ard was removed and replaced with the experimentally determined x - ray crystal structure of the human trpv4-ard ( pdbid 4dx2 , chain b , residues 148389 ) after alignment to residues 350389 ( ankyrin repeat 6 ) in coot . to relieve any resulting clashes , 10 rounds of geometry minimization of residues 390470 and 635666 were performed in phenix.refine with the 4-fold symmetry restrained . the c - terminal -strand ( residues 752762 in the 3j5p trpv1 structure ) was then added to the homology model to illustrate the position of this structural feature . hek293 t cells were cultured in dulbecco 's modified eagle 's medium supplemented with 10% ( vol / vol ) fetal calf serum at 37c with 6% co2 . cells were transfected with pcdna3.1-trpv4-flag ( wild - type [ wt ] or mutant ) constructs using lipofectamine ltx ( life technologies , grand island , ny ) and then replated onto poly - l - ornithine  coated coverslips 4.5 hours after transfection in the absence or presence of the trpv4-specific antagonist hc-067047 ( sigma - aldrich , st . 24 hours after transfection , cells were fixed in 4% paraformaldehyde in 0.1 m tris - buffered saline for 15 minutes at room temperature and then processed as described previously . hek293 t cells were lysed 24 hours after transfection in radioimmunoprecipitation assay buffer ( sigma - aldrich ) supplemented with protease inhibitors ( sigma - aldrich ) and sonicated . protein lysates were resolved on 4%15% tgx gels ( bio - rad laboratories , inc , hercules , ca ) and transferred to polyvinylidene fluoride membranes . primary antibodies used were rabbit anti - flag ( 1:1,000 ; cell signaling technology , danvers , ma ; # 2368 ) and rabbit anti--actin ( 1:1,000 ; cell signaling technology ; # 4970 ) , followed by a donkey anti - rabbit - hrp  conjugated secondary antibody ( 1:200,000 ; ge healthcare life sciences , pittsburgh , pa ) . membranes were developed using supersignal west femto maximum sensitivity substrate ( thermo scientific , waltham , ma ) . hek293 t and mn-1 ( mouse motor neuron  neuroblastoma fusion ) cells were cotransfected with pcdna3-mcherry and pcdna3.1-trpv4-flag ( wt or mutant ) constructs using lipofectamine ltx . calcium imaging was performed on a zeiss axio observer.z1 inverted microscope equipped with a lambda dg-4 ( sutter instrument company , novato , ca ) wavelength switcher . cells were bath - loaded with fura-2 am ( 8 m ; life technologies ) for 30 minutes at 37c in calcium - imaging buffer ( 150 mm nacl , 5 mm kcl , 1 mm mgcl2 , 2 mm cacl2 , 10 mm glucose , 10 mm hepes , ph 7.4 ) containing 0.04% pluronic f-127 . cell death assays were performed 24 hours after transfection using the cytotoxicity detection kit ( ldh ) kit ( roche diagnostics , indianapolis , in ) according to the manufacturer 's instructions . cytotoxicity in this assay is calculated by subtracting the absorbance of a background control ( normal medium ) from the absorbance of the experimental samples ( supernatant from transfected cells ) . results are expressed as a percentage relative to the absorbance of a high control ( supernatant from cells treated with lysis buffer ) . statistical significance was determined using the mann - whitney u test ( calcium imaging ) or unpaired student t test ( cytotoxicity assays ) . we examined 4 individuals from 2 families ( figure 1b ) exhibiting features consistent with the cmt2c phenotype , including distal limb weakness , hoarseness of voice , and vocal fold paresis ( table ) . , laryngoscopy revealed bilateral vocal fold paresis with vocal fold movement at 15%20% of normal . vocal fold emg in this individual confirmed a reduction in motor unit number , consistent with chronic denervation . family 2 has a 3-generation history of limb weakness and hoarseness on the maternal side ( figure 1b ) . summary of key clinical features of individuals with charcot - marie - tooth disease type 2c genetic testing of family 1 revealed a heterozygous missense mutation ( c.709c > g ) in exon 4 of trpv4 ( refseq accession number nm_021625.4 ) in both examined individuals . this nucleotide change results in an arginine to glycine substitution at amino acid 237 ( p.arg237gly ) of trpv4 . a heterozygous sequence change ( c.710g > t ) affecting amino acid 237 of trpv4 was also detected in the 2 examined individuals in family 2 , resulting in an arginine to leucine substitution ( p.arg237leu ) at this residue . neither nucleotide substitution is present in the nhlbi exome sequencing project exome variant server , the 1000 genomes project , exome aggregation consortium , or dbsnp . like most known neuropathy - causing trpv4 mutations , arg237 is located in the trpv4-ard ( third ankyrin repeat ; figure 1a ) and is highly conserved across vertebrate species ( figure 1c ) . in silico analyses predict both mutations to be deleterious ( polyphen-2 humvar scores : p.arg237gly , 0.785 ; p.arg237leu , 0.713 ) and disease - causing ( mutationtaster scores : p.arg237gly , 1 ; p.argr237leu , > 0.999 ) . ankyrin repeats comprise inner and outer helices followed by a connecting finger loop , and the 6 repeats of the trpv4-ard together form a hand - shaped domain with a concave palm surface and a convex surface analogous to the back of the hand . analysis of the high - resolution crystal structure of the human trpv4-ard reveals that arg237 is situated within the second finger loop , near 2 other arginine residues mutated in cmt2c ( arg232 and arg269 ; figure 2a ) . like other neuropathy - causing mutations in the trpv4-ard , arg237 is represented in magenta ; residues mutated in neuropathy , skeletal dysplasia , and osteoarthropathy are shown in blue ( r232 , r269 , r315 , r316 ) , yellow ( e183 , k197 , l199 , q239 , e278 , t295 , i331 , d333 , v342 , k407 , f471 , l523 , y591 , f592 , r594 , l596 , g600 , y602 , i604 , r616 , f617 , l618 , m625 , l709 , a716 ) , and orange ( g270 , r271 , f273 ) , respectively . mutated residues reported as causing mixed nerve / bone disease are shown in green ( a217 , k276 , e278 , s542 , v620 , t740 ) . ( a ) ribbon diagram of the trpv4-ard ( residues 148392 ) with disease - causing mutation positions marked with spheres . is located on the convex face of the ankyrin repeat domain ( ard ) within the previously identified cluster of arginine residues ( shown as sticks ) mutated in hereditary neuropathy . ( b ) homology model of human trpv4 based on the trpv1 structure . known disease - causing mutation positions marked with spheres . one subunit of the trpv4 tetramer is shown in cyan with the remainder shown in gray . the plasma membrane is denoted with black lines , and the arrow points to a cluster of skeletal dysplasia  causing mutations near the lower channel gate . as in figure 1 , indicates a mutation found in a family with charcot - marie - tooth disease type 2c and pronounced short stature and indicates a mutation described in a single patient with cmt2 and skeletal dysplasia , as well as in a single patient with axonal neuropathy and both the p.val620ile mutation and a second p.arg151trp trpv4 variant . ( c ) top - down view of the ion channel ( as seen from the extracellular side ) in surface representation . the cluster of neuropathy - causing mutations ( indicated in 1 subunit by the arrow ) is oriented toward the intracellular leaflet of the plasma membrane . ( d ) expanded view of the region indicated in panel b showing 1 subunit in cyan ribbon representation and the neighboring subunit in surface representation . the positioning of the ard relative to the rest of the assembled channel and the plasma membrane has not been previously investigated . like other trpv channels , we used the recently described electron cryomicroscopy structure of the trpv1 ion channel to generate a model of the human trpv4 homotetramer ( figure 2b ) that incorporates the experimentally determined trpv4-ard . an overall high sequence identity between trpv1 and trpv4 ( 45% ) allows us to map the positions of most disease - causing trpv4 mutations in other regions onto this model with high confidence . first , the convex face of the ard , on which neuropathy - causing mutations cluster , is on the outer surface of the trpv4 tetramer , facing toward the cytosol at an angle of 45 relative to the plasma membrane ( figure 2 , b and c ) . this orientation suggests that the ard convex face would likely be readily accessible for protein second , skeletal dysplasia  causing mutations form 2 main clusters within the channel ( figure 2 , b and d ) . the larger of these clusters is situated within the bottom half of the transmembrane region near the lower channel gate , consistent with the finding that many of these mutations increase constitutive channel activity . the second cluster occurs on the concave surface of the ard . in the tetrameric channel , this surface participates in intersubunit interactions connecting the ard of one protomer to a small -sheet structure arising from the ard - to - transmembrane domain linker and the c - terminus of the adjacent protomer ( figure 2d ) . the location of this second cluster suggests that disruption of this intersubunit interface may also increase constitutive channel activity . third , osteoarthropathy - causing mutants are clustered in the third finger loop of the ard , which interacts with the -sheet structure on one side but is exposed to the solvent on the other . although no clear conclusion can be drawn from our model , this loop can be flexible , and it is plausible that the mutations affect interactions with a regulator or trafficking chaperone that results in reduced cell surface localization , as previously observed . no clear localization patterns were evident for mutations thought to cause both nerve and bone disease ( green residues , figure 2 ) . to gain insights into the functional consequences of the p.arg237gly and p.arg237leu amino acid changes , we performed a series of in vitro studies examining the effects of heterologous expression of these mutant channels . to assess the magnitude of any observed effects , experiments were performed in parallel with known disease - causing trpv4 mutants ( neuropathy , p.arg269cys ; skeletal dysplasia , p.ile331phe ; osteoarthropathy , p.arg271pro ; the locations of these residues are shown in figure 2a ) . immunocytochemical analyses revealed that each of the neuropathy - causing mutants examined exhibited a subcellular distribution comparable to that of the wt channel , localizing to both the cytoplasm and the plasma membrane ( figure 3a ) . in contrast , the skeletal dysplasia and osteoarthropathy - causing mutants localized primarily to the cytoplasm ( figure 3avi ) . incubation of transfected cells in a trpv4 channel antagonist ( hc-067047 ) increased plasma membrane expression of both trpv4 , as described previously , and the mutant channels , including p.ile331phe and p.arg271pro ( figure 3b ) . to examine effects on channel function , we performed ratiometric calcium imaging to measure cytosolic calcium levels ( [ ca]i ) of transiently transfected mn-1 ( a motor neuron  like cell line ) and hek293 t cells 24 hours after transfection . expression of both p.arg237gly and p.arg237leu resulted in significant elevations of basal [ ca]i levels relative to the wt channel in both cell types , of comparable magnitude to known disease - causing trpv4 mutants ( figure 3 , c and d ) . hek293 t cells expressing p.arg237gly and p.arg237leu also exhibited significantly increased levels of cytotoxicity relative to trpv4-transfected cells ( figure 3e ) . both the effects on [ ca]i and cell viability were abrogated by application of hc-067047 ( figure 3 , c  e ) , consistent with a mutation - dependent increase in channel activity . it is interesting that despite causing significant elevations of basal [ ca]i , expression of the osteoarthropathy - causing mutant p.arg271pro was not associated with cytotoxicity ( figure 3e ) . to confirm that the elevations in [ ca]i and cytotoxicity caused by the neuropathy- and skeletal dysplasia  causing mutants did not result from increased expression levels , we performed immunoblot analyses of whole - cell lysates 24 hours after transfection . these analyses revealed reduced expression of these mutants relative to trpv4 ( figure 3f ) , likely reflecting their increased cytotoxicity . immunoblots performed on cells incubated in hc-067047 to abrogate toxicity , however , demonstrated equivalent protein expression levels across the wt and mutant channels ( figure 3 g ) . taken together , the above results suggest that the p.arg237gly and p.arg237leu mutants traffic normally to the plasma membrane but exhibit a gain of function in channel activity . ( a , b ) representative images of hek293 t cells transfected with wild - type ( wt ) or mutant trpv4-flag and labeled immunocytochemically for flag ( red ) to reveal the subcellular distribution of trpv4 . cells were costained for the transferrin receptor ( green ) and with dapi ( blue ) to delineate individual cells and nuclei , respectively . these studies included examination of known neuropathy  causing ( p.arg269cys ) , skeletal dysplasia  causing ( p.ile331phe ) , and osteoarthropathy - causing ( p.arg271pro ) trpv4 mutations . transfected cells were cultured in the absence ( a ) or presence ( b ) of the trpv4 antagonist hc-067047 ( 5 m ) . all images are shown at the same magnification , and the scale bar represents 20 m . ( c , d ) quantification of baseline fura-2 ratios in transiently transfected mn-1 ( c ) and hek293 t ( d ) cells reveals significant increases in basal [ ca]i with expression of each mutant channel that are blocked by the trpv4 antagonist hc-067047 ( 5 m ) . ( e ) quantification of cell death in hek293 t cells 24 hours after transfection using an ldh cytotoxicity assay . cells expressing trpv4 mutants , with the exception of p.arg271pro , show significantly increased levels of cytotoxicity relative to trpv4-expressing cells ; this increase is abrogated by hc-067047 ( 5 m ) . * * p < 0.001 , * * * p < 0.0001 . data in c and d are averaged from > 100 transfected cells , while data in e are averaged from 3 independent transfections . ( f , g ) immunoblotting of whole lysates from hek293 t cells 24 hours after transfection incubated in the absence ( f ) or presence ( g ) of hc-067047 ( 5 m ) . we examined 4 individuals from 2 families ( figure 1b ) exhibiting features consistent with the cmt2c phenotype , including distal limb weakness , hoarseness of voice , and vocal fold paresis ( table ) . , laryngoscopy revealed bilateral vocal fold paresis with vocal fold movement at 15%20% of normal . vocal fold emg in this individual confirmed a reduction in motor unit number , consistent with chronic denervation . family 2 has a 3-generation history of limb weakness and hoarseness on the maternal side ( figure 1b ) . summary of key clinical features of individuals with charcot - marie - tooth disease type 2c genetic testing of family 1 revealed a heterozygous missense mutation ( c.709c > g ) in exon 4 of trpv4 ( refseq accession number nm_021625.4 ) in both examined individuals . this nucleotide change results in an arginine to glycine substitution at amino acid 237 ( p.arg237gly ) of trpv4 . a heterozygous sequence change ( c.710g > t ) affecting amino acid 237 of trpv4 was also detected in the 2 examined individuals in family 2 , resulting in an arginine to leucine substitution ( p.arg237leu ) at this residue . neither nucleotide substitution is present in the nhlbi exome sequencing project exome variant server , the 1000 genomes project , exome aggregation consortium , or dbsnp . like most known neuropathy - causing trpv4 mutations , arg237 is located in the trpv4-ard ( third ankyrin repeat ; figure 1a ) and is highly conserved across vertebrate species ( figure 1c ) . in silico analyses predict both mutations to be deleterious ( polyphen-2 humvar scores : p.arg237gly , 0.785 ; p.arg237leu , 0.713 ) and disease - causing ( mutationtaster scores : p.arg237gly , 1 ; p.argr237leu , > 0.999 ) . ankyrin repeats comprise inner and outer helices followed by a connecting finger loop , and the 6 repeats of the trpv4-ard together form a hand - shaped domain with a concave palm surface and a convex surface analogous to the back of the hand . analysis of the high - resolution crystal structure of the human trpv4-ard reveals that arg237 is situated within the second finger loop , near 2 other arginine residues mutated in cmt2c ( arg232 and arg269 ; figure 2a ) . like other neuropathy - causing mutations in the trpv4-ard , arg237 is represented in magenta ; residues mutated in neuropathy , skeletal dysplasia , and osteoarthropathy are shown in blue ( r232 , r269 , r315 , r316 ) , yellow ( e183 , k197 , l199 , q239 , e278 , t295 , i331 , d333 , v342 , k407 , f471 , l523 , y591 , f592 , r594 , l596 , g600 , y602 , i604 , r616 , f617 , l618 , m625 , l709 , a716 ) , and orange ( g270 , r271 , f273 ) , respectively . mutated residues reported as causing mixed nerve / bone disease are shown in green ( a217 , k276 , e278 , s542 , v620 , t740 ) . ( a ) ribbon diagram of the trpv4-ard ( residues 148392 ) with disease - causing mutation positions marked with spheres . arg237 is located on the convex face of the ankyrin repeat domain ( ard ) within the previously identified cluster of arginine residues ( shown as sticks ) mutated in hereditary neuropathy . one subunit of the trpv4 tetramer is shown in cyan with the remainder shown in gray . the plasma membrane is denoted with black lines , and the arrow points to a cluster of skeletal dysplasia  causing mutations near the lower channel gate . as in figure 1 , indicates a mutation found in a family with charcot - marie - tooth disease type 2c and pronounced short stature and indicates a mutation described in a single patient with cmt2 and skeletal dysplasia , as well as in a single patient with axonal neuropathy and both the p.val620ile mutation and a second p.arg151trp trpv4 variant . ( c ) top - down view of the ion channel ( as seen from the extracellular side ) in surface representation . the cluster of neuropathy - causing mutations ( indicated in 1 subunit by the arrow ) is oriented toward the intracellular leaflet of the plasma membrane . ( d ) expanded view of the region indicated in panel b showing 1 subunit in cyan ribbon representation and the neighboring subunit in surface representation . the positioning of the ard relative to the rest of the assembled channel and the plasma membrane has not been previously investigated . we used the recently described electron cryomicroscopy structure of the trpv1 ion channel to generate a model of the human trpv4 homotetramer ( figure 2b ) that incorporates the experimentally determined trpv4-ard . an overall high sequence identity between trpv1 and trpv4 ( 45% ) allows us to map the positions of most disease - causing trpv4 mutations in other regions onto this model with high confidence . first , the convex face of the ard , on which neuropathy - causing mutations cluster , is on the outer surface of the trpv4 tetramer , facing toward the cytosol at an angle of 45 relative to the plasma membrane ( figure 2 , b and c ) . this orientation suggests that the ard convex face would likely be readily accessible for protein second , skeletal dysplasia  causing mutations form 2 main clusters within the channel ( figure 2 , b and d ) . the larger of these clusters is situated within the bottom half of the transmembrane region near the lower channel gate , consistent with the finding that many of these mutations increase constitutive channel activity . the second cluster occurs on the concave surface of the ard . in the tetrameric channel , this surface participates in intersubunit interactions connecting the ard of one protomer to a small -sheet structure arising from the ard - to - transmembrane domain linker and the c - terminus of the adjacent protomer ( figure 2d ) . the location of this second cluster suggests that disruption of this intersubunit interface may also increase constitutive channel activity . third , osteoarthropathy - causing mutants are clustered in the third finger loop of the ard , which interacts with the -sheet structure on one side but is exposed to the solvent on the other . although no clear conclusion can be drawn from our model , this loop can be flexible , and it is plausible that the mutations affect interactions with a regulator or trafficking chaperone that results in reduced cell surface localization , as previously observed . no clear localization patterns were evident for mutations thought to cause both nerve and bone disease ( green residues , figure 2 ) . to gain insights into the functional consequences of the p.arg237gly and p.arg237leu amino acid changes , we performed a series of in vitro studies examining the effects of heterologous expression of these mutant channels . to assess the magnitude of any observed effects , experiments were performed in parallel with known disease - causing trpv4 mutants ( neuropathy , p.arg269cys ; skeletal dysplasia , p.ile331phe ; osteoarthropathy , p.arg271pro ; the locations of these residues are shown in figure 2a ) . immunocytochemical analyses revealed that each of the neuropathy - causing mutants examined exhibited a subcellular distribution comparable to that of the wt channel , localizing to both the cytoplasm and the plasma membrane ( figure 3a ) . in contrast , the skeletal dysplasia and osteoarthropathy - causing mutants localized primarily to the cytoplasm ( figure 3avi ) . incubation of transfected cells in a trpv4 channel antagonist ( hc-067047 ) increased plasma membrane expression of both trpv4 , as described previously , and the mutant channels , including p.ile331phe and p.arg271pro ( figure 3b ) . to examine effects on channel function , we performed ratiometric calcium imaging to measure cytosolic calcium levels ( [ ca]i ) of transiently transfected mn-1 ( a motor neuron  like cell line ) and hek293 t cells 24 hours after transfection . expression of both p.arg237gly and p.arg237leu resulted in significant elevations of basal [ ca]i levels relative to the wt channel in both cell types , of comparable magnitude to known disease - causing trpv4 mutants ( figure 3 , c and d ) . hek293 t cells expressing p.arg237gly and p.arg237leu also exhibited significantly increased levels of cytotoxicity relative to trpv4-transfected cells ( figure 3e ) . both the effects on [ ca]i and cell viability were abrogated by application of hc-067047 ( figure 3 , c  e ) , consistent with a mutation - dependent increase in channel activity . it is interesting that despite causing significant elevations of basal [ ca]i , expression of the osteoarthropathy - causing mutant p.arg271pro was not associated with cytotoxicity ( figure 3e ) . to confirm that the elevations in [ ca]i and cytotoxicity caused by the neuropathy- and skeletal dysplasia  causing mutants did not result from increased expression levels , we performed immunoblot analyses of whole - cell lysates 24 hours after transfection . these analyses revealed reduced expression of these mutants relative to trpv4 ( figure 3f ) , likely reflecting their increased cytotoxicity . immunoblots performed on cells incubated in hc-067047 to abrogate toxicity , however , demonstrated equivalent protein expression levels across the wt and mutant channels ( figure 3 g ) . taken together , the above results suggest that the p.arg237gly and p.arg237leu mutants traffic normally to the plasma membrane but exhibit a gain of function in channel activity . ( a , b ) representative images of hek293 t cells transfected with wild - type ( wt ) or mutant trpv4-flag and labeled immunocytochemically for flag ( red ) to reveal the subcellular distribution of trpv4 . cells were costained for the transferrin receptor ( green ) and with dapi ( blue ) to delineate individual cells and nuclei , respectively . these studies included examination of known neuropathy  causing ( p.arg269cys ) , skeletal dysplasia  causing ( p.ile331phe ) , and osteoarthropathy - causing ( p.arg271pro ) trpv4 mutations . transfected cells were cultured in the absence ( a ) or presence ( b ) of the trpv4 antagonist hc-067047 ( 5 m ) . all images are shown at the same magnification , and the scale bar represents 20 m . ( c , d ) quantification of baseline fura-2 ratios in transiently transfected mn-1 ( c ) and hek293 t ( d ) cells reveals significant increases in basal [ ca]i with expression of each mutant channel that are blocked by the trpv4 antagonist hc-067047 ( 5 m ) . ( e ) quantification of cell death in hek293 t cells 24 hours after transfection using an ldh cytotoxicity assay . cells expressing trpv4 mutants , with the exception of p.arg271pro , show significantly increased levels of cytotoxicity relative to trpv4-expressing cells ; this increase is abrogated by hc-067047 ( 5 m ) . * * p < 0.001 , * * * p < 0.0001 . data in c and d are averaged from > 100 transfected cells , while data in e are averaged from 3 independent transfections . ( f , g ) immunoblotting of whole lysates from hek293 t cells 24 hours after transfection incubated in the absence ( f ) or presence ( g ) of hc-067047 ( 5 m ) . the structure of ards with a conserved hydrophobic core and variable exposed residues enables these domains to interact with binding partners ( inter- and intramolecular ) with high specificity and affinity . while these properties enable ards to perform diverse cellular functions , mutations in the ards of a number of proteins are associated with disease , including autism spectrum disorder ( shank3 ) , melanoma ( p16 ) , keratoderma ( kank2 ) , and bare lymphocyte syndrome ( rfxank ) . to date , 8 of 12 neuropathy - causing mutations in trpv4 occur within the trpv4-ard ( figure 1a ) . the identification in the present study of 2 additional trpv4-ard mutations ( p.arg237gly and p.arg237leu ) in 2 families with the cmt2c phenotype further underscores the importance of this domain in trpv4-mediated neuropathy . like previously described neuropathy - causing mutations in the trpv4-ard , p.arg237gly and p.arg237leu occur at an exposed arginine residue on the convex face of the ard . six of the 10 exposed arginine residues present on the convex face of trpv4-ard have now been associated with neuropathy . this cluster of arginine residues is highly conserved across vertebrate species but is not characteristic of the ards of other trpv channel family members . we previously determined experimentally that the neuropathy - causing mutations r232c , r269c , r269h , r315w , and r316c ( all surface - exposed arginine mutations ) do not substantially alter the fold or stability of the human trpv4-ard , which suggests that the functional consequence of these mutations may instead lie in interactions with binding partners . while the concave surface of the trpv4-ard has been shown to bind atp , calmodulin , and pi(4,5)p2 , no interactors of the ard convex face , we can better understand the functional consequences of these mutations by understanding them in the context of a structural model of the full channel . in this study , we showed that the ard convex face is directed toward the cytosol and would thus likely be readily accessible for protein furthermore , the orientation of this surface toward the plasma membrane suggests that the ard convex surface may interact principally with membrane - associated factors . as the trpv1 electron cryomicroscopy structure on which our model is based did not include regions n - terminal to the ard , it remains unclear how neuropathy - causing mutations in n - terminal regions of trpv4 ( figure 1a ) localize relative to the ard convex face . it is possible that the n - terminal region of trpv4 influences access to binding partners of the ard , as demonstrated for autism - associated mutations in the postsynaptic scaffolding protein shank3 . it is also likely that the channel adopts other conformations not yet seen experimentally , but the relative orientation of the ard in relation to the membrane is restrained by extensive interactions with other regions of the channel , suggesting that it is unlikely to be altered dramatically . generation of a model of the trpv4 homotetramer also enabled the mapping of trpv4 mutations associated with skeletal dysplasia and osteoarthropathy . skeletal dysplasia  causing mutations were found to occur primarily within 2 clusters : a large cluster situated within the bottom half of the transmembrane region near the lower channel gate and a second cluster on the concave surface of the ard that appears to participate in intersubunit interactions . together , these findings suggest that skeletal dysplasia  causing mutations may influence trpv4 channel activity by altering intramolecular interactions within the assembled channel . as described previously , osteoarthropathy - causing mutations localize to the tip of the ard third finger loop . in the tetrameric channel model , the corresponding residues are part of a potentially flexible loop and could be accessible to regulatory partners . in addition , a lack of any clear localization patterns for mutations thought to cause both nerve and bone disease ( green residues , figure 2 ) indicates that factors not accounted for by this model play an important role in trpv4 protein function . heterologous expression studies indicate that p.arg237gly and p.arg237leu exhibit a subcellular distribution equivalent to trpv4 , suggesting that these mutations do not influence channel assembly or trafficking . expression of these mutants , however , results in elevations of [ ca]i and significant cytotoxicity , both of which are abrogated by application of a trpv4 antagonist , consistent with increased channel activity . the magnitude of these effects is comparable to that of known disease - causing trpv4 mutants . together , these results suggest that both the p.arg237gly and p.arg237leu mutations confer a gain of function in channel activity . while heterologous expression of both skeletal dysplasia and osteoarthropathy - causing mutants ( p.ile331phe and p.arg271pro , respectively ) also resulted in significant increases in basal [ ca]i , this was not associated with significant cytotoxicity in p.arg271pro-expressing cells . similarly , expression of the osteoarthropathy mutant p.phe273leu in porcine chondrocytes results in elevations of [ ca]i but does not cause the channel activity  dependent increases in gene expression characteristic of skeletal dysplasia  causing mutants . together , these results suggest that factors in addition to , or independent of , increased ca influx may contribute to disease pathogenesis in some forms of trpv4 channelopathy . a clear understanding of how different mutations in trpv4 engender the diverse disease phenotypes characteristic of patients is only slowly emerging . elucidation of the etiologies underlying the different forms of trpv4 channelopathy is important because trpv4 is expressed at the plasma membrane and channel antagonism could therefore represent a therapeutic strategy , where appropriate . the identification of 2 novel mutations in the trpv4-ard highlights the key role of this domain in trpv4-mediated neuropathy . our findings suggest that molecular pathogenesis in these neuropathies may be driven by alterations in interactions of the ard convex face with as - yet - unidentified binding partners . identification of these interactors and characterization of their effects on channel function will be central to determining how mutations in trpv4 precipitate peripheral axonal degeneration . sullivan : study conceptualization and design , acquisition of data , analysis and interpretation , and drafting and revision of manuscript . c.m . r. gaudet : study conceptualization and design , analysis and interpretation , and revision of manuscript . sumner : primary investigator of the study , study conceptualization and design , analysis and interpretation , and drafting and revision of manuscript . supported by nih grant r21ns08757902 to c.j.s . , mda grant 277551 to c.j.s . , nih grant r01gm081340 to r.g . , and a postdoctoral fellowship from the jane coffin childs memorial fund for medical research to c.m.z . chen holds a patent for identifying genetic mutations in subjects susceptible to ataxia neurologic diseases ; has received research support from nih ( r01 ns069719 - 01 ) and national ataxia foundation ( fa100588 ) ; and has received license fee payments from athena diagnostics , inc . day has received travel funding from cytokinetics , biogen , roche inc , isis pharmaceuticals , the spinal muscular atrophy foundation , parent 's project muscular dystrophy , the myotonic dystrophy foundation , and the american association of pediatrics ; holds patents for myotonic dystrophy type 2 genetic testing and spinocerebellar ataxia type 5 genetic testing ; has been an employee of stanford university ; has been a consultant for isis pharmaceuticals , biogen , cytokinetics inc , and sarepta therapeutics ; has received research support from genzyme corporation , isis pharmaceuticals , sarepta pharmaceuticals , nih / ninds ( ns058901 ) , the muscular dystrophy association , the myotonic dystrophy foundation , and the spinal muscular atrophy foundation ; and has received royalty payments for dm2 genetic testing and sca5 genetic testing from athena diagnostics . bird holds a patent for genetic testing technology for cmt1c and sca14 and has received research support from the department of veterans ' affairs . siskind has received speaker honoraria from the national society of genetic counselors and invitae and has received research support from the muscular dystrophy association . european journal of physiology and the journal of molecular biology and has received research support from nih grant 1r01gm081340 and nih grant 2r01dc002281 . sumner has served on the scientific advisory boards of families of spinal muscular atrophy and avexis ; has received travel funding / speaker honoraria from the massachusetts general hospital , the european neuromuscular centre , thomas jefferson university , nyu school of medicine , carver college of medicine , and the university of california los angeles school of medicine ; has served on the editorial boards of experimental neurology and the annals of clinical and translational neurology ; coholds a patent for modulation of smn expression ; has been a consultant for avexis , pfizer , isis pharmaceuticals , roche , ptc pharmaceuticals , biogen idec , and the sma foundation ; and has received research support from nih / ninds r211ns087579 , muscular dystrophy association 2777551 , fight sma foundation and gwendolyn strong foundation emerging investigator award , spinal muscular atrophy research team ( smart ) , families of sma research award , and the spinal muscular atrophy foundation .
objective : to characterize 2 novel trpv4 mutations in 2 unrelated families exhibiting the charcot - marie - tooth disease type 2c ( cmt2c ) phenotype.methods:direct cmt gene testing was performed on 2 unrelated families with cmt2c . a 4-fold symmetric tetramer model of human trpv4 
 was generated to map the locations of novel trpv4 mutations in these families relative to previously identified disease - causing mutations ( neuropathy , skeletal dysplasia , and osteoarthropathy ) . 
 effects of the mutations on trpv4 expression , localization , and channel activity were determined by immunocytochemical , immunoblotting , ca2 + imaging , and cytotoxicity assays.results:previous studies suggest that neuropathy - causing mutations occur primarily at arginine residues on the convex face of the trpv4 ankyrin repeat domain ( ard ) . further highlighting the key role of this domain in trpv4-mediated hereditary neuropathy 
 , we report 2 novel heterozygous missense mutations in the trpv4-ard convex face ( p.arg237gly and p.arg237leu ) . 
 generation of a model of the trpv4 homotetramer revealed that while ard residues mutated in neuropathy ( including arg237 ) are likely accessible for intermolecular interactions , skeletal dysplasia  causing trpv4 mutations occur at sites suggesting disruption of intramolecular and/or intersubunit interactions . 
 like previously described neuropathy - causing mutations , the p.arg237gly and p.arg237leu substitutions do not alter trpv4 subcellular localization in transfected cells but cause elevations of cytosolic ca2 + levels and marked cytotoxicity.conclusions:these findings expand the number of ard residues mutated in trpv4-mediated neuropathy , providing further evidence of the central importance of this domain to trpv4 function in peripheral nerve .