shao et al (2016)

13
A Germline Variant in the PANX1 Gene Has Reduced Channel Function and Is Associated with Multisystem Dysfunction * Received for publication, January 27, 2016, and in revised form, April 12, 2016 Published, JBC Papers in Press, April 15, 2016, DOI 10.1074/jbc.M116.717934 Qing Shao ‡1 , Kristin Lindstrom §1 , Ruoyang Shi 1,2 , John Kelly , Audrey Schroeder**, Jane Juusola ‡‡ , Kara L. Levine ‡‡ , Jessica L. Esseltine , Silvia Penuela , Michael F. Jackson , and Dale W. Laird ‡3 From the Department of Anatomy and Cell Biology, The University of Western Ontario, London, Ontario N6A 5C1, Canada, the § Division of Genetics and Metabolism, Phoenix Children’s Hospital, Phoenix, Arizona 85016 , the Department of Pharmacology and Therapeutics, University of Manitoba, Winnipeg, Manitoba R3E 0Z3, Canada, the Kleysen Institute for Advanced Medicine, Health Sciences Centre, Winnipeg, Manitoba R3E 0Z3, Canada, the **Division of Genetics, University of Rochester Medical Center, Rochester, New York 14642, and ‡‡ GeneDx, Gaithersburg, Maryland 20877 Pannexin1 (PANX1) is probably best understood as an ATP release channel involved in paracrine signaling. Given its ubiquitous expression, PANX1 pathogenic variants would be expected to lead to disorders involving multiple organ systems. Using whole exome sequencing, we discovered the first patient with a homozygous PANX1 variant (c.650G3 A) resulting in an arginine to histidine substitution at position 217 (p.Arg217His). The 17-year-old female has intellectual disability, sensorineural hearing loss requiring bilateral cochlear implants, skeletal defects, including kyphoscoliosis, and primary ovarian failure. Her consanguineous parents are each heterozygous for this var- iant but are not affected by the multiorgan syndromes noted in the proband. Expression of the p.Arg217His mutant in HeLa, N2A, HEK293T, and Ad293 cells revealed normal PANX1 gly- cosylation and cell surface trafficking. Dye uptake, ATP release, and electrophysiological measurements revealed p.Arg217His to be a loss-of-function variant. Co-expression of the mutant with wild-type PANX1 suggested the mutant was not dominant- negative to PANX1 channel function. Collectively, we demon- strate a PANX1 missense change associated with human disease in the first report of a “PANX1-related disorder.” Pannexins are a new class of large-pore channels that were discovered early in the new millennium (1, 2). Members of the gene family (PANX1, PANX2, and PANX3) are expressed in numerous organs, tissues, and cells with PANX1 being the most prevalent (3). Rodent Panx1 is an 41– 48-kDa protein with its broad range in size due to the fact that it is post-translationally modified in what is now referred to as Gly0, Gly1, and Gly2 species to reflect the degree of glycosylation (4 – 6). PANX1 oligomerizes into a hexamer that contains a large pore func- tioning at the cell surface to allow the passage of small mole- cules below 1000 daltons in size (7, 8). Although the scope of molecules that pass through PANX1 pores is likely broad (9, 10), the functional consequence of ATP release via these chan- nels is best understood (11). For instance, PANX1 channels have been shown to release ATP in apoptotic immune cells as “find me” signals for the clearing of dying cells (12). In the last decade, PANX1 channels have become intimately linked to disease because of the fact that they are expressed in the vast majority of human cell types (13). Until this study, the link to disease has been associated with basal or elevated func- tional levels of PANX1, but the mechanisms involved remain poorly understood (13). In the first reported association with disease, PANX1 was linked to neuronal cell death in models of ischemia and stroke followed later by clear linkages to seizure severity and duration (14 –16). The abundant expression of PANX1 in enteric neurons led to the discovery that these chan- nels played vital roles in inflammatory bowel diseases, includ- ing ulcerative colitis and Crohn’s disease (17). Surprisingly, PANX1 channels can also be hijacked by viruses to facilitate infection as documented for HIV-1 (18). Furthermore, in mouse models, high levels of Panx1 in melanomas have been shown to facilitate disease progression, although Panx1 overex- pression has been shown to be tumor suppressive in glioblasto- mas, suggesting that pannexins are likely to have tumor-specific effects in cancer (19 –21). The list of connections between PANX1 and disease is extensive and continues to grow as there are elegant studies supporting a link between PANX1 and epi- lepsy (22, 23), glaucoma (24), migraines (25), Alzheimer disease (26), and diabetes (27). Although no disease-linked germline PANX1 variants have been identified prior to this study, Kwak and co-workers (28), including a member from our team, discovered through sequencing of 96 healthy patients that a single nucleotide poly- morphism (400A3 C) existed with a frequency of approxi- mately one-third 400A allele and two-thirds 400C allele. Although none exhibited overt disease, those homozygous for the 400C allele exhibited greater collagen-induced platelet aggregation, suggesting the possibility that there may be some variability in platelet reactivity among healthy individuals (28). In this study we report on the first patient with a PANX1 homozygous germline variant resulting in an arginine at posi- tion 217 being replaced with a histidine (p.Arg217His). This young female patient clinically presents with extensive disease that includes intellectual disabilities, severe hearing loss, and * This work was supported in part by Canadian Institutes of Health Research Grants 130530 (to D. W. L. and S. P.) and 125901 (to M. F. J.). The authors declare that they have no conflicts of interest with the contents of this article. This article was selected as a Paper of the Week. 1 These authors contributed equally to this work. 2 Recipient of a postdoctoral fellowship from Research Manitoba. 3 To whom correspondence should be addressed: Dept. of Anatomy and Cell Biology, University of Western Ontario, London, Ontario N6A-5C1, Canada. Tel.: 519-661-2111, ex. 86827; E-mail: [email protected]. crossmark THE JOURNAL OF BIOLOGICAL CHEMISTRY VOL. 291, NO. 24, pp. 12432–12443, June 10, 2016 © 2016 by The American Society for Biochemistry and Molecular Biology, Inc. Published in the U.S.A. 12432 JOURNAL OF BIOLOGICAL CHEMISTRY VOLUME 291 • NUMBER 24 • JUNE 10, 2016 at University of Manitoba Libraries on June 20, 2016 http://www.jbc.org/ Downloaded from

Upload: manitoba-neuroscience-network

Post on 03-Aug-2016

239 views

Category:

Documents


6 download

DESCRIPTION

A Germline Variant in the PANX1 Gene Has Reduced ChannelFunction and Is Associated with Multisystem Dysfunction 2016 Published, JBC Papers in Press, April 15, 2016, DOI 10.1074/jbc.M116.717934 Qing Shao‡1, Kristin Lindstrom§1, Ruoyang Shi¶1,2, John Kelly‡, Audrey Schroeder**, Jane Juusola‡‡, Kara L. Levine‡‡, Jessica L. Esseltine‡, Silvia Penuela‡, Michael F. Jackson¶, and Dale W. Laird‡3 From the ‡Department of Anatomy and Cell Biology, The University of Western Ontario, London, Ontario N6A 5C1, Canada, the §Division of Genetics and Metabolism, Phoenix Children’s Hospital, Phoenix, Arizona 85016 , the ¶Department of Pharmacology and Therapeutics, University of Manitoba, Winnipeg, Manitoba R3E 0Z3, Canada, the Kleysen Institute for Advanced Medicine, Health Sciences Centre, Winnipeg, Manitoba R3E 0Z3, Canada, the **Division of Genetics, University of Rochester Medical Center, Rochester, New York 14642, and ‡‡GeneDx, Gaithersburg, Maryland 20877

TRANSCRIPT

Page 1: Shao et al (2016)

A Germline Variant in the PANX1 Gene Has Reduced ChannelFunction and Is Associated with Multisystem Dysfunction*�

Received for publication, January 27, 2016, and in revised form, April 12, 2016 Published, JBC Papers in Press, April 15, 2016, DOI 10.1074/jbc.M116.717934

Qing Shao‡1, Kristin Lindstrom§1, Ruoyang Shi¶�1,2, John Kelly‡, Audrey Schroeder**, Jane Juusola‡‡,Kara L. Levine‡‡, Jessica L. Esseltine‡, Silvia Penuela‡, Michael F. Jackson¶�, and Dale W. Laird‡3

From the ‡Department of Anatomy and Cell Biology, The University of Western Ontario, London, Ontario N6A 5C1, Canada, the§Division of Genetics and Metabolism, Phoenix Children’s Hospital, Phoenix, Arizona 85016 , the ¶Department of Pharmacologyand Therapeutics, University of Manitoba, Winnipeg, Manitoba R3E 0Z3, Canada, the �Kleysen Institute for Advanced Medicine,Health Sciences Centre, Winnipeg, Manitoba R3E 0Z3, Canada, the **Division of Genetics, University of Rochester Medical Center,Rochester, New York 14642, and ‡‡GeneDx, Gaithersburg, Maryland 20877

Pannexin1 (PANX1) is probably best understood as an ATPrelease channel involved in paracrine signaling. Given itsubiquitous expression, PANX1 pathogenic variants would beexpected to lead to disorders involving multiple organ systems.Using whole exome sequencing, we discovered the first patientwith a homozygous PANX1 variant (c.650G3A) resulting in anarginine to histidine substitution at position 217 (p.Arg217His).The 17-year-old female has intellectual disability, sensorineuralhearing loss requiring bilateral cochlear implants, skeletaldefects, including kyphoscoliosis, and primary ovarian failure.Her consanguineous parents are each heterozygous for this var-iant but are not affected by the multiorgan syndromes noted inthe proband. Expression of the p.Arg217His mutant in HeLa,N2A, HEK293T, and Ad293 cells revealed normal PANX1 gly-cosylation and cell surface trafficking. Dye uptake, ATP release,and electrophysiological measurements revealed p.Arg217Histo be a loss-of-function variant. Co-expression of the mutantwith wild-type PANX1 suggested the mutant was not dominant-negative to PANX1 channel function. Collectively, we demon-strate a PANX1 missense change associated with human diseasein the first report of a “PANX1-related disorder.”

Pannexins are a new class of large-pore channels that werediscovered early in the new millennium (1, 2). Members of thegene family (PANX1, PANX2, and PANX3) are expressed innumerous organs, tissues, and cells with PANX1 being the mostprevalent (3). Rodent Panx1 is an �41– 48-kDa protein with itsbroad range in size due to the fact that it is post-translationallymodified in what is now referred to as Gly0, Gly1, and Gly2species to reflect the degree of glycosylation (4 – 6). PANX1oligomerizes into a hexamer that contains a large pore func-tioning at the cell surface to allow the passage of small mole-cules below 1000 daltons in size (7, 8). Although the scope of

molecules that pass through PANX1 pores is likely broad (9,10), the functional consequence of ATP release via these chan-nels is best understood (11). For instance, PANX1 channelshave been shown to release ATP in apoptotic immune cells as“find me” signals for the clearing of dying cells (12).

In the last decade, PANX1 channels have become intimatelylinked to disease because of the fact that they are expressed inthe vast majority of human cell types (13). Until this study, thelink to disease has been associated with basal or elevated func-tional levels of PANX1, but the mechanisms involved remainpoorly understood (13). In the first reported association withdisease, PANX1 was linked to neuronal cell death in models ofischemia and stroke followed later by clear linkages to seizureseverity and duration (14 –16). The abundant expression ofPANX1 in enteric neurons led to the discovery that these chan-nels played vital roles in inflammatory bowel diseases, includ-ing ulcerative colitis and Crohn’s disease (17). Surprisingly,PANX1 channels can also be hijacked by viruses to facilitateinfection as documented for HIV-1 (18). Furthermore, inmouse models, high levels of Panx1 in melanomas have beenshown to facilitate disease progression, although Panx1 overex-pression has been shown to be tumor suppressive in glioblasto-mas, suggesting that pannexins are likely to have tumor-specificeffects in cancer (19 –21). The list of connections betweenPANX1 and disease is extensive and continues to grow as thereare elegant studies supporting a link between PANX1 and epi-lepsy (22, 23), glaucoma (24), migraines (25), Alzheimer disease(26), and diabetes (27).

Although no disease-linked germline PANX1 variants havebeen identified prior to this study, Kwak and co-workers (28),including a member from our team, discovered throughsequencing of 96 healthy patients that a single nucleotide poly-morphism (400A3C) existed with a frequency of approxi-mately one-third 400A allele and two-thirds 400C allele.Although none exhibited overt disease, those homozygous forthe 400C allele exhibited greater collagen-induced plateletaggregation, suggesting the possibility that there may be somevariability in platelet reactivity among healthy individuals (28).

In this study we report on the first patient with a PANX1homozygous germline variant resulting in an arginine at posi-tion 217 being replaced with a histidine (p.Arg217His). Thisyoung female patient clinically presents with extensive diseasethat includes intellectual disabilities, severe hearing loss, and

* This work was supported in part by Canadian Institutes of Health ResearchGrants 130530 (to D. W. L. and S. P.) and 125901 (to M. F. J.). The authorsdeclare that they have no conflicts of interest with the contents of thisarticle.

� This article was selected as a Paper of the Week.1 These authors contributed equally to this work.2 Recipient of a postdoctoral fellowship from Research Manitoba.3 To whom correspondence should be addressed: Dept. of Anatomy and Cell

Biology, University of Western Ontario, London, Ontario N6A-5C1, Canada.Tel.: 519-661-2111, ex. 86827; E-mail: [email protected].

crossmarkTHE JOURNAL OF BIOLOGICAL CHEMISTRY VOL. 291, NO. 24, pp. 12432–12443, June 10, 2016

© 2016 by The American Society for Biochemistry and Molecular Biology, Inc. Published in the U.S.A.

12432 JOURNAL OF BIOLOGICAL CHEMISTRY VOLUME 291 • NUMBER 24 • JUNE 10, 2016

at University of M

anitoba Libraries on June 20, 2016

http://ww

w.jbc.org/

Dow

nloaded from

Page 2: Shao et al (2016)

multiple other multisystem defects. Her unaffected parents andsibling are heterozygous for the c.650G3A PANX1 variant.Generation and characterization of the R217H mutant revealedthat it is a loss-of-function variant as assessed by ATP release,dye uptake, and electrophysiological evaluation of the channelproperties. Although functional levels of PANX1 have beencorrelated to the onset and/or progression of over 10 diseases(13), disease-linked germline variants in the PANX1 gene havenot been previously reported. This study represents the firstreport of a patient harboring a disease-associated PANX1variant.

Experimental Procedures

Sequencing—Genomic DNA was extracted from whole bloodfrom the proband and her parents. Exome sequencing was per-formed on exon targets captured using the Agilent SureSelectHuman All Exon V4 (50 Mb) kit (Agilent Technologies, SantaClara, CA). The sequencing methodology and variant interpre-tation protocol has been previously described (29). Briefly,whole exome sequencing (WES)4 produced 18.2 Gb ofsequencing data for the proband. Mean coverage of capturedregions was �251� for the proband’s sample, with �99.17%covered with at least 10� coverage, an average of 89.82% of basecall quality of Q30 or greater, and an overall average mean qual-ity score of Q35. Filtering of common single nucleotide poly-morphisms (�10% frequency present in the 1000 Genomesdatabase) resulted in �4187 variants in the proband sample.After automated filtering of variants with a minor allele fre-quency of �10%, manual curation was performed to filter lesscommon variants with a minor allele frequency of 1–10% andsingle variants in genes inherited from unaffected parents andto evaluate predicted effects of rare variants and associatedhuman conditions.

Cells and Reagents—Normal rat kidney (NRK), mouseNeuro2A (N2A), and human embryonic kidney (HEK293T)cells were obtained from the American Type Culture Collec-tion (Manassas, VA). Ad293 cells (derivative of human embry-onic kidney 293 cells with improved cell adherence) wereobtained from Agilent Technologies (Palo Alto, CA). All cellswere grown in high glucose (4500 mg of glucose/liter) DMEM(Invitrogen) supplemented with 10% fetal bovine serum, 100units/ml penicillin, 100 �g/ml streptomycin, and 2 mM L-gluta-mine. Cells were cultured within a humidified environmentthat maintained 5% CO2 and a temperature of 37 °C. Probene-cid (catalog no. P36400) was purchased from Molecular Probes,and ATP (catalog no. PV3227) was obtained from Thermo-Fisher Scientific. Carbenoxolone (catalog no. C4790) and Dul-becco’s phosphate-buffered saline (catalog no. D1283) wereobtained from Sigma.

Mutant DNA Constructs—In humans, the gene encodingpannexin 1 is referred to as PANX1. Panx1 is used when refer-ring to the equivalent mouse gene, and the encoded proteins arenot italicized. An expression vector encoding human PANX1was purchased from InvivoGen (pUNO1-hPanx1). The R217H

PANX1 mutant encoding expression vector was generated byspecial order to Norclone Biotech Industries (London, Ontario,Canada). The fidelity of the PANX1- and R217H-encodingvectors was confirmed by DNA sequencing at the RobartsResearch Institute DNA Sequencing Facility (London, Ontario,Canada) using an Applied Biosystems (Foster City, CA) 3730analyzer.

Transfections and Immunofluorescent Labeling—Transienttransfections were performed using Lipofectamine2000 (LifeTechnologies, Inc.) according to the manufacturer’s instruc-tions. For some experiments, a GFP-encoding vector was co-transfected with the PANX1/R217H-encoding vector. Forstable transfections, cells were kept under G418/blasticidinselection pressure.

For electrophysiological experiments, pUNO1-PANX1 (2�g) or pUNO1-R217H (2 �g) plasmid DNA was co-transfectedwith pLB-GFP DNA (0.6 �g) and pCDNA3.1 (7 �g; carrierDNA) using JetPRIME� (Polyplus-transfection Inc., New York)according to the manufacturer’s instructions. In some cases,equal concentrations of plasmids encoding PANX1 and R217Hwere used in the transfection to determine whether the mutantaffected the function of wild-type PANX1. For these experi-ments, pUNO1-PANX1 (2 �g) plus pUNO1-R217H (2 �g)plasmid DNA was co-transfected with pLB-GFP DNA (0.6 �g)and pCDNA3.1 (7 �g; carrier DNA) using JetPRIME�.HEK293T cells not transfected with either PANX1 or R217Hserved as negative controls (untransfected). In this case, cellswere mock-transfected with only pLB-GFP DNA (0.6 �g) andpCDNA3.1 (7 �g; carrier DNA).

HEK293T cells, maintained as described above but with noantibiotics, were plated onto 100-mm dishes and transfectedwhen reaching 60 – 80% confluence. Transfected cells weresubsequently resuspended and seeded onto 35-mm dishes forelectrophysiological recordings. The remaining sister cells wereused for immunoblotting, as described below.

For immunolabeling studies, cells were fixed in 80% metha-nol, 20% acetone for 20 min at 4 °C and blocked for 30 min with2% BSA. For PANX1, we used an affinity-purified custom-maderabbit anti-human PANX1 polyclonal antibody (PANX1CT-412, 0.5 �g/ml), generated by Genemed Synthesis (SanFrancisco) against the C-terminal sequence of human PANX1(412NGEKNARQRLLDSSC426) (30). An anti-Cx43 monoclonalantibody (1:50 dilution; P4G9 from Dr. Paul D. Lampe, FredHutchinson Cancer Research Center, Seattle) was also used.Primary antibody labeling was followed by fluorescently taggedsecondary antibodies Alexa Fluor� 555 and Alexa Fluor� 488(Life Technologies, Inc.; diluted 1:500) for 1 h at room temper-ature. Cell nuclei were stained for 5 min with TO-PRO�-3Iodide (642/661) (Life Technologies, Inc.) and then rinsed indistilled H2O before mounting. All labeling was visualized withan LSM 510 META inverted confocal microscope equippedwith a 63� oil objective (Carl Zeiss, Jena, Germany).

Western Blot—Cells were solubilized using cell lysis buffer(1% Triton X-100, 150 mM NaCl, 10 mM Tris/HCl (pH 7.4), 1mM EDTA, 1 mM EGTA, and 0.5% Nonidet P-40, supplementedwith protease inhibitor mixture (CompleteMini, RocheApplied Science)) and phosphatase inhibitors (50 mM sodiumfluoride and 0.5 mM sodium orthovanadate). Protein concen-

4 The abbreviations used are: WES, whole exome sequencing; ANOVA, anal-ysis of variance; NRK, normal rat kidney; BFA, brefeldin A; CBX,carbenoxolone.

Disease-associated Loss-of-function Panx1 Variant

JUNE 10, 2016 • VOLUME 291 • NUMBER 24 JOURNAL OF BIOLOGICAL CHEMISTRY 12433

at University of M

anitoba Libraries on June 20, 2016

http://ww

w.jbc.org/

Dow

nloaded from

Page 3: Shao et al (2016)

trations were determined using a BCA protein determinationkit (Pierce). Cleared lysates were subjected to SDS-PAGE andimmunoblotted with a rabbit anti-PANX1 antibody (PANX1CT-412, 0.25 �g/ml) or a mouse anti-�-tubulin (0.4 �g/ml,Sigma, catalog no. T8328) antibody at 4 °C overnight. Primaryantibodies were detected using the fluorescently conjugatedanti-rabbit Alexa Fluor� 680 (1:10,000 dilution, LI-COR Biosci-ences) or anti-mouse IRDye 800 (1:10,000 dilution, RocklandImmunochemicals, Inc.) antibodies and scanned using theOdyssey Infrared Imaging System (LI-COR Biosciences).

Methods used by the University of Manitoba group weresimilar to that of the London group. Briefly, cells were solubi-lized in lysis buffer containing 1% Triton X-100, 0.1% SDS, 0.5%sodium deoxycholate, 150 mM NaCl, 50 mM Tris/HCl (pH 7.4),10 mM EDTA. An antibody recognizing �-actin (HRP conju-gated anti-�-actin, 1:10,000 dilution, Sigma) was used as a load-ing control. Immunoblots were imaged using a ChemiDoc MPsystem (Bio-Rad).

Cell Surface Biotinylation—Ad293 or N2A cells were tran-siently transfected with the cDNAs encoding wild-type PANX1or the R217H mutant. Forty eight hours after transfection, cellswere treated for 40 min with either DMEM � 5% fetal bovineserum (FBS) (control) or DMEM/FBS containing 140 mM

K�Glu and 100 mM K�Cl�. Cells were then placed on ice,washed in ice-cold Hanks’ balanced salt solution, and cell sur-face proteins labeled with 1.5 mg/ml EZLink Sulfo-NHS-SS-biotin (ThermoFisher catalog no. 21331) for 1 h. Biotin wassubsequently quenched with 100 mM glycine for 30 min, andcells were lysed. Cleared supernatant containing 150 –250 �g ofprotein was incubated with 25 �l of NeutrAvidin affinity beads(ThermoFisher catalog no. 29200) overnight, rotating at 4 °C toprecipitate biotin-labeled proteins. Following incubation, thebeads were washed twice with PBS, and precipitated proteinswere separated by SDS-PAGE, transferred to a nitrocellulosemembrane, and immunoblotted to identify biotinylatedPANX1 proteins using a primary rabbit anti-human PANX1antibody (1:1000 dilution). Total PANX1 and GAPDH levelswere determined by immunoblotting 20 �g of protein fromeach cell lysate used for biotinylation and the input.

ATP Release Assay—N2A cells were transfected to expresswild-type PANX1 or the R217H mutant and subcultured into24-well dishes. Forty eight hours post-transfection, the cellmedium was exchanged to medium with 5% FBS (heat-inactive)plus 20 �M ARL67156 (Santa Cruz Biotechnology, Santa Cruz,CA). ATP release was stimulated with 140 mM potassium glu-conate and 100 mM KCl (“High K�” solution) for 20 min. Inaddition, some cells were pre-treated with 70 �M carbenox-olone (CBX, Sigma) to block the opening of PANX1 channels(31, 32). Samples were taken and processed for ATP contentusing a bioluminescence ATP determination kit (Life Technol-ogies, Inc.). Briefly, 10 �l of the cell supernatants were trans-ferred to 96-well dishes with 90 �l of a luciferase standard reac-tion solution, according to the manufacturer’s protocol. ATPreleased into the medium was quantitatively measured by lumi-nometry. Although cells were plated at equal numbers, toaccount for any unexpected differences in cell numbers, lumi-nescence values were normalized to the total amount of proteinfor each well (as determined by a BCA assay, Pierce), and ATP

concentrations were then determined from an ATP standardcurve. Cell-free solutions were tested in the presence of differ-ent concentrations of ATP as a control. Plotted values aremeans � S.E. * � 0.05, ** � 0.01, and *** � 0.001 asdetermined by one-way ANOVA analysis followed by a Bonfer-roni corrected post hoc test (n � 3). All statistical analyses wereperformed using GraphPad version 4.1.

Dye Uptake Assay—Ad293 cells were transiently and/or sta-bly engineered to express GFP together with wild-type PANX1or the R217H mutant. Briefly, cells were sparsely subculturedon 35-mm glass-bottom dishes (MatTek Corp., Ashland, MA)for 1 day prior to the medium being replaced with 5% FBS(heat-inactive), DMEM containing 140 mM potassium gluco-nate, 100 mM KCl, and 10 �M ethidium bromide (EtBr) (7, 33,34). To block PANX1 channels, cells were treated with 1 mM

probenecid (35). Rapid time-lapse imaging was used to detectEtBr uptake and GFP fluorescence every 5 min for up to 40 minusing a Zeiss LSM 510 META imaging system. Mean fluores-cence intensity was measured using Zen lite (Zeiss). For eachtreatment, 10 –50 cells were analyzed for mean fluorescenceintensity at 0, 5, 30, and 40 min after exposure to the dye. Dataare presented as means � S.E. *, p 0.05; **, p 0.01; ***, p 0.001 PANX-expressing cells compared with wild-type orR217H mutant-expressing cells as determined by a one-wayANOVA followed by Tukey’s Multiple Comparison Test. Sta-tistical analysis was performed using GraphPad version 4.1.

Electrophysiology—Tight-seal whole-cell recordings wereconducted at room temperature 24 –72 h after transfectionusing an Axon MultiClamp 700A patch clamp amplifier andDigidata 1322A data acquisition system (Molecular Devices).On the day of experiments, recordings were performed using aninterleaved design from 293T cells expressing wild-typePANX1, R217H, or both prepared in parallel. Transfected cellswere visually selected for recording on the basis of GFP fluores-cence. Using a gravity-driven multi-barreled perfusion system,cells were continuously superfused with bath solution(extracellular fluid) containing 140 mM NaCl, 5.4 mM KCl, 2 mM

CaCl2, 1 mM MgCl2, 33 mM D-glucose, and 25 mM HEPES,adjusted to pH 7.4 (with NaOH), and osmolarity between 310and 320 mosM/liter. Patch pipettes were pulled from borosili-cate glass (World Precision Instruments, Inc., Sarasota, FL) andhad a resistance of 4 –5 megohms when filled with a pipettesolution (intracellular fluid) containing 150 mM cesium gluco-nate, 2 mM MgCl2, and 10 mM HEPES, adjusted to pH 7.3 (withCsOH), and osmolarity between 290 and 300 mosM/liter. Forthe high potassium treatment, a modified bath solution wasapplied containing 90 mM NaCl, 50 mM potassium gluconate,5.4 mM KCl, 2 mM CaCl2, 1 mM MgCl2, 33 mM D-glucose, and 25mM HEPES, adjusted to pH 7.4 (with NaOH), and osmolaritybetween 310 and 320 mosM/liter.

In cells voltage clamped at �60 mV, PANX1 and R217Hcurrents were recorded in response to 500-ms voltage ramps(�100 mV, 1/10 s). PANX1 and R217H ramp currents werefirst recorded in normal bath solution (or high potassium solu-tion) for 5 min and then inhibited by a bath solution (normal orhigh potassium) containing 100 �M carbenoxolone. Signals, fil-tered at 2 kHz and sampled at 10 kHz, were collected and ana-lyzed using Clampex 9.2 and Clampfit 9.2 software (Molecular

Disease-associated Loss-of-function Panx1 Variant

12434 JOURNAL OF BIOLOGICAL CHEMISTRY VOLUME 291 • NUMBER 24 • JUNE 10, 2016

at University of M

anitoba Libraries on June 20, 2016

http://ww

w.jbc.org/

Dow

nloaded from

Page 4: Shao et al (2016)

Devices). Data are presented as means � S.E. Statistical analy-ses were conducted using GraphPad Prism 5.0 software(GraphPad Software, Inc., La Jolla, CA). Comparisons betweenPANX1 and R217H groups were made using two-way ANOVAanalyses with Bonferroni corrected post hoc test. Comparisonsbetween PANX1, R217H, and the co-expression groups weremade using one-way ANOVA analyses with Bonferroni cor-rected post hoc test. A difference was considered significant at*, p 0.05; **, p 0.01; ***, p 0.001.

In other experiments on the day of recordings, 293T cellsexpressing wild-type PANX1 or R217H were first pretreatedwith brefeldin A (BFA) (5 �g/ml; Tocris Bioscience, UK)-con-taining medium for 6 h at 37 °C, a treatment regimen shownpreviously to prevent ion channel forward trafficking (36).Later, the culture medium was removed and replaced by a bathsolution containing 5 �g/ml BFA right before the recording.

Results

Clinical Presentation—We report on a 17-year-old femalewho initially presented for clinical genetics evaluation at 15years of age with multiple concerns, including primary ovarianfailure, intellectual disability, sensorineural hearing loss, andkyphosis. She had significant speech delay that led to the diag-nosis of severe sensorineural hearing loss at 15 months of age,requiring bilateral cochlear implants at age 6. Despite theimplants, her receptive and expressive language remainsdelayed with nearly absent speech and the ability to use onlybasic signs. She is unable to read or to count past 30. Formalpsychological testing at 16 years of age revealed both verbalcomprehension index and perceptual reasoning indices of 45(less than 2nd percentile) on the Wechsler Intelligence Scale forChildren (WISC-IV), commensurate with a child who is 6 yearsold, and academic skills at a first grade level, although it wasnoted that this test is not specifically designed for assessingstudents who are deaf or hard-of-hearing. She is mainly inde-

pendent with basic activities of daily living but does requiresome help with bathing, cooking, oral care, managing medica-tions, and, occasionally, toileting. Primary ovarian failure wasdiagnosed via elevated follicle-stimulating hormone (108.6mIU/ml), luteinizing hormone (23.7 mIU/ml), and low estra-diol ( 5 pg/ml), in the setting of primary amenorrhea withnormal pelvic ultrasound and secondary sexual characteristics.Further endocrine evaluation revealed that she had a delayedbone age, hypothyroidism, and ultrasound evidence of fattyliver disease associated with mild transaminitis of unclear eti-ology. Kyphosis was first noticed in early adolescence andimproved, but did not resolve, with physical therapy. Otherinvestigations included a normal echocardiogram, renal ultra-sound, and ophthalmic exam, although her family reports somesuspected issues with night vision. Physical examination at theage of 16 years revealed mild short stature with a height of 150.2cm (3rd percentile) and weight of 46.8 kg (12th percentile). Shewas not facially dysmorphic and generally resembled her imme-diate family, although she did have kyphosis and bilateral 2–3toe syndactyly. The family declined the inclusion of patientphotos in this report.

Family History and Gene Sequencing—Family history wasnotable for one sister, 2 years her elder, with mild short stature(height 150 cm) and hypothyroidism, her mother with mildshort stature (height 155 cm), and her father having hypothy-roidism and fatty liver disease (Fig. 1). The proband’s parentsare known to be first cousins, originally from Turkey. The fam-ily is not aware of any other consanguineous relationships fur-ther back in the pedigree. Genetic evaluation of the probanddemonstrated a normal chromosome analysis. Chromosomemicroarray revealed no microdeletions or duplications but didshow 139.4 Mb of homozygosity spread over seven chromo-somes, consistent with the known consanguinity. Given hercomplex phenotype, the possibility of a new genetic syndrome

FIGURE 1. Three generation pedigree demonstrating consanguinity, with the proband’s parents as first cousins. Selected clinical conditions are noted.Full shading represents the homozygous R217H variants, and half-shading represents heterozygous R217H PANX1 variants. Arrow indicates proband; “�”indicates wild-type allele.

Disease-associated Loss-of-function Panx1 Variant

JUNE 10, 2016 • VOLUME 291 • NUMBER 24 JOURNAL OF BIOLOGICAL CHEMISTRY 12435

at University of M

anitoba Libraries on June 20, 2016

http://ww

w.jbc.org/

Dow

nloaded from

Page 5: Shao et al (2016)

was considered, and whole exome sequencing of the patientand her parents was performed. A homozygous missense vari-ant in PANX1, c.650G3A (R217H) was identified, with eachparent and her sister found to be heterozygous. These findingswere confirmed by Sanger sequencing. This variant is predictedto be deleterious by PhyloP, PolyPhen2, SIFT, CADD, andMutationTaster and is present in the Exome Aggregation Con-sortium (ExAC) Browser with an allele frequency of 1/10,000(6.65e-05). The PANX1 gene is within one of her regions ofhomozygosity, on chromosome 11.

Expression and Localization of the R217H Mutant—Giventhat arginine at position 217 is conserved in vertebrates, and insilico analysis predicts the motif containing this residue is likelyof structural and/or functional importance, we engineered andsequence-confirmed this mutation into the pUNO1-hPANX1expression vector. Expression of the R217H mutant in mouseneuroblastoma (N2A) and NRK cells revealed that the mutanttrafficked to the cell surface (as revealed by the location of theCx43 gap junction protein) with seemingly identical efficacy aswild-type PANX1 (Fig. 2A). The R217H mutant was glycosy-lated to the well documented Gly1 and Gly2 species (4, 30, 37)of PANX1 when expressed in N2A, 293T, and Ad293-human

embryonic kidney cells (Fig. 2B). This was not unexpected giventhat the topological location of the mutation is within the intra-cellular loop of PANX1 (Fig. 2C), whereas glycosylation occurson the second extracellular loop (4, 30). Thus, through the useof several cell lines to eliminate any potential cell type differ-ences, these studies strongly suggest that the R217H mutant istrafficking-competent and appropriately glycosylated.

Functional Analysis of the R217H Mutant—Because PANX1channels are large-pore channels (7, 9, 32) suitable for theuptake of small fluorescent dyes (38, 39), we assessed whetherchannels formed from the R217H mutant were functionallycompromised. Ad293 cells expressing wild-type PANX1 or theR217H mutant (Fig. 3A) were assessed for ethidium bromide(EtBr) dye uptake (9) after channels were induced open in highpotassium medium (Fig. 3B) (7, 34, 40). Quantitative assess-ment of EtBr uptake revealed that cells expressing the R217Hmutant exhibited significantly reduced dye uptake over a timecourse of 5– 40 min (Fig. 3B). Confirming the involvement ofPANX1-based channels, the pannexin channel blocker probe-necid (35) was found to block dye uptake in cells expressingPANX1. Moreover, in untransfected Ad293 cells, high potas-sium treatment did not stimulate dye uptake. These studies

FIGURE 2. Disease-linked R217H mutant exhibits no characteristic difference in cellular localization or glycosylated isoforms in comparison withwild-type PANX1. A, N2A and NRK cells were engineered to express wild-type PANX1 or the R217H mutant and immunostained for the location of PANX1 (red)or the gap junction protein, Cx43 (green). Arrow indicates the R217H mutant at the cell surface with no apposing cells. Nuclei were stained with TO-PRO�-3(blue). Bars, 10 �m. B, untransfected N2A, 293T, and Ad293 cells or cells expressing wild-type PANX1 or the R217H mutant were immunoblotted for PANX1 orthe gel loading control �-tubulin. Note that all glycosylated species of wild-type PANX1 and the R217H mutant (Gly0, Gly1, and Gly2) are expressed. Molecularmass markers are shown, and the experiments were repeated across a minimum of three cell lines to eliminate any cell type differences that might exist. C,model of PANX1 illustrating the approximate topological position of the R217H variant (red sphere).

Disease-associated Loss-of-function Panx1 Variant

12436 JOURNAL OF BIOLOGICAL CHEMISTRY VOLUME 291 • NUMBER 24 • JUNE 10, 2016

at University of M

anitoba Libraries on June 20, 2016

http://ww

w.jbc.org/

Dow

nloaded from

Page 6: Shao et al (2016)

strongly suggest that channels formed from the R217H mutanthad impaired ability to uptake a small fluorescent dye.

As PANX1 channels are probably best known as ATP releasechannels (7, 12, 41), we next determined whether channelsassembled from the R217H mutant had reduced ability torelease ATP. To eliminate any confounding problems thatmight be due to ATP release from connexin-based hemichan-nels (42), we expressed the R217H mutant and wild-typePANX1 in connexin- and pannexin-deficient N2A cells (43)and assessed channel activity under physiological levels of cal-cium. As predicted, upon exposure to high potassium, there

was a surge of ATP release in N2A cells expressing wild-typePANX1 but not when cells expressed the R217H mutant (Fig.3C). To confirm that cell surface channels were indeed respon-sible for ATP release, the channel blocker CBX (12, 44) elimi-nated the surge of ATP release induced by high potassium. Var-iability in ATP release in control cells with and without PANX1or mutant may represent depletion of intracellular stores ofATP due to baseline activity of PANX1 channels. Nevertheless,these studies support the dye uptake data and strongly suggestthat the R217H variant greatly attenuates channel function.

To determine whether the R217H variant would alter ioniccurrent flow through the channel, we performed whole-cellvoltage clamp recordings. For these experiments, we made useof 293T cells, devoid of endogenously expressed CBX-sensitivecurrents (45, 46), and we assessed pannexin function by record-ing membrane currents generated by voltage ramps from �100to �100 mV. In 293T cells expressing R217H, ramp currentsrecorded at �100 mV were reduced by �50% compared withwild-type PANX1 (Fig. 4, A and B). The reversal potentials ofR217H (�71.77 � 4.415, n � 6) and PANX1 currents(�61.83 � 3.508, n � 6) were comparable, suggesting thatreduced currents in R217H-expressing cells could not be attrib-uted to a change in ionic permeability. Consistent with previousreports (7, 34, 40), PANX1-mediated currents were augmentedby treatment with high potassium, most notably at negativeholding potentials (Fig. 4C). High potassium augmented rampcurrents in R217H-expressing cells were reduced by �50%when compared with wild-type PANX1 (Fig. 4C). Ramp cur-rents recorded from R217H- and PANX1-expressing cells, incontrol or high potassium-containing solutions, were sup-pressed by CBX (Fig. 4, A–C). The absence of comparable CBX-sensitive currents in mock-transfected 293T cells (Fig. 4B) con-firms the specific involvement of pannexin channels under ourrecording conditions. To exclude the possibility that the loss-of-channel function was due to a dramatic reduction in theexpression of the R217H mutant, PANX1 immunoblottingconfirmed that both the mutant and wild-type PANX1 wereexpressed at equal levels in 293T cells prepared for electro-physiological measurements (Fig. 4D).

To further assess whether the R217H mutant was dominantto the functional channel properties of PANX1, we co-ex-pressed equal quantities of plasmids encoding both wild-typeand mutant PANX1. The ramp currents recorded at �100 mVwere reduced by �50% for the mutant compared with wild-typePANX1, although currents were not significantly reduced whenboth mutant and wild-type PANX1 were co-expressed (Fig. 5, Aand B). Similar findings were observed when channel functionwas augmented by high potassium (Fig. 5C) suggesting that theR217H mutant was not dominantly inhibiting the function ofPANX1. As before, carbenoxolone was effective in blockingPANX1 channel function as well as the residual mutant channelfunction (Fig. 5C).

Collectively, our functional assays monitoring dye, ATP, andion flux support that R217H represents a loss-of-channel-func-tion variant. Loss-of-function was evident in cells maintainedunder basal (i.e. reduced current amplitude in “normal” potas-sium) and high potassium-stimulated conditions. Loss-of-function with variants targeting channels that operate at the

FIGURE 3. R217H mutant exhibits defective dye uptake and ATP release.A, untransfected Ad293 cells or cells expressing wild-type PANX1 or theR217H mutant were immunolabeled for PANX1 (red), and all cells were coun-terstained for TO-PRO�-3 (blue). Bar, 10 �m. B, untransfected (UNTR) Ad293cells or cells expressing wild-type PANX1 or R217H, or expressing wild-typePANX1 and treated with probenecid were subjected to ethidium bromide(EtBr) dye uptake over a period of 40 min. Mean fluorescent measurementsrevealed that wild-type PANX1-expressing cells were capable of dye uptake,whereas R217H-expressing, untreated cells, or cells expressing wild-typePANX1 and treated with probenecid exhibited significantly reduced dyeuptake. *, p 0.05; ***, p 0.001, n � 3. C, untransfected (UNTR) N2A cells orN2A cells expressing wild-type PANX1 or the R217H mutant were assayed forATP release upon treatment with a high potassium (K�) medium containingor lacking the channel blocker CBX. ns, not significant. **, p 0.01, n � 3.

Disease-associated Loss-of-function Panx1 Variant

JUNE 10, 2016 • VOLUME 291 • NUMBER 24 JOURNAL OF BIOLOGICAL CHEMISTRY 12437

at University of M

anitoba Libraries on June 20, 2016

http://ww

w.jbc.org/

Dow

nloaded from

Page 7: Shao et al (2016)

cell surface can result from changes in channel function and/ordefects in folding and cell surface expression. Evidence demon-strating that R217H expression, glycosylation, and cell surfacetrafficking is unaltered suggests that reduced current ampli-tudes observed in the absence of high potassium stimulationcan be attributed to reduced channel function. However, withextended high potassium stimulation an additional mechanismcould be recruited, namely reduced stimulated delivery of pan-nexin channels to the cell surface. Although high potassium isreported to allosterically augment pannexin function in a rap-idly reversible manner (7, 47), a parallel increase in pannexinsurface expression via increased forward trafficking could alsocontribute to augmented pannexin function. In this context,reduced R217H function could be attributed in part to a deficitin stimulated forward trafficking despite unaltered steady statesurface expression. To address this possibility, we used cell sur-face biotinylation to first examine whether high potassiumtreatment altered the surface expression of R217H relative tothat of PANX1. As might be expected, high potassium did not

increase the amount of wild-type PANX1 or the R217H mutantat the cell surface of Ad293 or N2A cells (Fig. 6A). Furthermore,we undertook a series of whole-cell recordings from 293T cellsexpressing PANX1 or R217H, which were pretreated with BFA,to block protein transport from the endoplasmic reticulum tothe cell surface. Although current amplitude in cells expressingeither R217H or wild-type PANX1 channels was reduced byBFA treatment, likely reflecting reduced surface expression,R217H functional deficits at rest and in the presence of highpotassium were maintained (Fig. 6, B and C).

Discussion

The pannexin family of channel-forming glycoproteins hasgrown in importance given their documented roles in ischemia,stroke, overactive bladder, HIV infections, Crohn’s disease,platelet aggregation, and over a half-dozen other diseases (13,28, 45, 48, 49). In most of these cases, PANX1 was identified asbeing the key pannexin linked to the disease, but the causalmechanism of how PANX1 large-pore channels are associated

FIGURE 4. R217H mutant shows a dramatic reduction in channel function when expressed in HEK293T cells. A, representative current-voltage relation-ships recorded in the absence or presence of CBX in cells expressing wild-type PANX1 (top) and the R217H mutant (bottom). B, summary of ramp currentsrecorded at �100 mV from wild-type PANX1 and R217H mutant expressing or mock-transfected (UNTR) HEK293T cells. C, ramp currents at �60 mV recordedin HEK293T cells expressing wild-type PANX1 and the R217H mutant, before (control) and after treatment with high potassium (High K�) or high potassium andCBX. D, representative Western blot reveals comparable cellular expression of wild-type PANX1 and R217H channels in whole-cell lysates prepared from sisterHEK293T cells used in electrophysiological recordings (position of molecular mass standards are shown). For all panels, the number of cells recorded isindicated in parentheses. p values were calculated in comparison with PANX1 groups using two-way ANOVA analyses with Bonferroni post-tests (B and C), ***,p 0.001.

Disease-associated Loss-of-function Panx1 Variant

12438 JOURNAL OF BIOLOGICAL CHEMISTRY VOLUME 291 • NUMBER 24 • JUNE 10, 2016

at University of M

anitoba Libraries on June 20, 2016

http://ww

w.jbc.org/

Dow

nloaded from

Page 8: Shao et al (2016)

with disease susceptibility remains poorly understood (13).Given the breadth of diseases linked to PANX1 and its ubiqui-tous distribution, we predicted that loss- or gain-of-functionpathogenic variants in the PANX1 gene would likely be causallyrelated to disease, and the disease may involve many organs. Tothe best of our knowledge, this report is the first and only germ-line variant identified in any of the three pannexin-encodinggenes that has been associated with inherited human disabili-ties and disease. As anticipated, the proband has extensive andsevere multi-organ involvement that has affected her cognition,hearing, skeleton, and reproductive organs. These clinical pre-sentations are consistent with the current literature on Panx1expression and function in animal studies (3). One of the organswith the highest expression of Panx1 is the brain (21, 50). Neu-rons and glial cells of the central nervous system have beenshown to express Panx1 at early stages of development (51, 52),and therefore, the expression of a functionally impaired chan-nel may have an effect on neuronal development and differen-tiation. Similarly, Panx1 expression in the cochlea, primarily inepithelial cells of the organ of Corti, and neurons of the spiral

ganglia (49, 53), would predict a potentially important functionfor these channels in hearing. Consistent with severe hearingloss found in the proband, hearing loss was recently reported ina Panx1 conditional knock-out (KO) mouse from the cochlea(54, 55). At present, there is evidence of Panx1 expression inosteoblasts (56), but because Panx3 has been the most studiedpannexin in bone and cartilage (57), our knowledge of the roleof Panx1 in skeletal development remains limited. Likewise,although Panx1 has been reported in the ovaries and malereproductive organs, its functional importance is poorly under-stood (50, 58). However, based on the proband in this study, wesuggest that PANX1 plays a role in both the skeleton and repro-ductive organs, although it likely is not accountable for the pro-band’s short stature as her mother and sister have short statureas well. Also, in the absence of additional patients harboringhomozygous PANX1 gene mutations, we cannot definitivelyassign causal relationships to all the clinical presentations of theproband to PANX1 as this awaits further confirmation.

The severity of developmental abnormalities is in strikingcontrast to the lack of overt phenotype noted in homozygous

FIGURE 5. R217H mutant is not dominant-negative to co-expressed PANX1. A, representative current-voltage relationships recorded in HEK293T cellsexpressing PANX1, R217H, or both. B, summary of ramp currents recorded at �100 mV from wild-type PANX1, R217H, or PANX1/R217H co-expressing cells. C,ramp currents at �60 mV recorded in HEK293T cells expressing wild-type PANX1, the R217H mutant, or both, before (control) and after treatment with highpotassium (High K�) or high potassium and CBX. For all panels, the number of cells recorded is indicated in parentheses. *, p 0.05; **, p 0.01. ns, notsignificant.

Disease-associated Loss-of-function Panx1 Variant

JUNE 10, 2016 • VOLUME 291 • NUMBER 24 JOURNAL OF BIOLOGICAL CHEMISTRY 12439

at University of M

anitoba Libraries on June 20, 2016

http://ww

w.jbc.org/

Dow

nloaded from

Page 9: Shao et al (2016)

and heterozygous global Panx1 KO mice (3, 59) suggesting thatreduced expression by 50% or more does not phenocopy thehuman R217H variant. However, the lack of overt phenotypesin global Panx1 KO mice may be due, at least in part, to com-pensation by other pannexins. For example, when Panx1 wasconstitutively deleted in a Panx1 KO mouse, there was anincrease in Panx3 expression in dorsal skin (60). A similar up-regulation of Panx3 expression was observed in the wall of tho-racodorsal arteries of Panx1 KO mice compared with C57Bl/6controls (61). In another case, both Panx1 and Panx2 needed tobe deleted to observe neuroprotective effects in a mouse modelof ischemia because it appeared that one pannexin compen-sated for the other (62). It should also be noted that the expres-sion of the loss-of-function PANX1 variant that reduces chan-

nel function does not equate to the loss of PANX1 expression.For example, we have observed in the connexin channel fieldthat genetically modified mice heterozygous for the GJA1(Cx43) gene do not exhibit disease, whereas heterozygous miceharboring the I130T mutant, reducing overall Cx43 function to50% of controls, phenocopy the human disease known as ocu-lodentodigital dysplasia (63– 65).

Our multidimensional molecular analysis of the R217Hmutant revealed that PANX1 channels formed from the mutanthave an overall functional capacity of only 50% compared withcontrols. In this context, it is relevant to consider whether thedevelopmental abnormalities are strictly attributed to loss-of-function or whether, alternatively, a gain-of-toxic-function(irrespective of reduced channel flux) could contribute. Of

FIGURE 6. A, cell surface biotinylation of wild-type PANX1 and the R217H mutant in the presence and absence of high potassium. Cells were not transfected (NT)or transiently transfected with cDNA encoding either wild-type PANX1 or the R217H mutant. Forty eight hours after transfection, cells were untreated (�) ortreated (�) with 140 mM K�Glu in DMEM for 40 min. Cells were placed on ice, washed in ice-cold Hanks’ balanced salt solution, and cell surface proteinsbiotinylated. Biotinylated proteins were precipitated using neutravidin-conjugated beads, subjected to SDS-PAGE, transferred to nitrocellulose, and immu-noblotted (IB) for PANX1 or GAPDH. Note the abundance of cell surface biotinylated PANX1 and the R217H mutant, although the lack of biotinylated GAPDHindicates that only cell surface proteins were biotinylated. Position of molecular mass standards are shown. B, R217H mutant showed a dramatic reduction inchannel function when expressed in HEK293T cells pretreated with BFA for 6 h while CBX effectively blocked channel function. C, after pre-treatment with BFAfor 6 h, ramp currents at �60 mV were recorded in HEK293T cells expressing wild-type PANX1 or the R217H mutant, before (control) and after treatment withhigh potassium (High K�) or high potassium and CBX. n � 6 for each panel. ***, p 0.001.

Disease-associated Loss-of-function Panx1 Variant

12440 JOURNAL OF BIOLOGICAL CHEMISTRY VOLUME 291 • NUMBER 24 • JUNE 10, 2016

at University of M

anitoba Libraries on June 20, 2016

http://ww

w.jbc.org/

Dow

nloaded from

Page 10: Shao et al (2016)

note, expression of the R217H mutant in five distinct cell lineswas well tolerated with no apparent detrimental effect on cellviability or morphology, suggesting that this latter scenario isunlikely. Thus, we propose that the R217H mutant disruptssome essential function of pannexin channels critical for devel-opment in a variety of tissue types. Because the proband’s par-ents, as well as her sister, are heterozygous for R217H andexhibit no symptoms that can be linked to the variant, it wouldsuggest that the disease is autosomal recessive in nature.Although the allele frequency of the R217H variant is not specifi-cally known in the Turkish population, it has been reported in3/8596 European alleles in the Exome Sequencing Project and7/66,102 European alleles and 1/16,412 South Asian alleles in theExome Aggregation Consortium. Consistent with the proband’sparent and sister not exhibiting the clinical symptoms of the pro-band, we found that the mutant was not dominant to the functionof PANX1 when co-expressed in cultured cells.

Given that this patient is the first reported individual withbiallelic PANX1 gene variants, and that WES in any individualwill reveal variants in numerous genes, we assessed the evi-dence that the PANX1 variants are responsible for this individ-ual’s features. A review of the full WES findings reveals variantsin three other genes known to be related to a human diseasephenotype as follows: a single pathogenic variant in the auto-somal recessive RDX gene associated with a form of nonsyn-dromic hearing loss, inherited from her unaffected mother; amissense variant in FGFR3, an autosomal dominant generesponsible for various forms of skeletal dysplasias, inheritedfrom her unaffected father; and a single missense variant in theautosomal recessive POR gene, associated with disorders of ste-roidogenesis, inherited from her asymptomatic mother. Noneof the phenotypes associated with these genes are similar to theproband’s presentation, and two of the three genes associatedwith autosomal recessive inheritance are not located in ourpatient’s regions of loss of heterozygosity, and have only onevariant found. In addition, despite the large runs of homozygos-ity seen on chromosome microarrays, WES only identifiedthree other genes of interest with homozygous variants in addi-tion to PANX1 (R217H): PHF12 (R765W), MRPL49 (R88C),and NRXN2 (L53F). PHF12 encodes a PHD finger protein thathas been proposed to be associated with the regulation of intra-ocular pressure, which is not associated with our proband’sphenotype (66). MRPL49 encodes the mitochondrial ribosomalprotein L49, which is involved in protein synthesis (67). Thisgene is associated with glossopharyngeal neuralgia, a conditioncharacterized by repeated episodes of severe pain in the tongue,throat, ear, and tonsils, conditions not experienced by the pro-band in this study. The NRXN2 gene is expressed in the brainand encodes a synaptic organizing protein that helps to mediatethe differentiation of certain synapses. At least one loss-of-function truncating mutation of this gene has been identified inthe heterozygous state in a family with autism spectrum disor-der, language delay, and a family history of schizophrenia, sug-gesting autosomal dominant inheritance (68). The autosomaldominant mode of inheritance proposed by this and other stud-ies does not fit in this family as both parents are carriers of themissense change and are unaffected. Furthermore, the NRXN2proband did not present with the scope of organ involvement

noted in the proband of this study. Although this provides acompelling argument to rule out the involvement of NRXN2, pro-tein structure/function studies also provide supportive evidenceagainst a pivotal role for NRXN2. In brief, NRXN2 encodes a type Itransmembrane protein with six conserved interacting LNSdomains (laminin/neurexin/sex hormone) (69–71). The LNSdomains are key for interactions between neurexin and severalpostsynaptic binding partners that have been described (e.g. neu-roligins, leucine-rich repeat transmembrane protein, and dystro-glycan). Whereas LNS6 is the most important domain in terms ofbinding, the proband’s variant is positioned in the first LNS region,with no known interacting partners. Therefore, LNS1 is predictedto not be a functionally relevant domain.

This analysis leaves us with PANX1 as the best gene candi-date for the proband’s multi-organ involvement, althoughgiven the fact that other homozygous and heterozygous vari-ants exist in several genes, we await substantiation of this casestudy by the identification of other patients that harbor PANX1variants. A PANX1 gene search of the Online Mendelian Inher-itance in Man (OMIM) database did not reveal any PANX1gene mutations linked to any known disease. In addition,OMIM was searched for conditions that include all of the majorand most striking features of the proband’s phenotype, namelyintellectual disability, primary ovarian insufficiency, and senso-rineural hearing loss, to make sure that the genes associatedwith these other conditions had good coverage on WES and didnot contain any variants of uncertain significance. The mainconditions that matched the proband’s phenotype on thissearch included the Perrault syndromes and Woodhouse-Sataki syndrome. No suspicious variants were found in any ofthese genes associated with these conditions on WES. Overall,our findings fit well with the broad spectrum of PANX1 expres-sion in all of the proband’s affected organs (13) and to the factthat a new conditional mouse model of Panx1 ablation in thecochlea revealed severe hearing loss (54, 55). We suggest thatPANX1 be considered as a rare cause of intellectual disability,particularly when accompanied by other systemic dysfunction,especially sensorineural hearing loss and premature ovarianfailure. Finally, as this is the first report of a disease-associatedPANX1 germline variant, it is possible that a broader contribu-tion of PANX1 to human disease has previously been over-looked. Accordingly, this study may raise awareness of PANX1as a candidate gene contributing to the genetic basis of disease.

Author Contributions—K. L. and A. S. identified the patient,obtained informed consent, and worked closely with the family toacquire all the clinical information. J. J. and K. L. L. evaluated all thegene sequencing. Q. S., J. K., J. L. E., and S. P. performed the expres-sion, ATP release, biotinylation, and dye uptake studies. R. S. andM. F. J. performed all the electrophysiology studies. D. W. L. over-saw the project and wrote the manuscript with assistance from allauthors, especially K. L., J. J., and M. F. J.

Acknowledgments—We thank the patient and family for their partic-ipation. We also thank Natalie Lavine for technical assistance andJamie Simek for the artwork in Fig. 1C. Informed consent was obtainedfrom the patient and her family to use her information forpublication.

Disease-associated Loss-of-function Panx1 Variant

JUNE 10, 2016 • VOLUME 291 • NUMBER 24 JOURNAL OF BIOLOGICAL CHEMISTRY 12441

at University of M

anitoba Libraries on June 20, 2016

http://ww

w.jbc.org/

Dow

nloaded from

Page 11: Shao et al (2016)

References1. Panchin, Y., Kelmanson, I., Matz, M., Lukyanov, K., Usman, N., and Luky-

anov, S. (2000) A ubiquitous family of putative gap junction molecules.Curr. Biol. 10, R473– 474

2. Sosinsky, G. E., Boassa, D., Dermietzel, R., Duffy, H. S., Laird, D. W.,MacVicar, B., Naus, C. C., Penuela, S., Scemes, E., Spray, D. C., Thompson,R. J., Zhao, H. B., and Dahl, G. (2011) Pannexin channels are not gapjunction hemichannels. Channels 5, 193–197

3. Bond, S. R., and Naus, C. C. (2014) The pannexins: past and present. Front.Physiol. 5, 58

4. Boassa, D., Ambrosi, C., Qiu, F., Dahl, G., Gaietta, G., and Sosinsky, G.(2007) Pannexin1 channels contain a glycosylation site that targets thehexamer to the plasma membrane. J. Biol. Chem. 282, 31733–31743

5. Penuela, S., Simek, J., and Thompson, R. J. (2014) Regulation of pannexinchannels by post-translational modifications. FEBS Lett. 588, 1411–1415

6. Gehi, R., Shao, Q., and Laird, D. W. (2011) Pathways regulating the traf-ficking and turnover of pannexin1 protein and the role of the C-terminaldomain. J. Biol. Chem. 286, 27639 –27653

7. Wang, J., Ambrosi, C., Qiu, F., Jackson, D. G., Sosinsky, G., and Dahl, G.(2014) The membrane protein Pannexin1 forms two open-channel con-formations depending on the mode of activation. Sci. Signal. 7, ra69

8. Ambrosi, C., Gassmann, O., Pranskevich, J. N., Boassa, D., Smock, A.,Wang, J., Dahl, G., Steinem, C., and Sosinsky, G. E. (2010) Pannexin1 andPannexin2 channels show quaternary similarities to connexons and dif-ferent oligomerization numbers from each other. J. Biol. Chem. 285,24420 –24431

9. Pelegrin, P., and Surprenant, A. (2006) Pannexin-1 mediates large poreformation and interleukin-1� release by the ATP-gated P2X7 receptor.EMBO J. 25, 5071–5082

10. Pelegrin, P., and Surprenant, A. (2007) Pannexin-1 couples to maitotoxin-and nigericin-induced interleukin-1� release through a dye uptake-inde-pendent pathway. J. Biol. Chem. 282, 2386 –2394

11. Dubyak, G. R. (2009) Both sides now: multiple interactions of ATP withpannexin-1 hemichannels. Focus on “a permeant regulating its perme-ation pore: inhibition of pannexin 1 channels by ATP”. Am. J. Physiol. CellPhysiol. 296, C235–C241

12. Chekeni, F. B., Elliott, M. R., Sandilos, J. K., Walk, S. F., Kinchen, J. M.,Lazarowski, E. R., Armstrong, A. J., Penuela, S., Laird, D. W., Salvesen,G. S., Isakson, B. E., Bayliss, D. A., and Ravichandran, K. S. (2010) Pannexin1 channels mediate ’find-me’ signal release and membrane permeabilityduring apoptosis. Nature 467, 863– 867

13. Penuela, S., Harland, L., Simek, J., and Laird, D. W. (2014) Pannexin chan-nels and their links to human disease. Biochem. J. 461, 371–381

14. Thompson, R. J. (2015) Pannexin channels and ischaemia. J. Physiol. 593,3463–3470

15. Thompson, R. J., Jackson, M. F., Olah, M. E., Rungta, R. L., Hines, D. J.,Beazely, M. A., MacDonald, J. F., and MacVicar, B. A. (2008) Activation ofpannexin-1 hemichannels augments aberrant bursting in the hippocam-pus. Science 322, 1555–1559

16. Thompson, R. J., Zhou, N., and MacVicar, B. A. (2006) Ischemia opensneuronal gap junction hemichannels. Science 312, 924 –927

17. Gulbransen, B. D., Bashashati, M., Hirota, S. A., Gui, X., Roberts, J. A.,MacDonald, J. A., Muruve, D. A., McKay, D. M., Beck, P. L., Mawe, G. M.,Thompson, R. J., and Sharkey, K. A. (2012) Activation of neuronal P2X7receptor-pannexin-1 mediates death of enteric neurons during colitis.Nat. Med. 18, 600 – 604

18. Séror, C., Melki, M. T., Subra, F., Raza, S. Q., Bras, M., Saïdi, H., Nardacci,R., Voisin, L., Paoletti, A., Law, F., Martins, I., Amendola, A., Abdul-Sater,A. A., Ciccosanti, F., Delelis, O., et al. (2011) Extracellular ATP acts onP2Y2 purinergic receptors to facilitate HIV-1 infection. J. Exp. Med. 208,1823–1834

19. Penuela, S., Gyenis, L., Ablack, A., Churko, J. M., Berger, A. C., Litchfield,D. W., Lewis, J. D., and Laird, D. W. (2012) Loss of pannexin 1 attenuatesmelanoma progression by reversion to a melanocytic phenotype. J. Biol.Chem. 287, 29184 –29193

20. Bao, B. A., Lai, C. P., Naus, C. C., and Morgan, J. R. (2012) Pannexin1 drivesmulticellular aggregate compaction via a signaling cascade that remodels

the actin cytoskeleton. J. Biol. Chem. 287, 8407– 841621. Lai, C. P., Bechberger, J. F., Thompson, R. J., MacVicar, B. A., Bruzzone, R.,

and Naus, C. C. (2007) Tumor-suppressive effects of pannexin 1 in C6glioma cells. Cancer Res. 67, 1545–1554

22. Mylvaganam, S., Ramani, M., Krawczyk, M., and Carlen, P. L. (2014) Rolesof gap junctions, connexins, and pannexins in epilepsy. Front. Physiol. 5,172

23. Carlen, P. L. (2012) Curious and contradictory roles of glial connexins andpannexins in epilepsy. Brain Res. 1487, 54 – 60

24. Kurtenbach, S., Kurtenbach, S., and Zoidl, G. (2014) Emerging functionsof pannexin 1 in the eye. Front. Cell Neurosci. 8, 263

25. Sarrouilhe, D., Dejean, C., and Mesnil, M. (2014) Involvement of gap junc-tion channels in the pathophysiology of migraine with aura. Front. Physiol.5, 78

26. Orellana, J. A., Froger, N., Ezan, P., Jiang, J. X., Bennett, M. V., Naus, C. C.,Giaume, C., and Sáez, J. C. (2011) ATP and glutamate released via astro-glial connexin 43 hemichannels mediate neuronal death through activa-tion of pannexin 1 hemichannels. J. Neurochem. 118, 826 – 840

27. Schenk, U., Westendorf, A. M., Radaelli, E., Casati, A., Ferro, M., Fum-agalli, M., Verderio, C., Buer, J., Scanziani, E., and Grassi, F. (2008) Puri-nergic control of T cell activation by ATP released through pannexin-1hemichannels. Sci. Signal. 1, ra6

28. Molica, F., Morel, S., Meens, M. J., Denis, J. F., Bradfield, P. F., Penuela, S.,Zufferey, A., Monyer, H., Imhof, B. A., Chanson, M., Laird, D. W., Fon-tana, P., and Kwak, B. R. (2015) Functional role of a polymorphism in thePannexin1 gene in collagen-induced platelet aggregation. Thromb. Hae-most. 114, 325–336

29. Tanaka, A. J., Cho, M. T., Millan, F., Juusola, J., Retterer, K., Joshi, C.,Niyazov, D., Garnica, A., Gratz, E., Deardorff, M., Wilkins, A., Ortiz-Gon-zalez, X., Mathews, K., Panzer, K., Brilstra, E., et al. (2015) Mutations inSPATA5 are associated with microcephaly, intellectual disability, seizures,and hearing loss. Am. J. Hum. Genet. 97, 457– 464

30. Penuela, S., Bhalla, R., Gong, X. Q., Cowan, K. N., Celetti, S. J., Cowan, B. J.,Bai, D., Shao, Q., and Laird, D. W. (2007) Pannexin 1 and pannexin 3 areglycoproteins that exhibit many distinct characteristics from the connexinfamily of gap junction proteins. J. Cell Sci. 120, 3772–3783

31. Benfenati, V., Caprini, M., Nicchia, G. P., Rossi, A., Dovizio, M., Cervetto,C., Nobile, M., and Ferroni, S. (2009) Carbenoxolone inhibits volume-regulated anion conductance in cultured rat cortical astroglia. Channels 5,323–336

32. Bruzzone, R., Barbe, M. T., Jakob, N. J., and Monyer, H. (2005) Pharma-cological properties of homomeric and heteromeric pannexin hemichan-nels expressed in Xenopus oocytes. J. Neurochem. 92, 1033–1043

33. Bao, L., Locovei, S., and Dahl, G. (2004) Pannexin membrane channels aremechanosensitive conduits for ATP. FEBS Lett. 572, 65– 68

34. Silverman, W. R., de Rivero Vaccari, J. P., Locovei, S., Qiu, F., Carlsson,S. K., Scemes, E., Keane, R. W., and Dahl, G. (2009) The pannexin 1 chan-nel activates the inflammasome in neurons and astrocytes. J. Biol. Chem.284, 18143–18151

35. Silverman, W., Locovei, S., and Dahl, G. (2008) Probenecid, a gout remedy,inhibits pannexin 1 channels. Am. J. Physiol. Cell Physiol. 295, C761–C767

36. Zadeh, A. D., Cheng, Y., Xu, H., Wong, N., Wang, Z., Goonasekara, C.,Steele, D. F., and Fedida, D. (2009) Kif5b is an essential forward traffickingmotor for the Kv1.5 cardiac potassium channel. J. Physiol. 587, 4565– 4574

37. Boassa, D., Qiu, F., Dahl, G., and Sosinsky, G. (2008) Trafficking dynamicsof glycosylated pannexin 1 proteins. Cell Commun. Adhes. 15, 119 –132

38. Hansen, D. B., Ye, Z. C., Calloe, K., Braunstein, T. H., Hofgaard, J. P.,Ransom, B. R., Nielsen, M. S., and MacAulay, N. (2014) Activation, per-meability, and inhibition of astrocytic and neuronal large pore (hemi-)channels. J. Biol. Chem. 289, 26058 –26073

39. Penuela, S., Bhalla, R., Nag, K., and Laird, D. W. (2009) Glycosylationregulates pannexin intermixing and cellular localization. Mol. Biol. Cell20, 4313– 4323

40. Scemes, E., and Spray, D. C. (2012) Extracellular K� and astrocyte signal-ing via connexin and pannexin channels. Neurochem. Res. 37, 2310 –2316

41. Sandilos, J. K., Chiu, Y. H., Chekeni, F. B., Armstrong, A. J., Walk, S. F.,Ravichandran, K. S., and Bayliss, D. A. (2012) Pannexin 1, an ATP releasechannel, is activated by caspase cleavage of its pore-associated C-terminal

Disease-associated Loss-of-function Panx1 Variant

12442 JOURNAL OF BIOLOGICAL CHEMISTRY VOLUME 291 • NUMBER 24 • JUNE 10, 2016

at University of M

anitoba Libraries on June 20, 2016

http://ww

w.jbc.org/

Dow

nloaded from

Page 12: Shao et al (2016)

autoinhibitory region. J. Biol. Chem. 287, 11303–1131142. Sáez, J. C., and Leybaert, L. (2014) Hunting for connexin hemichannels.

FEBS Lett. 588, 1205–121143. Bunse, S., Schmidt, M., Hoffmann, S., Engelhardt, K., Zoidl, G., and Der-

mietzel, R. (2011) Single cysteines in the extracellular and transmembraneregions modulate pannexin 1 channel function. J. Membr. Biol. 244, 21–33

44. Ma, W., Hui, H., Pelegrin, P., and Surprenant, A. (2009) Pharmacologicalcharacterization of pannexin-1 currents expressed in mammalian cells.J. Pharmacol. Exp. Ther. 328, 409 – 418

45. Romanov, R. A., Bystrova, M. F., Rogachevskaya, O. A., Sadovnikov, V. B.,Shestopalov, V. I., and Kolesnikov, S. S. (2012) The ATP permeability ofpannexin 1 channels in a heterologous system and in mammalian tastecells is dispensable. J. Cell Sci. 125, 5514 –5523

46. Billaud, M., Chiu, Y. H., Lohman, A. W., Parpaite, T., Butcher, J. T.,Mutchler, S. M., DeLalio, L. J., Artamonov, M. V., Sandilos, J. K., Best,A. K., Somlyo, A. V., Thompson, R. J., Le, T. H., Ravichandran, K. S.,Bayliss, D. A., and Isakson, B. E. (2015) A molecular signature in the pan-nexin1 intracellular loop confers channel activation by the alpha1 adreno-receptor in smooth muscle cells. Sci. Signal. 8, ra17

47. Dahl, G. (2015) ATP release through pannexon channels. Philos. Trans. R.Soc. Lond. B Biol. Sci. 370, 1672

48. Prochnow, N., Abdulazim, A., Kurtenbach, S., Wildförster, V., Dvoriant-chikova, G., Hanske, J., Petrasch-Parwez, E., Shestopalov, V. I., Dermietzel,R., Manahan-Vaughan, D., and Zoidl, G. (2012) Pannexin1 stabilizes syn-aptic plasticity and is needed for learning. PLoS ONE 7, e51767

49. Tang, W., Ahmad, S., Shestopalov, V. I., and Lin, X. (2008) Pannexins arenew molecular candidates for assembling gap junctions in the cochlea.Neuroreport 19, 1253–1257

50. Baranova, A., Ivanov, D., Petrash, N., Pestova, A., Skoblov, M., Kelmanson,I., Shagin, D., Nazarenko, S., Geraymovych, E., Litvin, O., Tiunova, A.,Born, T. L., Usman, N., Staroverov, D., Lukyanov, S., and Panchin, Y.(2004) The mammalian pannexin family is homologous to the inverte-brate innexin gap junction proteins. Genomics 83, 706 –716

51. Vogt, A., Hormuzdi, S. G., and Monyer, H. (2005) Pannexin1 and Pan-nexin2 expression in the developing and mature rat brain. Brain Res. Mol.Brain Res. 141, 113–120

52. Wicki-Stordeur, L. E., Dzugalo, A. D., Swansburg, R. M., Suits, J. M., andSwayne, L. A. (2012) Pannexin 1 regulates postnatal neural stem and pro-genitor cell proliferation. Neural Dev. 7, 11

53. Wang, X. H., Streeter, M., Liu, Y. P., and Zhao, H. B. (2009) Identificationand characterization of pannexin expression in the mammalian cochlea.J. Comp. Neurol. 512, 336 –346

54. Chen, J., Zhu, Y., Liang, C., Chen, J., and Zhao, H. B. (2015) Pannexin1channels dominate ATP release in the cochlea ensuring endocochlearpotential and auditory receptor potential generation and hearing. Sci. Rep.5, 10762

55. Zhao, H. B., Zhu, Y., Liang, C., and Chen, J. (2015) Pannexin 1 deficiencycan induce hearing loss. Biochem. Biophys. Res. Commun. 463, 143–147

56. Penuela, S., Celetti, S. J., Bhalla, R., Shao, Q., and Laird, D. W. (2008)Diverse subcellular distribution profiles of pannexin 1 and pannexin 3.Cell Commun. Adhes. 15, 133–142

57. Moon, P. M., Penuela, S., Barr, K., Khan, S., Pin, C. L., Welch, I., Attur, M.,

Abramson, S. B., Laird, D. W., and Beier, F. (2015) Deletion of Panx3prevents the development of surgically induced osteoarthritis. J. Mol.Med. 93, 845– 856

58. Turmel, P., Dufresne, J., Hermo, L., Smith, C. E., Penuela, S., Laird, D. W.,and Cyr, D. G. (2011) Characterization of pannexin1 and pannexin3 andtheir regulation by androgens in the male reproductive tract of the adultrat. Mol. Reprod. Dev. 78, 124 –138

59. Penuela, S., Gehi, R., and Laird, D. W. (2013) The biochemistry and func-tion of pannexin channels. Biochim. Biophys. Acta 1828, 15–22

60. Penuela, S., Kelly, J. J., Churko, J. M., Barr, K. J., Berger, A. C., and Laird,D. W. (2014) Panx1 regulates cellular properties of keratinocytes and der-mal fibroblasts in skin development and wound healing. J. Invest. Derma-tol. 134, 2026 –2035

61. Lohman, A. W., and Isakson, B. E. (2014) Differentiating connexin hemi-channels and pannexin channels in cellular ATP release. FEBS Lett. 588,1379 –1388

62. Bargiotas, P., Krenz, A., Hormuzdi, S. G., Ridder, D. A., Herb, A., Barakat,W., Penuela, S., von Engelhardt, J., Monyer, H., and Schwaninger, M.(2011) Pannexins in ischemia-induced neurodegeneration. Proc. Natl.Aca. Sci. U.S.A. 108, 20772–20777

63. Laird, D. W. (2014) Syndromic and non-syndromic disease-linked Cx43mutations. FEBS Lett. 588, 1339 –1348

64. Stewart, M. K., Gong, X. Q., Barr, K. J., Bai, D., Fishman, G. I., and Laird,D. W. (2013) The severity of mammary gland developmental defects islinked to the overall functional status of Cx43 as revealed by geneticallymodified mice. Biochem. J. 449, 401– 413

65. Kalcheva, N., Qu, J., Sandeep, N., Garcia, L., Zhang, J., Wang, Z., Lampe,P. D., Suadicani, S. O., Spray, D. C., and Fishman, G. I. (2007) Gap junctionremodeling and cardiac arrhythmogenesis in a murine model of oculo-dentodigital dysplasia. Proc. Natl. Acad. Sci. U.S.A. 104, 20512–20516

66. Chen, F., Klein, A. P., Klein, B. E., Lee, K. E., Truitt, B., Klein, R., Iyengar,S. K., and Duggal, P. (2015) Exome array analysis identifies CAV1/CAV2as a susceptibility locus for intraocular pressure. Invest. Ophthalmol. Vis.Sci. 56, 544 –551

67. Jung, S. J., Seo, Y., Lee, K. C., Lee, D., and Roe, J. H. (2015) Essentialfunction of Aco2, a fusion protein of aconitase and mitochondrial ribo-somal protein bL21, in mitochondrial translation in fission yeast. FEBSLett. 589, 822– 828

68. Gauthier, J., Siddiqui, T. J., Huashan, P., Yokomaku, D., Hamdan, F. F.,Champagne, N., Lapointe, M., Spiegelman, D., Noreau, A., Lafrenière,R. G., Fathalli, F., Joober, R., Krebs, M. O., DeLisi, L. E., Mottron, L., et al.(2011) Truncating mutations in NRXN2 and NRXN1 in autism spectrumdisorders and schizophrenia. Hum. Genet. 130, 563–573

69. Tanaka, H., Nogi, T., Yasui, N., Iwasaki, K., and Takagi, J. (2011) Structuralbasis for variant-specific neuroligin-binding by �-neurexin. PLoS ONE 6,e19411

70. Sheckler, L. R., Henry, L., Sugita, S., Südhof, T. C., and Rudenko, G. (2006)Crystal structure of the second LNS/LG domain from neurexin 1�: Ca2�

binding and the effects of alternative splicing. J. Biol. Chem. 281,22896 –22905

71. Rudenko, G., Hohenester, E., and Muller, Y. A. (2001) LG/LNS domains:multiple functions– one business end? Trends Biochem. Sci. 26, 363–368

Disease-associated Loss-of-function Panx1 Variant

JUNE 10, 2016 • VOLUME 291 • NUMBER 24 JOURNAL OF BIOLOGICAL CHEMISTRY 12443

at University of M

anitoba Libraries on June 20, 2016

http://ww

w.jbc.org/

Dow

nloaded from

Page 13: Shao et al (2016)

Dale W. LairdJuusola, Kara L. Levine, Jessica L. Esseltine, Silvia Penuela, Michael F. Jackson and

Qing Shao, Kristin Lindstrom, Ruoyang Shi, John Kelly, Audrey Schroeder, JaneAssociated with Multisystem Dysfunction

Gene Has Reduced Channel Function and IsPANX1A Germline Variant in the

doi: 10.1074/jbc.M116.717934 originally published online April 15, 20162016, 291:12432-12443.J. Biol. Chem. 

  10.1074/jbc.M116.717934Access the most updated version of this article at doi:

 Alerts:

  When a correction for this article is posted• 

When this article is cited• 

to choose from all of JBC's e-mail alertsClick here

  http://www.jbc.org/content/291/24/12432.full.html#ref-list-1

This article cites 71 references, 29 of which can be accessed free at

at University of M

anitoba Libraries on June 20, 2016

http://ww

w.jbc.org/

Dow

nloaded from