novel mutations in the connexin 43(gja1) may contribute to nonsyndromic hearing loss. by
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Novel Mutations in the Connexin 43(GJA1) may contribute to Nonsyndromic Hearing Loss. By Asha Kiran Akula Master of Research. Gap Junctions Intercellular communication channels. Gap junctions allow the selective permeability to ions and small molecules. - PowerPoint PPT PresentationTRANSCRIPT
Novel Mutations in the Connexin 43(GJA1) may contribute to Nonsyndromic Hearing Loss.
By Asha Kiran Akula
Master of Research
Gap Junctions
Intercellular communication channels. Gap junctions allow the selective permeability to ions and small molecules.Movement through these channels is passive and non specific.Gap junctions are made up of clusters of closely packed connexonsThe structural unit of gap junction is Connexon.
Connexon
Consist of pairs of transmembrane channels.The connexon hemi channel in one cell membrane docks with a connexon hemi channel in an adjacent cell.Hexameric: they consist of arrays of 6 connexin protein subunitsDifferent connexin isoforms have been identified.Homomeric or heteromeric
FunctionsGap junctions involve in regulation of
Tissue homeostasis Regulation of cell growth
Embryonic development Electrical and metabolic coupling
The loss of connexins, or the existence of mutations affecting their normal functions, has been implicated in a variety of diseases and disorders, including cancers.
Gap Junctions play a major role in intercellular calcium signalling.
Gap junctional opening is controlled by the intracellular concentration of calcium.
The low intracellular calcium concentration enables the gap junction channels to open and vice versa.
Controlled gating of gap junction channels may be responsible for the normal functioning of the cell.
Connexins Show overlapping
tissue expression patterns, most tissues expressing more than one connexin type.
Each connexin contains 4 TM domains, with two extracellular and three cytoplasmic regions.
Both N- and C-termini –face the cytoplasm The third TM domain - amphipathic in nature
forms the lining of the formed channel.
Amino acid sequence identity between the isoforms is ~50-80%, with the TM domains being well conserved.
Both extracellular loops – contain conserved cysteine residues, which likely form intramolecular disulphide bonds.
Single putative intracellular loop (between TM domains 2 and 3) and the cytoplasmic C terminus are highly variable among the family members. Six connexins associate to form a hemi-channel, or connexon. Two connexons then interact (likely via the extracellular loops of their connexins) to form the complete gap junction channel.
Connexin Gene Multigene family comprising 20 in mouse and 21 genes in human genome (Cardiovascular Research, 2010). α and β gene families.The "Gja/Gjb" nomenclature-adopted by the NCBI data base. Cells express multiple types of connexin-potentially associate to form gap junction channels containing more than one type of connexin.
MutationsAlterations in the gap junction, hemichannel, or general functions of the connexins. Cause various human diseases like skin diseases, nonsyndormic and syndromic deafness, cataracts, Oculodentodigital Dysplasia (ODDD), cancers etc.
Mutations in Cx32 - Charcot-Marie-Tooth disease. Cx26 -deafness and skin disease.
Cx30, Cx30.3, and Cx31- hearing loss and skin disorders.
Hearing Loss
Affects 1 in 1000 newborns. Syndromic and nonsyndromic70% of genetically related hearing loss-nonsyndromic.Two types-
Conductive hearing lossSensorineural Hearing Loss
Mutations in Cx26- common cause of congenital bilateral non-syndromic sensorineural hearing loss.
HearingThe ear is made up of three different sections:
1.the outer earExternal auditory canalTympanic membrabe
2.the middle ear-bones3.the inner ear
CochleaVestibular system.
CochleaMain auditory portion of the inner ear.Core component-organ of cortiOrgan of corti- sensory organ of hearing.Organ of corti comprises - hair cells
supporting cells Endolymph
Hair cells-two typesInner hair cellsOuter hair cells
Connexins are present in supporting cells.
Sound waves
Tympanic membrane(Outer ear)
Bones maleus, incus and
stapes(middle ear)
Movement of stapes
Pressure waves (fluid filled inner
ear)
hairs to move in the inner
ear
stimulate the auditory
nerve
General mechanism of hearing
Organ of Corti
Outer hair cellsInner hair cells
Tectorial membrane
Basilar membrane
Supporting cellsSpiralLimbus
Stria Vascularis
Endolymph
Epithelial tissue
CX43- Four predicted membrane-spanning segments (M1–M4) linked by two extracellular (E1–E2)and one cytoplasmic loop.
Amino and carboxyl tails faced intracellularly.Expressed in non sensory epithelial cells of the inner ear.
CX43
Three novel missense mutations have been identified in the GJA1 gene - related to hearing loss.
The three missense mutations c.205T>C (p.S69P) - Extracellular loopc.932delC - C-terminal region c.977C>T (p.T326I) - C-terminal cytoplasmic
domain. Studied the intracellular distribution, assembly and the effects of the three Cx43 mutants with the
wild type Cx43.
Plasmid construction with the mutations
Permanent transfected HeLa-CX43 cell line
Immunostaining
Dye Transfer
Plasmid was constructed using CX43WT cDNA and cloned into pCDNA3.1 vector
Constructed plasmid-expression in HeLa cells via transfection- lipofectamine method.
Addition of G418- isolation of stable transfectantsRT-PCR – success of transfection and
expression of transfected genes.PCR products- Gel electrophoresis
CX43WT-Positive control. HeLa and water-Negative control β-actin-internal control
Expression analysis of GJA1 mRNA in four stable transfectedHeLa cells by RT-PCR. RT-PCR analysis of total RNA from HeLacells expressing CX43 WT, CX43 S69P, CX43 932delC and CX43T326I confirms expression of the corresponding mRNAs in stablytransfected HeLa cell lines (upper panel). -actin served as a reference for the loading amount of total RNA for each sample (lower panel).Mock HeLa and water were used as negative controls
Electrophoresis -Results
confirm the expressed CX43 mutant proteins
Primary antibody- monoclonal anti-CX43 antibody
Secondary antibody- HRP-conjugated anti-mouse IgG
GAPDH-Internal ControlMock HeLa cells- Negative control
Western Blot-Results
ImmunostainingTransfected cells - washed and fixed.
Primary antibodiesMouse anti-pan-cadherin antibody (anti-CH19) – cell membraneMouse anti-CX43-epitope of CX43 protein
Secondary AntibodiesAlexa Fluor 488 andAlexa Flour 594
Nuclei-stained with DAPI
Immunostaining -Results
Localization analysis of CX43 WT in stably transfected HeLa cells by immunocytochemistry using anti-CX43 and pan-cadherin antibody. Analysis of fluorescence microscopy on HeLa cells expressing CX43 WT reveals localization of the CX43 protein in the plasmamembranes. CX43 proteins are indicated by arrows. The cells were counterstained with 4-6-diamidino-2-phenylindole, DAPI, to highlight the nuclei. Scale bars 10 m
Dye TransferFunctionality of gap junction formed.Transfected HeLa cells-microinjected with Lucifer Yellow.
Cell line Dye-filled Number of Total numberneighbor injections of cell (n)showing cell number (n) dye transfer(mean ± SE)
HeLa-Cx43 WT 4.17 ±1.621 30 100HeLa-Cx43 S69P 0 30 0HeLa-Cx43 T326I 0 30 0HeLa-Cx43 A311V 0.54 ±1.471 50 86HeLa 0 30 0
Lucifer Yellow transfer stably expressed with WT or mutant
Cx43 HeLa cells
DiscussionWith the above results, these three mutations-risk factor for the development of hearing.Three mutations-loss of function of CX43 –hearing loss.CX43WT- found localized to the cell membranes at the point of contact between adjacent expressing cells.Membrane localization-confirmed by colocalization with pan-cadherin.
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