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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