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 connexons
The 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 subunits
Different connexin isoforms have been identified.
Homomeric or heteromeric
Functions
Gap 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.


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 connexinpotentially associate to form gap junction channels
containing more than one type of connexin.
Mutations
Alterations 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 nonsyndromic
70% of genetically related hearing lossnonsyndromic.
Two typesConductive hearing loss
Sensorineural Hearing Loss
Mutations in Cx26- common cause of congenital
bilateral non-syndromic sensorineural hearing loss.
Hearing
The ear is made up of three different sections:
1.the outer ear
External auditory canal
Tympanic membrabe
2.the middle ear-bones
3.the inner ear
Cochlea
Vestibular system.
Cochlea
Main auditory portion of the inner ear.
Core component-organ of corti
Organ of corti- sensory organ of hearing.
Organ of corti comprises - hair cells
supporting cells
Endolymph
Hair cells-two types
Inner hair cells
Outer hair cells
Connexins are present in supporting cells.
General mechanism of hearing
Sound
waves
Tympanic
membrane
(Outer ear)
stimulate the
auditory
nerve
hairs to move
in the inner
ear
Bones maleus,
incus and
stapes
(middle ear)
Movement of
stapes
Pressure
waves (fluid
filled inner
ear)
Organ of Corti
Endolymph
Stria
Vascularis
Tectorial membrane
Outer hair cells
Inner hair cells
Spiral
Limbus
Supporting cells
Basilar membrane
Epithelial tissue
CX43
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.
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 loop
c.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 transfectants
RT-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
Electrophoresis -Results
Expression analysis of GJA1 mRNA
in four stable transfected
HeLa cells by RT-PCR. RT-PCR
analysis of total RNA from HeLa
cells expressing CX43 WT, CX43
S69P, CX43 932delC and CX43
T326I confirms expression of the
corresponding mRNAs in stably
transfected 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
Western Blot-Results
confirm the expressed
mutant proteins
Primary antibodyanti-CX43 antibody
CX43
monoclonal
Secondary
antibodyconjugated anti-mouse IgG
HRP-
GAPDH-Internal Control
Mock HeLa cells- Negative control
Immunostaining
Transfected cells - washed and fixed.
Primary antibodies
Mouse anti-pan-cadherin antibody (anti-CH19) – cell
membrane
Mouse anti-CX43-epitope of CX43 protein
Secondary Antibodies
Alexa Fluor 488 and
Alexa 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 Transfer
Functionality
of
gap
junction formed.
Transfected HeLa cellsmicroinjected with Lucifer
Yellow.
Lucifer Yellow transfer stably expressed with WT or
mutant
Cx43 HeLa cells
Cell line
Dye-filled
Number of
neighbor
injections
cell number
(n)
(mean ± SE)
HeLa-Cx43 WT
4.17 ±1.621
HeLa-Cx43 S69P
0
HeLa-Cx43 T326I
0
HeLa-Cx43 A311V 0.54 ±1.471
HeLa
0
30
30
30
50
30
Total number
of cell (n)showing
dye transfer
100
0
0
86
0
Discussion
With 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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