Gap Junctions Flashcards
The connexin family
21 family members, 21 genes Number gives molecular weight Little splicing Present in all chordates Not present in pre chordates
Innexin/pannexin family
Gap junctions in pre chordates
8 genes in drosophila, 25 in c.elegans.
Splicing can produce different variants e.g. ShakB lethal, neural and neural +16
Other family members named numerically
Pannexins- innexins that were kept in chordates
Original members found in the innexin/pannexin families
DmshakB- Defective escape
Dm-ogre- small optic lobes
Ce-unc7 and unc9- uncoordinated movement
Ce-eat5- feeding defect
Membrane topology
4 TM domains
Two extra cellular loops
Single intracellular loop
No sequence homology- example of convergent evolution
Structure of gap junction
6 protein subunits
TM3 lines the pore
Extra cellular fixed in conformation by disulphide bonds
Interdigitation
Gap junction TM3
Channel narrows from 40A to 15A
This is because TM3 tilts
Opening and closing of gap junction channel
Rotation and tilting of TM3
Movement of either small or bulky AAs in the pore
N terminus may possibly swing and block the pore
Biochemical regulation
Ph- protonation of histidine residues in c terminus or I loop
Ca- Ca:H transporter makes the cell more acidic.
Activates phospholipase C and A2 -> DAG and arachidonic acid
These integrate into membrane around pores
Calmodulin can bind to gap junction proteins and prevent them entering membranes
Different connexins have different c-termini so can respond differently to Ca changes
How would you tell if a region is calcium sensitive?
Removed region and measure sensitivity
Phosphorylation
Ser, thr or tyr kinases on c tail
This can alter signalling between channels
Kinases activated by growth factors and neurotransmitters
Voltage
N terminus AAs sense membrane potential
Conserved Pro in TM2 gives a 15-20 link and can block pore
Transcriptional
The gap junction proteins have a very short half life
3-4 hour turnover
Size and charge selectivity
Different sequences in pore lining regions
Different permeability- prefer anions or cations
Max 2kDa but some only 500Da
Assembly
Not all combinations work together
Homomeric or heteromeric hemichannel
Homotypic or hetrotypic typic channel
Cx43 and 40 are incompatible- can be used to create compartments in embryos
Analysing channel function
Dye transfer- cascade blue injected
Voltage clamp- micro electrodes in both cell can send and receive voltage
Patch clamp inserted into membrane can measure single channel
Chemical vs electrical synapses
Electrical is x5 faster (0.1ms)
Electrical can be uni or bi directional
Electrical are generally excitatory
Neuronal gap junctions- types of synapse
Cx36 is major, also 45 and 57
Bidirectional/self rectifying- either direction, sync neutrons for memory
Unidirectional/non rectifying- one direction, when lots of neutrons fire. Escape responses
Giant fibre physiology
Two GFs from each side of brain connected by GCI (giant commissural interneuron)
GF synapses
TTMn -> tergotrochanteral muscle motor neuron
PSI peripherally synapsing neuron -> DLMn dorsal longitudinal flight muscle
Experiment to prove shakb in gap junctions
Shakb RNA injected
Cell pairs voltage clamped, those with shakb showed voltage transmission
Alternative splicing of shakb
Shakb neural- in the developing CNS not GFS
Shakb neural +16- primarily GFS
Shakb lethal- CNS inc GFS and gut and heart
Where is shakb neural + 16 found?
Found in the GF system
Is rectification due to heterotypic gap junctions?
Rectifying electrical synapse is a heterotypic junction
Composed of neural 16 (pre) -> lethal (post)
Found in TTMn and PSI
Asymmetrically hated by voltage-
N+16 is relatively positive and lethal negative
Supports a positive to negative flow
Shakb (n+16) Homotypic channels show little voltage sensitivity, likely to be gated in post synaptic lethal
Gap junctions mutations in the ocular lens
Lens fibres no organelles depend on epithelial
NaKATPase in epithelial gives a translenticular potential and current flow
Cx 43 + 50- epithelium
46 50 - lens fibres
46 and 50 give different phenotypes although same place
50 deletion gives slow growth and microphthalmia
Both give nuclear cataracts
Female infertility
Cx37
No ovulation
Follicle stops growing as Antrum formed
Growth promoting factors from follicular cells per ate through gap junctions to make ooycytes mature
Charcot Marie tooth
10-20% are cx32 X linked
Autosomal- peripheral myelin protein 22 or p0
Weak distal muscles, reduced touch, CNS defects less severe
X100 faster from peri nuclear to periaxonal
Myelin dies
Recycling of K+
Role of cx
1 epithelial network- organ of corti
2 connective tissue network- spiral ligament and stria vascularis
Cx26 might couple cells or supply glucose for survival
Cx in hearing loss
Two types
Non syndromic and syndromic
Cx26 -> non syndromic but can cause skin alone, 50% of cases
Most cx26 non syndromic are recessive but all syndromic cx26 are dominant
May be more redundant in the skin and compensated for
Role of Cx in the skin
Expression of different connexin markers as cells move up from the stratum basal
No junctions are found in the stratum corneum
Cx26 may control keratinocytes differentiation, if a mutation causes over proliferation this gives flaking skin
Communication departments in cell proliferation
Vonwinkel syndrome
Cx26 Dominant Palmoplanter hyperkeratosis Auto amputation Hearing loss
Keratitis ichthyosis deafness (Kid)
Mutations in NT of ECL1 of cx26 Determines pore size and sensitivity Keratitis (inflamed cornea) blindness Ichthyosis (dry thick scaly skin) prone to infection Widespread skin Hair and hearing loss
Connexin expression in the heart
Speed of the connexins
SA- 45,40 AV- 45,40 (0.05m/s) Atria- 40,43 His- 40,43 Purjunke- 40,43 (5/ms) Bundle- 45 Ventricle- 43
40- large channels (0.8-1 across atria)
43- medium channels
45- low conductance channels
Atrial fibrillation
Atria don’t contract, blood pooling
Thrombosis risk of embolic stroke
Can be acquired after hypertension, arterial disease
Idiopathic is autosomal dominant mutations in cx40, 10-20%
Ventricular fibrillation
Causes pump failure
Ventricles twitch instead of contracting
Cx43
Atrial standstill
Loss of electrical and contraction in atria
Polymorphism in cx40 and mutation in SCN5A for Na+ channel
Sudden infant death syndrome
Cx43
Lone AF
Ventricular tachyarrythmias (fast rate)
Non junctional functions of connexins
Can act independent
Open hemichannels can release ATP to act on receptors on other cells
Regulation stops leak of molecules
Types of taste buds
1- glial like
2- receptor, sweet bitter umami
3- sour and salty, release serotonin onto neuron
Pannexin in taste buds
T1R and T2R GPCRs on type 2 cells activated
Activates PLCB2 raising Ca
TRPM5 calcium gatedchannels open, NA+ entry
Depolarisation opens more NA channels
Pannexin 1 channels open- regulated by voltage and Ca
ATP released
ATP acts on P2X on type 3 to depolarise
Type 3 release serotonin
Why not conventional transmission in taste buds?
Receptor cells continuously replaced
Release of ATP means that synaptic contacts aren’t made and broken
TTMN
GCI
PSI
DLMN
Tergotrochanteral
Commissural interneuron
Peripherally synapsing
Dorsal longitudinal
