Lecture 10 - B subunits Flashcards
Beta subunits
are cytoplasmic/integral membrane proteins which regulate trafficking and Po
KCNE family
vary in size 103-177aa
small mr
one TMD
KCNQ1 changes it properties by…
depending on different Beta subunit effecting it
Effect of KCNE1 on KCNQ1
E1 enhances Q1 function, the current is larger when both are expressed
Also reaches steady state faster
KCNE1 and renal function
KCNE1 is on the apical membrane of the proximal tubule, important for glucose transport
Proximal tubule proteins (3)
Apical = KCNE1 Basolateral = Na+/K+ATPase and K+/glucose cotransporter
Clearance studies in vivo
anaesthetise mouse on heated pad, use rectal thermometer
cannulate carotid artery, jugular vein and ballder
Why cannulate carotid artery?
BP measurement and blood samples
Why cannulate jugular vein?
fluid replacement
Why cannulate the bladder?
collecting urine for analysis
E1 KO mice
no change in sodium or chloride plasma conc. decreased glucose conc. GFR normal
Fractional excretion calculation
rate excretion/rate filtration
E1 KO fractional excretion
sodium and chloride increase with water
no change in glucose
E1 KO mice shows that…
E1 is most likely in the late proximal tubule therefore sodium channel is not working and causes a reduction in the sodium driving force
Chromanol 293b
specific KCNQ1 inhibitor
Chromanol 293b in WT
mimics an E1 KO
but no effect on KO
Q1 KO clearance studies
no difference between KO and WT in sodium and water fractional excretion
Bigger the chromosome-sensitive current…
greater channel function
Patch clamp studies on E1 and Q1
WT does not show voltage-dependence so is not regulated by E1
therefore E1 does not regulate Q1
Parietal cell basolateral membrane proteins
Na+/H+ exchanger
Na+/K+ATPase
Basolateral K+ channel
HCO3-/Cl- exchanger
Parietal cell apical membrane proteins
chloride transporter
K+ channel
H+ATPase
Parietal cell mechanisms
Co2 and H2O diffuse into the cell under carbonic anhydrase influence to produce HCO3- and H+
Chloride driving force for net secretion of HCL
ATP used to exchange K+ into the cell (recycled across the membrane) and H+ out to form HCL
Parietal cell stimulants
acetylcholine
Histamine
Gastrin
Acetylcholine stimulates…
M3
Histamine stimulates…
H2
Gastrin stimulates…
CCKB
Ammonium pulse technique
ammonium added to EC solution, dissociates to NH3 and H+
NH3 enters cell and mops H+ up inside so pH increases
Take away EC ammonium so NH3 dissociates from H+ and moves out of cell, increased acidification in the cell
Ammonium pulse technique shows
the rate of pH recovery of the cells to remove H+
stimulate carbochol/histamine then remove EC Na+ and measure rate in absence of a protein
In H+/K+ATPase KO
no recovery seen
KCNE2 KO
achlorhydic
pH is already very high under control conditions and does not react with added histamine
KCNE2 KO ammonium pulse technique
histamine stimulated
KP secretes less H+
lack of function of K+/H+ATPase due to KCNE2 loss
apical membrane has Q1/E2 complex
Q1 location
regulates KCNE2 ont he apical membrane of parietal cells
