Lecture 09 Potassium Channels Flashcards
What is the function of potassium channels?
the channel maintains a negative resting membrane potential which helps to regulate the cell volume
What are the main 3 potassium channel families?
How many transmembrane domains and pore regions in each subunit? How many subunits are required to make one functional channel?
Give an example
voltage gated potassium channels (Kv) - 4 TMD 1P - 4 subunits - KCNQ1; KCNA10; calcium activated Kv channels - SK4; BK
inwardly rectifying potassium channels (Kir) - 2TMD 1P - 4 subunits - Kir1.1 (ROMK)
two pore potassium channels - 4TMD 2P - 2P - 2 subunits - TWIK-1; TASK-2
What ion channel helps to drive chloride secretion?
K+ selective channel in the basolateral membrane
increasing activity results in a greater negative potential which increases chloride secretion
Describe the KCNQ1 or Q1 channel
How can we study it’s function?
KCNQ1 or Q1 is a Kv channel in basolateral membrane of upper airway epithelial cells
it can be inhibited by chromanol 293B - this can be used as a pharmacological tool to look at Q1 function
What is a disadvantage of chromanol 293B?
alone chromanol 293B is not sufficient to be linked to Q1 expression as they can still be partially functional in the presence of the inhibitor
resolve this using mRNA or shRNA expression studies
How was the expression of the Q1 protein proven in upper respiratory epithelia?
PCR and Southern Blot of Q1 RNA
showed expression of Q1 in all upper respiratory cells including non-CF patients, CF patients and in human bronchial epithelia (HBE) cell lines
How was the function of the Q1 protein linked to chloride secretion?
What else did this experiment show?
Ussing chamber technique
added chromanol 293B in increasing amounts resulting in an inhibition of the chloride SCC - inhibiting potassium transport decreases the driving force for chloride secretion so Vm becomes less negative allowing indirect measurement of Q1 function
did not block all potassium transport - still functioning potassium channel is barium-sensitive
What experiment was completed to show the chloride secretion in cystic fibrosis patients in terms of potassium channel Q1?
compared non-CF and CF tissues - Ussing chamber
293B sensitive channels
non-CF - small secretion of chloride, increased secretion when stimulated with IBMX which increases cAMP leading to increased Q1 and CFTR function
CF - CFTR channels not functional no matter with or without stimulation leading to increased cAMP
barium sensitive channels
non-CF - small increase in chloride secretion when stimulated with IBMX
CF - some potassium transport driven by barium-sensitive channels driving chloride secretion but not as much as normal
What beta subunit regulates Q1?
KCNE3 (E3)
What are the other functionally linked potassium and chloride channels in the upper respiratory tract?
What is there function?
hSK4 - calcium activated potassium channel in the basolateral membrane
CaCC - calcium activated chloride channel in the apical membrane
What can be used to inhibit hSK4?
clotrimazole
What can be used to activate CaCC?
UTP (purinoceptor activation)
What effect does adding UTP have on epithelial cells?
small but significant hyperpolarising shift in epithelial potential resulting in increased chloride secretion through activation of hSK4 and CaCC
What effect does 293B have on UTP induced SCC in the absence of cAMP? Is this what is expected?
little-to-no effect on UTP SCC
not surprising as no cAMP present for Q1 function
What effect does Clotrimazole have on UTP induced SCC in the absence of cAMP? Is this what is expected?
SCC is lost on addition of clotrimazole
clotrimazole inhibits hK4 which is functionally linked to CaCC through changes in intracellular calcium
calcium stimulation is required for chloride secretion and so no UTP SCC is detected
