Lecture 4 Flashcards
How would you use patch clamp to measure recordings from a channel?
Use the on-cell conformation, so apply the patch clamp to form a gigaohm seal then take recordings.
How would you use a patch clamp to take recordings from the whole cell membrane?
You would use the whole-cell method which involves applying the clamp to form a gigaohm seal then applying suction causing the cell to rupture.
How would you use a patch clamp to see how a molecule affects the intracellular side of a channel?
You would use the inside-out conformation, so attach the patch clamp to form a gigaohm seal then pull
How would you use a patch clamp to record how a channel is affected exteacellularly?
You would use the outside out conformation. So you’d attach the patch clamp to form a gigaohm seal then suck then pull.
What tool can be used to record the activity of a single channel?
Patch clamp
Describe single channel current events
They are extremely small about 10-12A to 10-10A and have a short duration
What can single channel recordings be used for?
To measure the probability of a channel being open
Measure single channel current amplitude and conductance
Describe the key features of Na channel current
They are stochastic
Single current amplitude is about the same from channel to channel
Opening of the channel is very brief, but duration does vary slightly
They have a very short latency
The Popen initially rapidly increases upon current application then falls shortly after
Describe the key features of the potassium channel current
Higher latency
open longer and more often
Popen slowly increases then plateaus for a long time period and drops rapidly
Why does sodium channel open probability decrease with with prolonged depolarisation?
Sodium channels have three conformations, controlled by the activation gate and the inactivation gate.
Closed - activation gate closed
Open - both gates open
Inactivated - inactivation gate closed
The probability of channel going from closed to open is increases with depolarisation, causing the activation gate to open. There is then a short delay before the inactivation gate closes making the channel inactivated.
On a molecular level describe why a refractory period occurs after an AP?
When depolarisation occurs the Na channels open transiently, they then enter the inactive state as the inactivation gate closes. In order to return to the open conformation the channel must recover, and the membrane potential has to repolarize before this can occur. Hence, the absolute refractory period.
The relative refractory period occurs as the cell repolarizes and some channels have recovered whilst others have not. This means that a larger threshold potential is required as there are fewer sodium channels in the closed conformation.
Meanwhile the potassium Popen remains high, and the potassium channels close slowly therefore a larger depolarising stimulus is required to reach threshold as hyperpolarisation occurs
What are ion channels molecularly?
Membrane spanning glycoproteins that form pores through the cdll membrane
Which type of ion channel is the most structurally and functionally diverse?
Potassium channels with 90 different genes encoding them
Outline the basic structure of a voltage gated
potassium channel
They are made up of four identical subunits
Each subunit contains 6 a
Pha helical segments that span the membrane
P-loop between S5 and S6 contains ion selectivity filter
S5-S6 = pore forming domain
P loop located in the centore
S1-s4 is the voltage sensing domain
How can s4 region of the voltage gated potassium channel detect voltage changes?
It has positively charged arginines or lysines at every third position that detect changes in TM potential
Outline the structure of a sodium channel
Pore formin peptide subunit is large and has 4 homologous repear domains connected by intracellular linkers
Only one subunit is required to form the channel
Each homologous domain has 6 TM segments
The intracellular linker between III and IV forms the inactivation gate
WhAt can bs used to cleave the inactivation gate preventing inactivation of a sodium ion channel?
Pronase or binding antibodies
