10/4/16 Review.

10/4/16 Review

Ih HCN channels Hyperpolarization activated Cyclic nucleotide gated Cation nonselective: reversal potential ≈ -40mV What would the activation curve for a hyperpolarization activated channel look like? If it is cyclic nucleotide gated (more active in the presence of cyclic nucleotides), how would the curve shift in the presence of cyclic nucleotides?

Calcium-activated potassium channels (Kca)
Calcium and voltage-gated IAHP BK: big conductance. Large, medium-fast AHP SK: small conductance. Slower, longer lasting AHP; Both are slowly or non-inactivating depending on subunit composition Slow to activate and require calcium influx. Activate during repetitive spiking

Apamin blocks SK Spike frequency adaptation

If this is the activation curve for a Kca channel at one calcium concentration, what would the curve look like at a lower calcium concentration? At a higher calcium concentration?

Inward rectifier K+ channels ‘clamp’ neuron
at resting membrane potential Ach IM M2 muscarinic Ach receptor EK G Gi EM Vm GIRK: G-protein coupled inwardly rectifying K+ channel

L-type Ca2+ channel Voltage-gated: higher voltage-activated than T-type Non-inactivating or slowly inactivating L-type: Large conductance, Long lasting T-type: Transient Erev ≈ ECa Responsible for cardiac action potentials Conductance increases in presence of Adrenaline Conductance decreases in presence of Acetylcholine Often calcium source for activating Kca channels

L-type Ca 2+ channels Delayed onset long-lasting effect Bean et al.

Other calcium channels
N-type P/Q-type High voltage activated Usually involved in neurotransmitter release

What would Katz say is going on
B Evoked EPP Spontaneous mEPP

early evidence for excitation by glutamate
1. What kind of recordings are these? How can you tell? 2. Why does L-Glut have units of Amperes instead of Moles? 3. Why did they include the trace on the bottom right?

Brief aside on extracellular recordings
1. Which recordings are intracellular and which are extracellular? 2. How can you tell for sure? 3. How many cells are they recording from(probably) in each case? 4. What would it look like if they were recording from multiple cells extracellularly? Here is a zoomed in view of a spike from an EC recording. Why do you see a downward deflection first and why is it larger than the upward deflection if action potentials are positive spikes in membrane potential?

glutamate receptor pharmacology
NMDA AMPA kainate agonists glutamate glutamate glutamate NMDA AMPA kainate aspartate quisqualate domoate kainate (AMPA) domoate antagonists ________ _________ ______ GYKI53655 permeability Na, K, Ca Na, K (Ca) Na, K, (Ca) co-agonist _______ channel _____ block MK-801 So common they are worth knowing

glutamate receptor pharmacology
NMDA AMPA kainate agonistsglutamate glutamate glutamate NMDA AMPA kainate aspartate quisqualate domoate kainate (AMPA) domoate antagonists APV CNQX CNQX GYKI53655 permeability Na, K, Ca Na, K (Ca) Na, K, (Ca) co-agonist glycine channel Mg block MK-801

orientation of KcsA versus iGluR

I-V relation for whole cell current through NMDA receptors
What is the approximate reversal potential for NMDA receptors? What about for AMPA receptors? Kainate? with Mg2+ without Mg2+ Nowak et al., 1984

Why do you not see these short-lived blocked/closed states here?
Magnesium concentration Nowak and Ascher, 1988

What are they showing you here?
I thought they were called NMDA receptors. Why is this glutamate evoked current so much larger? What are they showing you here? Johnson and Ascher, 1987

agonist-dependent desensitization of AMPA receptors
Patneau and Mayer, 1991

Things relevant to tomorrow’s discussion paper

I-V relations for the peak and decay phase
How did they get this curve? What does it look exactly like? What about this curve? Why is it linear? Hestrin et al., 1990

What is APV? Hestrin et al., 1990
Where do the white circles come from? Why linear? Why do they almost just go straight across the x-axis? What is APV? Where do these white triangles come from? Hestrin et al., 1990

Where do the white dots come from?
Hestrin et al., 1990

Why hold at +20mV? Why a larger current in –Mg2+? What are these? Why do the white circles have a steeper slope? Hestrin et al., 1990

Why is the NMDA receptor-mediated component slow
Why is the NMDA receptor-mediated component slow? (I think he really means long lasting) Lester et al., 1990

Why are NMDA receptors sometime called “coincidence detectors”

Which of these are pentameric
NMDA receptors AMPA receptors Kainate receptors Nicotinic Acetylcholine Receptors

Which of these are tetrameric

Which of these have a pore loop domain
NMDA receptors AMPA receptors Kainate receptors Nicotinic Acetylcholine Receptors Metabotropic glutamate receptors Voltage-gated potassium channels

Which of these are Ca2+ permeable and experience voltage-dependent magnesium block

RNA editing controls permeability in

Which of these are G-protein coupled
NMDA receptors AMPA receptors Kainate receptors Nicotinic Acetylcholine Receptors Metabotropic glutamate receptors Voltage-gated potassium channels

Which is the main target of altered trafficking in LTP/LTD

What you are looking at in a single-channel recording
CLOSED OPEN 6 pA 5 ms Single channel opens channel closes channel reopens You are measuring the current flowing through one ion channel Please note that throughout this lecture, channel openings are shown as upward deflections, regardless of the actual direction of current flow.

Closed Open k1 k-1 What is the mean duration of closed?
What is the mean duration of open?

What is the mean open time for the channel
Closed Open Blocked 100s-1 5000s-1 What is the mean open time for the channel

What is the mean open time for the channel
Closed Open Blocked 100s-1 5000s-1 What is the mean open time for the channel 1/(100s s-1) = s or .909ms The time spent in any state is just 1/the sum of the rate constants leaving that state What is the average blocked time 1/5000s-1 = .0002s or .2ms What is the average closed time 1/200s-1 = .005s or 5ms

What is the mean number of openings per burst
Lets call a burst a period where the channel opens and never closes for more than 1ms 200s-1 1000s-1 Closed Open Blocked 100s-1 5000s-1 What is the mean number of openings per burst

What is the mean number of openings per burst 1+(1000s-1/100s-1) = 11
Lets call a burst a period where the channel opens and never closes for more than 1ms. Note: this is not the official definition for a burst. I made it up for this example. He will define a burst for you if he asks this question. 200s-1 1000s-1 Closed Open Blocked 100s-1 5000s-1 What is the mean number of openings per burst 1+(1000s-1/100s-1) = 11

If the channel enters this state, it will only be blocked for a fraction of a millisecond on average. Entering this state will not end the burst If the channel enters this state, it will stay here for 5ms on average, ending the burst (see previous question) Lets call a burst a period where the channel opens and never closes for more than 1ms 200s-1 1000s-1 Closed Open Blocked 100s-1 5000s-1

If the channel enters this state, it will only be blocked for a fraction of a millisecond on average. Entering this state will not end the burst If the channel enters this state, it will stay here for 5ms on average, ending the burst (see previous question) Lets call a burst a period where the channel opens and never closes for more than 1ms 200s-1 1000s-1 Closed Open Blocked 100s-1 5000s-1 To calculate the number of openings then all we need to know is how many times on average the channel transitions from “open” to “blocked” before it transitions to the “closed” state. To calculate this, all you have to do is divide the rate constant for the “open” to “blocked” transition (1000s-1) by the rate constant for the “open” to “closed” transition (100s-1) (1000s-1/100s-1) = 10

If the channel enters this state, it will only be blocked for a fraction of a millisecond on average. Entering this state will not end the burst If the channel enters this state, it will stay here for 5ms on average, ending the burst (see previous question) Lets call a burst a period where the channel opens and never closes for more than 1ms 200s-1 1000s-1 Closed Open Blocked 100s-1 5000s-1 To calculate the number of openings then all we need to know is how many times on average the channel transitions from “open” to “blocked” before it transitions to the “closed” state. To calculate this, all you have to do is divide the rate constant for the “open” to “blocked” transition (1000s-1) by the rate constant for the “open” to “closed” transition (100s-1) (1000s-1/100s-1) = 10 Then, to get to the final answer, you just have to remember that any burst must begin with the channel open, so you always add 1.

What is the mean number of openings per burst 1+(1000s-1/100s-1) = 11
If the channel enters this state, it will only be blocked for a fraction of a millisecond on average so entering this state will not end out burst If the channel enters this state, it will stay here for 5ms on average, ending the burst (see previous question) Lets call a burst a period where the channel opens and never closes for more than 1ms 200s-1 1000s-1 Closed Open Blocked 100s-1 5000s-1 What is the mean number of openings per burst 1+(1000s-1/100s-1) = 11 Every burst must start with the channel open The open channel will make 10 transitions between open and blocked on average for each transition it makes to closed.

What is the mean open time per burst
Closed Open Blocked 100s-1 5000s-1 What is the mean open time per burst

Closed Open Blocked 100s-1 5000s-1 What is the mean open time per burst Hopefully the answer to this is obvious after the previous two questions. You have already calculated the mean open time and the mean number of opening per burst, so all you have to do multiply those together to get the answer

Closed Open Blocked 200s-1 100s-1 1000s-1 5000s-1 What is the mean open time per burst Hopefully the answer to this is obvious after the previous two questions. You have already calculated the mean open time and the mean number of opening per burst. All you have to do multiply those together to get the answer open time = #openings * duration of opening ≈ 11*.909ms ≈ 10ms If he asked for the total burst duration, you would need to add the blocked time to the above answer During a burst, you would enter the blocked state 10 times on average. The average time spent in the blocked state is .2ms. During a burst, the channel would spend on average 2ms in the blocked state. 10ms + 2ms = 12ms would be the total burst duration

Calculate the number of ions that go through the channel during a burst
If we say that when the channel is open a 5pA current flows through it 5pA is 5*10-12A or 5*10-12C/s The channel is open for 10ms during a burst The number of ions would be (5*10-12C/s) * (10*10-3s) = 5*10-14 Coulombs

The channel has made 9 transitions from open to blocked
The channel has made 9 transitions from open to blocked. What is the probability that the next transition from open will be to blocked again instead of closed? Closed Open Blocked 200s-1 100s-1 1000s-1 5000s-1

The channel has made 9 transitions from open to blocked
The channel has made 9 transitions from open to blocked. What is the probability that the next transition from open will be to blocked again instead of closed? The channel is still 10 times more likely to transition from open to blocked again. This is what he is talking about when he talks about Markovian or memoryless processes. The previous history of the channel does not affect the transition probability. Closed Open Blocked 200s-1 100s-1 1000s-1 5000s-1

mean duration of opening = a-1 mean duration of burst = a-1 (1+b/k-2)
cluster burst openings (A2O) A2C When b>k-2, we observe several openings per burst. On the other hand, with low efficacy agonists that have low beta, bursts contain a single opening. mean duration of opening = a-1 mean duration of burst = a-1 (1+b/k-2) mean life time of A2C = (b+k-2)-1 number of openings per burst = 1+ b/k-2 You should be able to look at this and tell him these I think this should say mean open time during burst.

two receptors simultaneously active
Like these big gaps and stuff Why do these looks so different? two receptors simultaneously active Currents were simulated with 5 ion channels at two different agonist concentrations.

2 # channels open 1 Burst Short closings/blocks within a burst
# channels open Burst Short closings/blocks within a burst End of burst channel closes and stays closed

Transition rate from A2C to A2O
burst A2C openings (A2O) Closed 1 agonist molecule bound but still closed 2 agonist molecules bound but still closed Channel open Transition rate from A2C to A2O Transition rate from A2O to A2C

A common question is: how long is an average channel opening?
Rate at which open channels close A common question is: how long is an average channel opening? The rate constants tell you the rate at which the channel transitions between states. In this case, the only rate constant that leaves the open state is alpha. Rate constants will have units of s-1 or per second. If, for example, alpha is 1000s-1, there would be 1000 transitions from open to closed each second. The average open time during a single opening of the channel would therefore be 1/1000s-1 or .001s or 1ms.

We can ask the same question of our doubly-liganded closed state: how long on average does the channel spend in this state once it enters. The basic procedure is the same, but there are now two ways the channel can exit this state: it can reopen (move to A2O) or one of the ligands can unbind (move to AC). Average time in A2C: 1/(beta + k-2)

k2 Now for bursting. Lets say that the transition from A2C to A2O is extremely fast (beta = s-1) and the transition from AC to A2C is extremely slow (K2 = 1s-1). We will keep k-2 and alpha in between (1000s-1). Another common Gustav question is to ask how long an average “burst” is. By burst he means a period over which the channel is open, may switch to short-lived closed states, but not a fully closed state. He will usually define a burst for you. For our example lets say a burst ends if the channel is closed for more than 1ms. If you calculate the time spent in each state in the mechanism above, you will see that the average time in A2C is less than 1ms, while the average time in AC is much greater than 1ms. This means that if we enter AC the burst is over. To calculate the burst duration all we need to know is the average amount of time spent in A2C and A2O and on average how many transitions we will make between these two states for each transition between A2C and AC. Fortunately, this is easy to calculate. beta = s-1 k-2 = 1000s-2 so, if we are in A2C, on average we are 100 times more likely to make a transition from A2C to A2O than we are to AC.

mean duration of opening = a-1 mean duration of burst = a-1 (1+b/k-2)
cluster burst openings (A2O) A2C When b>k-2, we observe several openings per burst. On the other hand, with low efficacy agonists that have low beta, bursts contain a single opening. mean duration of opening = a-1 mean duration of burst = a-1 (1+b/k-2) mean life time of A2C = (b+k-2)-1 number of openings per burst = 1+ b/k-2

1000 s-1 10 s-1 CLOSED OPEN CLOSED2 1) In the model above, what is the mean duration of an opening, what are the mean durations of closed times? 2) If a "burst" is an episode of activity with the mean closed time between openings under 100 ms, what is the mean number of openings per burst? (Hint: it may help to draw what activity would look like.) 3) What is the mean duration of a burst? 4) What is the net number of monovalent ions that move through the channel during a burst if the amplitude of the open state is 1 pA? 1000 s-1 0.1 s-1

1000 s-1 10 s-1 CLOSED OPEN CLOSED2 1) In the model above, what is the mean duration of an opening, what are the mean durations of closed times? Open time: 1/(1000 s-1+10 s-1) = 0.99 ms; Closed times: 1/1000 s-1 = 1 ms; 1/0.1 s-1 = 10 s 1000 s-1 0.1 s-1

Burst must start with opening
CLOSED OPEN CLOSED2 2) If a "burst" is an episode of activity with the mean closed time between openings under 100 ms, what is the mean number of openings per burst? (Hint: it may help to draw what activity would look like.) The mean lifetime of Closed2 is 10 s, the mean lifetime of Closed1 is 1 ms. The question then becomes how often does the receptor enter Closed1 from the Open state before it is terminated by entry to Closed2. Number of openings per burst = 1+(1000 s-1/10 s-1) = 101 1000 s-1 0.1 s-1 Burst must start with opening

(1/1010)*101 = (101/1010) = .1 seconds = 100 milliseconds
CLOSED OPEN CLOSED2 3) What is the mean duration of a burst? From previous question, a burst contains 101 openings, that are separated by 100 dwells in Closed1. The mean duration of burst is then 101 x 0.99 ms x 1 ms = 200 ms. 1000 s-1 0.1 s-1 Always 1 less than the number of openings Rounded Duration of opening Number of openings exact (1/1010)*101 = (101/1010) = .1 seconds = 100 milliseconds Total open time

1000 s-1 10 s-1 CLOSED OPEN CLOSED2 4) What is the net number of monovalent ions that move through the channel during a burst if the amplitude of the open state is 1 pA? First, let's determine the number of ions moving during an opening. 1 coulomb = 1 ampere x 1 sec. The charge movement during an opening is 1 pA x 1 ms = C 1 C = 6.2 x 1018 elementary charges, C = 6200 elementary charges In a burst containing 101 openings, the number of monovalent ions conducted is ~600,000. 1000 s-1 0.1 s-1 10^ ^-3 Per opening Rounded ions per burst

K1 = 1000/sec K2 = 500/sec K-1 = 1000/sec K-2 = 500/sec
cluster burst openings (A2O) A2C K1 K2 K--1 K1 = 1000/sec K2 = 500/sec K-1 = 1000/sec K-2 = 500/sec From AC, how many transitions ( on average) will you make to C for each transition to A2C? 2 What is the probability of a transition to C from AC? What about a transition to A2C? 66, 33% When b>k-2, we observe several openings per burst. On the other hand, with low efficacy agonists that have low beta, bursts contain a single opening. mean duration of opening = mean duration of burst = mean life time of A2C = number of openings per burst = Mean duration of AC =

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