What can the cell-attached configuration tell us?
Gives us real-time, label-free measurements on an isolated single protein molecule
Why use cell-attached configuration/single channel recordings?
Contains more information than macroscopic records (allows us to understand channels in terms of mechanisms)
Activation of a single channel in the right conditions is equal to synaptic activations
Can reveal biophysical properties, stoichiometry, and even structural aspects of the cell
What conditions do we need to satisfy for single-channel recordings?
Channel has large amplitude (>1 pA) or slow openings (5 ms), otherwise it is very difficult to record
Can practically reach 100 fA quite easily, so need to keep amplifier limits in mind
What kind of filter needs to be used to make data from single channel recordings visible? Why?
low pass
Increases signal-to-noise ratio/reduces noise
But information from frequencies above the cutoff are lost forever, so always record with an open filter and do more filtering after if necessary
How to filter
- Obtain low noise recording conditions
- Record long stretches of single channel activity
- Idealize the data (need to do reconstruction)
- Look at amplitude histograms
- Look at dwell time histograms and fit them
- Fit the data w/ mechanisms like maximum likelihood to obtain the rate constants
Why should we record threshold crossing?
Always need to define events that are too fast to detect
Something above threshold may be important, but may be lost through filtering
What does time course fitting give us?
Get amplitude and duration information
What do dwell time distributions show us?
Show number of shut times where channels were shut
Number of components in the distribution corresponds to the number of states (= a way of counting how many different shut states there are, how fast they are, etc.)
- Linear scale dwell time distributions show us all observations
- In a Sigworth-Sine plot each component has a peak at tau
Software for idealization/fitting/etc.
QUB (automatic, but info about fast events is lost), DCPROGS (manual fitting, get most complete info), Bruxton, AXON, SCATE (single channel fitting on iPad), ASCAM
What does the Q matrix give us?
the kinetic mechanism of the channel
Can estimate values in the matrix which fit single channel data
Why are single channel recordings important for physiology?
- Synaptic transmission is largely driven by packets of NT and receptor activation occurs mostly at 0 transmitter
- Channels open in bursts (are especially important for synaptic transmission at low agonist concentrations; states between open/shut/inactive are important to look at)
- Can ascertain channel kinetics, which set the decay of the synaptic current
- Can help identify type of receptor
Insights gained from single channel recordings
Multiple conductances of Glu receptor → more than one kind of Glu Receptor
- ->For the NMDAR at positive potential the response in Mg free and Mg block are approximately the same (need strong depolarization to remove block), but if there’s a negative potential, receptor will shut again
- -> AMPAR are more complicated, and have staircase activation, revealing 4 identical binding sites (tetramer) leading to partially opened states (→ 3 intermediate states)
- -> CII toxin binding to AMPAR led to much longer activation, but instability of the open state
Partial agonism
→ Taurine is a partial agonist for the Glycine/nicotining receptor superfamily
→ taurine requires more activation energy to go from resting to open
→ glycine strongly favors the flipped (shut) and open states, because it is initially at a higher energy level
What are TARPS?
= Transmembrane AMPAR regulatory proteins
They’re proteins that change the single channel properties of AMPARS
What does the 1st latency period describe?
the minimum number of shut states that the receptor must transit to open
