Synapses are specialized structures where nerve cells communicate chemically. The presynaptic cell releases a chemical neurotransmitter, which then binds to receptors in the postsynaptic cell membrane. These receptors are proteins known as ligand-gated ion channels.
Neurotransmitter release by pre-synaptic membranes is triggered by action potentials, and typically the concentration of neurotransmitter in the synapse rises to over 1 mM (millimolar) in tens of microseconds.
Figure 1: Schematic of an excitatory synapse. From Campagna et al, NEJM 2003;348:2110
To simulate this event experimentally, we use a piezo-actuated quad (2´2) array of capillary tubes that carry solutions superfusing a voltage-clamped outside-out membrane patch. The voltage-clamped membrane patch contains a number of ligand-gated ion channels that are identical to the neurotransmitter receptors in post-synaptic membranes. By altering the DNA sequence encoding ion channel proteins, we can also study mutated or chimeric channels that may display altered sensitivity to neurotransmitter or to various anesthetics.
Currents carried by ions passing across the voltage-clamped membrane patch are monitored using a patch-clamp amplifier and a specialized computer interface for recording these rapidly varying currents. Small whole cells can also be studied using patch-clamp electrophysiology, but the speed of solution switching is slower (> 2 ms) when whole cells are used.
The piezo elements in our experiment can actuate rapid movements, so that concentration jumps are achieved in under 1 ms (millisecond). The quad array allows us to rapidly change the concentration of neurotransmitter and/or anesthetic at the membrane patch. We can also sequentially change these drugs to, for instance, observe the speed of anesthetic effects after channels have been opened, or to determine whether pre-exposure to anesthetics before channel activation alters the resulting current when neurotransmitters are added. By adding arrays of solution reservoirs and upstream selection valves, we can also use this device for concentration-response studies.
One particularly useful experiment is the internally controlled single sweep experiment. For this, we expose the ion channels first to a low agonist concentration for a duration that allows maximal current to evolve. Then the superfusate is switched to an internal control, usually a maximal agonist concentration. The resulting current trace contains two peak currents at one experimental and one control agonist concentration. The control peak is used to normalize the peak response at low concentrations. The resulting concentration-response curve is very similar to that obtained using traditional methods where every other sweep is a control sweep. However, in certain cases, such as when mutant channels activate slowly and desensitize rapidly, there can be significant differences between the internally controlled peak ratio and the traditional experiment (e.g. the α1S270I mutant channel shown in Figure 3). This is because the internal control of the single-sweep experiment represents a mixture of ion channel states that contains more desensitized channels relative to a single rapid jump into high agonist.
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