Dear Haushe,

If you are seeing currents in GABA absence and also in presence of GABA blockers, it is unlikely that the channel you are recording from is a GABA-gated channel. Which cell type are you recording from? Possible that you are recoreding from other endogenous channels such as mechano-gated channels (?) What is the reversal potential and conductance of the single-channel currents you are recording?

If possible include an axample of the recording

best wishes,

Refik

Refik Kanjhan I am recording from pyramidal neurons, in the painted turtle cerebral cortex, I have included an example trace, reversal potential is seemingly around - 20 mV, I further went onto use other blockers such as AP5 and CNQX in my electrode in addition with gabazine, and also am using TTX 1uM in the bath as well... not sure what other channels may be possible - I never thought about mechanics-gated channels really..

Dear Haushe,

From the traces the currents through the channel seem to be reversing close to 0 mV, and thus suggesting it is a non-selective cation current. If you are encountering this very frequently, it means that the membranes has plenty of these channel embedded. Unfortunately it is not a straight forward answer, and the cell type you are studying is not extensively studied previously. It is also obscure that whether it is a ligand-gated channel? You may need to use different blockers to identify what type of non-selective cation current it is. Does it respond to divalent cation concentrations in your pipette solution?

best wishes,

Refik

Nicolas Paradis-Isler in single channel recording, you are sealing around a single channel on the membrane, and still applying the drugs (inside the recording pipette) extracellularly without rupturing the membrane. Although you are supposed to record from single channels sensitive to the agonist, sometimes by chance you can also record from channels such as mechanosensitive channels, that are activated by the mechanical stimulation of the membrane during suction and seal formation.

best wishes,

Refik

Hi Nicolas Paradis-Isler it is not really necessary to add antagonists for other ligand-gated channels such glutamate-gated channels. It is costly, time-consuming, and also may indirectly or non-specifically effect the membrane properties; and certainly does not make it any safer. Single ligand-gated ion channels are usually studied by adding the agonist in the patch pipette. In addition to antagonists, properties such as channel conductance and reversal potential of currents help to characterize and confirm the identity of the channel.

Most channels are well characterized in expression systems such as oocytes or HEK cells, which do not have many native channels but can express high levels of channel of interest after introduction of its mRNA. In native cells, it is more challenging as these can express many different channels and also at different locations such as soma, proximal and distal dendrites, or axon. Therefore such experiments can be time consuming, since you blindly seal to the membrane until you get response to the agonist in the pipette solution. If the channel is not expressed or at very low levels at the soma then finding a channel for recording can be alot harder.

Best wishes,

Refik