Methods

Question 1

Neuro2A cells express MA currents

Answer 1

1. Patch-clamp recordings of whole cell currents in whole cell configurations.
2. Patch clamp recording of single channel currents in cell-attached configuration.
   From this, found that N2A displayed consistent MA currents w/ relatively fast adaptation in response to stimuli.

Question 2

Piezo1 (Fam38A) is required for MA currents of N2A cells

Answer 2

1. Silenced the genes coding the different protein candidates using siRNA knockdown in N2A cells. Measured MA currents by doing patch-clamp recordings in whole-cell configuration while applying pressure with glass probe.
2. Quantitative PCR assays used to confirm that siRNAs worked in decreasing transcription.
   a. Confirming that these siRNAs targeted the intended gene and protein.
3. Transfected N2A cells with TRPV1 cDNA (so that the N2A cells can now respond to capsaicin, a chemical stimulus) and added scrambled or Piezo1 siRNA.
   a. Did this to check that the general cell signalling is not impaired, and that that isn’t the reason MA currents have been decreased. This experiment showed that the cell can still respond to other forms of stimuli, so impact on MA current response is specific to mechanical stimuli and Fam38A is involved.
4. Patch-clamp recordings to see if MA currents are also diminished in response to patch-membrane stretch when siRNAs target Piezo1.
   a. MA currents in response to membrane-stretch were also diminished when Piezo1 was targeted. So Piezo1 is also required for response to membrane-stretch.

Question 3

Piezo1 induces MA currents in various cell types

Answer 3

1. Cloned full length Piezo1 into a vector also encoding GFP. Did patch-clamp recordings in whole-cell configuration of GFP+ cells 12-48hrs after transfection. Transfected and did patch-clamp recordings with different cell types – N2A, HEK293T, C2C12.
2. Blocking Piezo1 channels by adding EC non permeant cation NMDG blocked inward MA current.
3. Patch-clamp recordings with IC solution lacking cations and adding different combinations of cations to EC solution.
4. Membrane stretch though patch-clamp recordings in cell-attached configuration of cells used to assay Piezo1 transfected cells. Measured MA currents in response to negative pressure.

Question 4

MA currents in cells overexpressing Piezo2

Answer 4

1. Cloned full-length Piezo2 from DRG neurons into a vector that also encoded GFP. Transfected HEK293T cells with this vector. Cotransfected N2A cells with this vector as well as Piezo1 siRNA to suppress endogenous MA currents, isolating current generated by Piezo2.
2. Piezo2-dependent currents were suppressed by NMDG.
   a. Suggests nonselective cationic conductance.

Question 5

Piezo1 is detected at the plasma membrane

Answer 5

1. Generated an antibody against mouse Piezo1, which would only recognise Piezo1-transfected HEK293T cells. Transfected cells with Piezo1 and PM ion channel TRPA1, and observed some staining overlap on the cell surface.

This indicates that Piezo1 protein can be localised at/near PM.

Question 6

Requirements of Piezo2 for rapidly adapting MA currents in DRG neurons

Answer 6

1. Performed in situ hybridisation on adult mouse DRG sections.
2. siRNA transfection – scrambled and Piezo2 as well as GFP – and recorded whole-cell currents. These were divided into 4 classes according to inactivation kinetics and trends were determined.
3. RNA interference on DRG neurons validated on TRPA1 – expressed in DRG neurons.