Neuro_Evans_4

After proteins are run on SDS-Polyacrylamide gel electrophoresis (PAGE) gels under denaturing conditions the use of PPAPA allows cross linking of receptor SUs so that the MW of the endogenous complex can be viewed. Under increasing concentrations of PPAPA from 0-100µM, bands of three increasing sizes were viewed on a western blot, indicating that the receptor exists as a trimer.

Heteromerisation of receptor SUs produces composite channels of intermediate phenotype e.g. nAChRs often formed of a2ß2y. Also, some native P2X phenotypes don’t correspond to homomers. Many P2X SUs have been observed in a range of tissues.

In order to elucidate the native structure of a P2X receptor, and to identify whether channels can heteromerise, the P2X response was recorded in nodose sensory neurons. The response to ATP and aß-meATP was found to be identical in nodose neurons. This was compared to P2X2 expressing cells and P2X3 expressing cells. P2X2 expressing cells did not respond to aß-meATP, and P2X3 cells showed only a transient response, both in contrast to the nodose cells. P2X2/3 expressing cells showed same properties as the nodose cells indicating that the channels can heteromerise.

Co-immunoprecipitation studies have shown which SU combinations are possible. Tagging receptors with different tags e.g. P2X1-FLAG and P2X2-HA is required. Co-express SUs of interest in a heterologous cell, lyse the cell and use Ab to pull out on of the tags e.g. FLAG and run on western blot. Then stain with anti-HA Ab - band indicates multimerisation of the two SUs. P2X7 only forms homomer. P2X6 only forms heteromer.

P2X6 forms channels with P2X1 but not P2X3. Making chimeric channels e.g. 1.3.1, 3.1.3 and expressing with P2X6 allows the determination of which region of the SU is responsible for SU assembly. 1.3.1 chimeras co-assembled with P2X6, suggesting the intracellular termini are important for SU assembly. 

P2X receptors like P2X2 possess a consensus site for phosphorylation by PKC at the N-terminus - S/T-X-K/R. To test whether this is a site for regulation by PKC, a T18A mutant was produced. P2X2 WT and T18A channels were expressed and ran on a gel. An anti-phosphothreonine Ab was used to stain, showing that the WT was phosphorylated but the mutant was not - indicating this is a site for regulation. The T18A mutant was found to desensitise quickly compared to WT, indicating that the phosphorylation site is crucial for regulation of the time course of the channel.

Alternative splicing can also regulate channel properties. P2X2b differs from P2X2a in that its C-terminal is truncated. P2X2b channels display faster desensitisation. For example, growth factors could be implicated here, favouring expression of different splice variants to regulate the time course of a cell. 

P2X receptors may interact with other ion channels. Though ACh does not affect P2X receptors, and ATP does not affect nAChRs, the depolarisation response in cells coexpressing the two channels is not as expected. ACh application results in an inward current, and ATP application results in a larger inward current. However, co-application does not result in summation of the response i.e. depolarisation does not exceed the ATP response when ACh is also added. This implies cross-inhibition is occurring. 

The difference between the predicted ATP/ACh current assuming summation and the actual current observed upon co-application can be accounted for by total inhibition of the ACh current. To test whether P2X activation was inhibiting nAChRs the T18A mutant was used due to it’s desensitising kinetics. ACh was initially added to a cell expressing both channels. The expected inward current was observed, but upon application of ATP, the ACh current was abolished for a short time until the P2X current was seen, showing that P2X activation inhibits the nAChR.

To determine whether P2X interacts with other proteins, proteins from cells expressing P2X7 ran on gel and stained with anti-P2X7 Ab. (and any associated proteins). This was ran on a gel. Staining identified proteins. Mass spectroscopy was used to identify the proteins. P2X7 was found to be associated with cytoskeletal proteins and tyrosine phosphatase ß (regulation)