Neuro_Evans_2
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Lernkarten
2.1) P2X receptor family info
The P2 family comprises the ionotropic, cation conducting P2X receptors and the metabotropic P2Y receptorsThe P2X receptor family are the simplest ion channel family. The were first cloned in 1994 and were found to possess 2TM domains. Various SU types are expressed widely throughout the body. The channels play a role in synaptic transmission and immune cell functioning.
2.2) Initial evidence for ATP NT activity
Purines were found to display signalling properties in the 50s. Evidence for ATP acting as a neurotransmitter came from e.g. injecting rabbits with ATP was found to increase blood pressure. P2X receptors are important in the regulation of smooth muscle contraction.ATP depolarises neurons and has been found to be co-released with every other major neurotransmitter. .
2.3) Further evidence for ATP NT activity
Application of NA to an artery causes depolarisation. Intrestingly application of the alpha-adrenergic antagonist, phentolamine, does not completely abolish the response, with excitatory junction potentials (EJPs) persisting. Desensitisation of P2X receptors by aß-methyleneATP abolishes the EJPs and co-application with phentolamine completely abolishes the response.
2.4) Cloning-Expression and screen library to isolate target protein cDNA
Early attempts to clone P2X based on an assumed similarity to nAChRs failed. As a result expression cloning was used. Expression -cloning involves tissues highly expressing the channel e.g. rat vas deferens or PC12 cells . mRNA is extracted and reverse transcribed to cDNA. cDNA molecules were inserted into vectors e.g. E. coli cells. Grow clones to make cDNA library.
Next, the library is screened to identify the mRNA encoding the channel. RNA is extracted from the vectors and subdivided e.g. into 10 pools and injected into xenopus oocytes. Application of ATP to the oocytes will elucidate which pool contains the P2X channel. Pools are further subdivided until the mRNA of interest is isolated. DNA clone then sequenced
2.5) P2X receptor subtypes
P2X1 receptors were isolated from rat vas deferens, and P2X2 from PC12 cells. The DNA sequences were used to screen other tissues, and 7 P2X receptor subtypes were identified, with splice variants.
2.6) Norhtern analysis, in situ hybridisation and immunoshistochemical localisation
Northern analysis was used to screen tissues for P2X. The process involves extraction of RNA from tissues which is then ran on agarose gel, and radiolabelled cDNA for P2X is added. Stains indicate the presence of P2X1 cDNA e.g. was found in bladder but not the brain.
In situ hybridisation was used to assess the tissue distribution of P2X4 receptors, as an example. Anti-sense RNA probes were applied to tissue sections of the brain. High expression was found in the cerebellum.
Immunohistochemical localisation can also be used to assess distribution. This is done by identifying a unique amino acid sequence in the protein, immunising an organism e.g. rabbit with a protein containing this sequence and then isolating antibodies produced against the sequence. The Ab can then be applied to a tissue section and a second 2’ Ab coupled to a fluorophore like GFP will bind to epitope-bound antibodies, thus fluorescing in sections containing the P2X receptor.
2.7) Hydrophobicity
DNA isolation permits elucidation of the amino acid sequence which allows a hydrophobicity analysis to be carried out (scale of AAs ranging from -4.5 - 4.5 based on solubility properties).Hydrophobicity analysis identified two stretches of ~20 amino acids of considerable hydrophobicity (the number of AAs in an a-helix required to traverse the PM). This implied the P2X receptor is 2TM. The characteristic sequence of an extracellular N-terminus was screened for but not found, implying an intracellular N-terminus.
