Prologue

What is a P2X4 receptor?

• An ATP gated non selective cation channel (transport of K+, Na+, Ca2+)
• Found in eukaryotes only (unusual!)
• A member of the purinigenic receptor family (7 members in total)
• Roles in synaptic transmission, taste sensation, control of smooth muscle, inflammation and pain sensation
• Different family members show different desensitisation speeds and respond to a whole range of allosteric modulators.

Prior to this study, what did Hattori and Gouaux already know?

• Before 2009, the highest resolution P2X receptor structure was 20 angstroms
• Structural predictions were made using atomic force microscopy (AFM) and high resolution cryogenic electron microscopy.



An example of structural predictions made for the P2X4 receptor trimer using electron microscopy data
'Molecular shape, architecture, and size of P2X4 receptors determined using fluorescence resonance energy transfer and electron microscopy.'-Young et al, 2008



• Without atomic resolution data, a molecular understanding of ATP binding, gating, and allosteric modulation remained elusive.
• But, in 2009 Kawate et al published the first crystal structure of the zebrafish P2X4 receptor at 3.1angstrom resolution:

'Crystal structure of the ATP-gated P2X4 ion channel in the closed state'-Kawate et al, 2009


• Mark T. Young has written a good review that evaluates previous structural predictions in light of the new crystal structure. He really highlights the excitement felt by the P2X research cohort over these new structures: 'P2X receptors: dawn of the post structure era'-Mark T. Young

 So if the structure had already been solved by Kawate, why did Hattori and Gouaux bother?

• Kawate et al’s structure shows the channel in a non-ATP bound, closed state (the ‘apo’ protein).
• From this structure, predictions were made that 3 non-canonical (ie pretty unsual) ATP binding sites were present but the structure didn’t provide enough information to truly get to grips with these sites and how the channel opens.
• The true nature of these sites and the activation mechanism would only become clear if an ATP bound structure could be made.

Understanding a mechanism can't be that important can it?

• Activation of P2X4 channels is thought to be responsible for the development of neuropathic pain after injury:

P2X Channels on microglia are activated by ATP. This causes release of BDNF which acts on neurones to reverse inhibitory GABA Cl- current resulting in hypersensitivity in the neurone

• Neuropathic pain is a condition in which sufferers continue to experience extreme pain to innocuous stimuli like raindrops.
• Pharmaceutical companies want to develop new drugs to block P2X4 in the treatment of pain and inflammation.
• So we need to know about the mechanism!

So, Hattori and Gouaux's first challenge was to successfully crystalise an ATP bound form of the P2X4 receptor...