RT Journal Article
SR Electronic
T1 P2X7 Receptor Channels Allow Direct Permeation of Nanometer-Sized Dyes
JF The Journal of Neuroscience
JO J. Neurosci.
FD Society for Neuroscience
SP 3557
OP 3566
DO 10.1523/JNEUROSCI.2235-12.2013
VO 33
IS 8
A1 Liam E. Browne
A1 Vincent Compan
A1 Laricia Bragg
A1 R. Alan North
YR 2013
UL http://www.jneurosci.org/content/33/8/3557.abstract
AB P2X receptors are widely distributed in the nervous system, and P2X7 receptors have roles in neuropathic pain and in the release of cytokines from microglia. They are trimeric membrane proteins, which open an integral ion channel when ligated by extracellular ATP. This channel is preferentially permeable to small cations (sodium, potassium, calcium) but also allows permeation of larger cations such as N-methyl-d-glucamine. ATP also leads to entry of fluorescent dyes in many cells expressing P2X7 receptors, but controversy persists as to whether such large molecules pass directly through the open ion channel or enter the cell by a different route. We measured cellular fluorescence and membrane currents in individual human embryonic kidney cells expressing rat P2X7 receptors. Introduction of positive side chains by mutagenesis into the inner half of the pore-forming second transmembrane domain of the receptor (T348K, D352N, D352K) increased relative permeability of chloride ions. It also promoted entry of the large (>1 nm) negative dye fluorescein-5-isothiocyanate while decreasing entry of the structurally similar but positive dye ethidium. Furthermore, larger cysteine-reactive methanethiosulfonates [sulforhodamine-methanethiosulfonate and 2-((biotinoyl)amino)ethyl methanethiosulfonate] reduced both ATP-evoked currents and dye entry when applied to open P2X7[G345C] receptors. The results demonstrate that the open channel of the P2X7 receptor can allow passage of molecules with sizes up to 1.4 nm.