N-methyl-D-glucamine and propidium dyes utilize different permeation pathways at rat P2X(7) receptors.
Activation of membrane P2X(7) receptors by extracellular ATP [or its analog 2',3'-O-(4-benzoylbenzoyl)-ATP] results in the opening within several milliseconds of an integral ion channel that is permeable to small cations. If the ATP application is maintained for several seconds, two further sequelae occur: there is a gradual increase in permeability to the larger cation N-methyl-d-glucamine and the cationic propidium dye quinolinium, 4-[(3-methyl-2(3H)-benzoxazolylidene)methyl]-1-[3-(triethylammonio)propyl]diiodide (YO-PRO-1) enters the cell. The similarity in the time course of these two events has led to the widespread view that N-methyl-d-glucamine and YO-PRO-1 enter through a common permeation pathway, the "dilating" P2X(7) receptor pore. Here we provide two independent lines of evidence against this view. We studied single human embryonic kidney cells expressing rat P2X(7) receptors with patch-clamp recordings of membrane current and with fluorescence measurements of YO-PRO-1 uptake. First, we found that maintained application of the ATP analog did not cause any increase in N-methyl-d-glucamine permeability when the extracellular solution contained its normal sodium concentration, although YO-PRO-1 uptake was readily observed. Second, we deleted a cysteine-rich 18-amino acid segment in the intracellular juxtamembrane region of the P2X(7) receptor. This mutated receptor showed normal YO-PRO-1 uptake but had no permeability to N-methyl-d-glucamine. Together, the clear differential effects of extracellular sodium ions or of mutation of the receptor strongly suggest that N-methyl-d-glucamine and YO-PRO-1 do not enter the cell by the same permeation pathway.
Jiang
Lin-Hua
LH
Institute of Molecular Physiology, University of Sheffield, Western Bank, Sheffield S10 2TN, UK.
Rassendren
Francois
F
Mackenzie
Amanda
A
Zhang
Yi-Hong
YH
Surprenant
Annmarie
A
North
R Alan
RA
D001583:Benzoxazoles; D002460:Cell Line; D002462:Cell Membrane / Q000502:physiology; D045604:Extracellular Fluid / Q000737:chemistry / Q000502:physiology; D005456:Fluorescent Dyes / Q000378:metabolism; D006801:Humans; D008536:Meglumine / Q000378:metabolism*; D008564:Membrane Potentials / Q000502:physiology; D010539:Permeability; D011419:Propidium / Q000378:metabolism*; D017434:Protein Structure, Tertiary; D011806:Quinolinium Compounds; D018048:Receptors, Purinergic P2 / Q000502:physiology*; D058486:Receptors, Purinergic P2X7; D012964:Sodium / Q000502:physiology
D001583:Benzoxazoles; D005456:Fluorescent Dyes; C546468:P2RX7 protein, human; C546470:P2rx7 protein, rat; D011806:Quinolinium Compounds; D018048:Receptors, Purinergic P2; D058486:Receptors, Purinergic P2X7; C089813:YO-PRO 1; D011419:Propidium; D008536:Meglumine; D012964:Sodium