Molecular cloning and functional expression of Xenopus laevis oocyte ATP-activated P2X4 channels

All cells contain mechanosensitive ion channels, yet the molecular identiti es of most are unknown. The purpose of our study was to determine what enco des the Xenopus oocyte's mechanosensitive cation channel. Based on the idea that homologues to known channels might contribute to the stretch channels , we screened a Xenopus oocyte cDNA library with cation channel probes. Whe reas other screens were negative, P2X probes identified six isoforms of the P2X4 subtype of ATP-gated channels. From RNase protection assays and RT-PC R, we demonstrated that Xenopus oocytes express P2X4 mRNA. In expression st udies, four isoforms produced functional ATP-gated ion channels; however, o ne, xP2X4c, had a conserved cysteine replaced by a tyrosine and failed to g ive rise to functional channels. By changing the tyrosine to a cysteine, we showed that this cysteine was crucial for function. We raised antibodies a gainst a Xenopus P2X4 C-terminal peptide to investigate xP2X4 protein expre ssion. This affinity purified anti-xP2X4 antibody recognized a 56 kDa glyco sylated Xenopus P2X4 protein expressed in stably transfected HEK-293 cells and in P2X4 cDNA injected oocytes overexpressing the cloned P2X4 channels; however, it failed to recognize proteins in control, uninjected oocytes. Th is suggests that P2X4 channels and mechanosensitive cation channels are not linked. Instead, oocyte P2X4 mRNA may be part of the stored pool of stable maternal mRNA that remains untranslated until later developmental stages. (C) 2001 Elsevier Science B.V. All rights reserved.