It has been proposed that the pI(Cln) protein forms a nucleotide-sensitive plasma membrane anion channel with a GXGXG motif being an essential component of the extracellular nucleotide-binding site. To evaluate this hypothesis, we have performed voltage-clamp experiments on Xenopus laevis oocytes injected with RNA encoding a rat mutant pI(Cln) in which the three glycines of the putative nucleotide-binding site have been changed into alanines (G54A; G56A; G58A). The injected oocytes displayed outwardly rectifying anion currents, which were voltage-dependently blocked by extracellular cAMP, but which were not affected by removal of extracellular Ca2+. Furthermore, the mutation did not affect the voltage-dependent inactivation. We therefore conclude that there is no evidence in favour of an extracellular nucleotide-binding site in pI(Cln).