Functional and molecular evidence for P2X receptors in LLC-PK1 cells

Extracellular ATP affects a wide variety of cells via purinergic membrane receptors. One class of purinergic receptors, P2X, consists of ATP-gated, calcium-permeable, cation-selective channels. We performed whole cell patch-clamp studies, intracellular calcium concentration ([Ca2+]i) measurements, and reverse transcription-polymerase chain reaction (RT-PCR) to determine whether P2X receptors are expressed in LLC-PK1 cells. First, in patch-clamp studies, 100 microM ATP depolarized the cell membrane and increased the whole cell conductance of LLC-PK1 cells. This response was dose dependent and inhibited by 100 microM suramin, a P2 receptor antagonist. The ATP-induced conductance was cation selective but did not discriminate between Na+ and K+. ADP, alpha,beta-methylene-ATP, and beta,gamma-methylene-ATP had no effect on the whole cell conductance. Next, 10 microM ATP caused a rapid rise in [Ca2+]i in LLC-PK1 cells. This effect of ATP was inhibited by the absence of extracellular calcium and by suramin but not by pretreatment with pertussis toxin. ADP and beta,gamma-methylene-ATP had little or no effect on [Ca2+]i. Finally, RT-PCR produced a 330-bp fragment from LLC-PK1 cell RNA, whose sequence was 80% identical to the rat P2X1 receptor. We conclude that LLC-PK1 cells express purinergic receptors of the P2X class, which mediate depolarization and calcium entry when activated.