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 μM ATP depolarized the cell membrane and increased the whole cell conductance of LLC-PK₁ cells. This response was dose dependent and inhibited by 100 μM suramin, a P2 receptor antagonist. The ATP-induced conductance was cation selective but did not discriminate between Na⁺ and K+. ADP, α,β-methylene-ATP, and β,γ-methylene-ATP had no effect on the whole cell conductance. Next, 10 μM 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 β,γ-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.