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

Dragana M. Filipovic, Olugbenga A. Adebanjo, Mone Zaidi, W. Brian Reeves

Research output: Contribution to journalArticle

29 Scopus citations

Abstract

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.

Original languageEnglish (US)
Pages (from-to)F1070-F1077
JournalAmerican Journal of Physiology - Renal Physiology
Volume274
Issue number6 43-6
StatePublished - Jun 1 1998
Externally publishedYes

Keywords

  • Adenosine 5'-triphosphate
  • Intracellular calcium
  • Patch clamp
  • Purinergic receptors
  • Renal epithelial cells

ASJC Scopus subject areas

  • Physiology
  • Urology

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