NCI-H295R cell line as in vitro model of hyperaldosteronism lacks functional KCNJ5 (GIRK4; Kir3.4) channels

Mol Cell Endocrinol. 2015 Sep 5:412:272-80. doi: 10.1016/j.mce.2015.05.013. Epub 2015 May 18.

Abstract

As a major cause of aldosterone producing adenomas, numerous gain-of-function mutations in the KCNJ5 gene (encoding the K(+) channel subunit GIRK4) have been identified. The human adrenocortical carcinoma cell line NCI-H295R is the most frequently used cellular model for in vitro studies related to regulation of aldosterone-synthesis. Because of the undefined role of KCNJ5 (GIRK4) in regulating synthesis of aldosterone, we aimed at identifying basal and G protein-activated GIRK4 currents in this paradigmatic cell line. The GIRK-specific blocker Tertiapin-Q did not affect basal current. Neither loading of the cells with GTP-γ-S via the patch-clamp pipette nor agonist stimulation of an infected A1-adenosine receptor resulted in activation of GIRK current. In cells co-infected with KCNJ5, robust activation of basal and adenosine-activated inward-rectifying current was observed. Although GIRK4 protein can be detected in Western blots of H295R homogenates, we suggest that GIRK4 in aldosterone-producing cells does not form functional G(βγ)-activated channels.

Keywords: Adrenocortical cell line; Electrophysiology; GIRK4; KCNJ5; Primary aldosteronism.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Cell Line, Tumor
  • Cell Membrane / metabolism
  • G Protein-Coupled Inwardly-Rectifying Potassium Channels / genetics*
  • G Protein-Coupled Inwardly-Rectifying Potassium Channels / metabolism
  • Humans
  • Hyperaldosteronism / genetics
  • Hyperaldosteronism / metabolism*
  • Membrane Potentials
  • Protein Subunits / genetics
  • Protein Subunits / metabolism
  • Protein Transport

Substances

  • G Protein-Coupled Inwardly-Rectifying Potassium Channels
  • KCNJ5 protein, human
  • Protein Subunits