Identification of G protein-coupled, inward rectifier potassium channel gene products from the rat anterior pituitary gland

Karen A. Gregerson, Thomas P. Flagg, Thomas J. O'Neill, Mark Anderson, Oanh Lauring, Jill S. Horel, Paul A. Welling

Research output: Contribution to journalArticle

Abstract

Dopamine (DA) is a physiological regulator of PRL secretion, exerting tonic inhibitory control. DA activates an inward rectifier K+ (IRK) channel in rat lactotropes, causing membrane hyperpolarization and inhibition of Ca2+-dependent action potentials. Both the activation of this effector K+ channel and the inhibition of PRL release are mediated by D2-type receptor activation and pertussis toxin-sensitive G proteins. To study the molecular basis of this physiologically relevant channel, a homology-based PCR approach was employed to identify members of the IRK channel family expressed in the anterior pituitary gland. Nondegenerate primers corresponding to regions specific for IRK channels known to be G protein activated (GIRKs; gene subfamily Kir 3.0) were synthesized and used in the PCR with reverse transcribed female rat anterior pituitary messenger RNA as the template. PCR products of predicted sizes for Kir 3.1, 3.2, and 3.4 were consistently observed by ethidium bromide staining after 16 amplification cycles. The identities of the products were confirmed by subcloning and sequencing. Expression of each of these gene products in anterior pituitary was confirmed by Northern blot analysis. Functional analysis of the GIRK proteins was performed in the heterologous expression system, Xenopus laevis oocytes. Macroscopic K+ currents were examined in oocytes injected with different combinations of Kir 3.0 complementary RNA (cRNA) and G protein subunit (β1γ2) cRNA. The current-voltage relationships demonstrated strong inward rectification for each individual and pairwise combination of GIRK channel subunits. Oocytes coinjected with any pair of GIRK subunit cRNA exhibited significantly larger inward K+ currents than oocytes injected with only one GIRK channel subtype. Ligand-dependent activation of only one of the GIRK combinations (GIRK1 and GIRK4) was observed when channel subunits were co-expressed with the D2 receptor in Xenopus oocytes. Dose-response data fit to a Michaelis-Menten equation gave an apparent Kd similar to that for DA binding in anterior pituitary tissue. GIRK1 and GIRK4 proteins were coimmunoprecipitated from anterior pituitary lysates, confirming the presence of native GIRK1/GIRK4 oligomers in this tissue. These data indicate that GIRK1 and GIRK4 are excellent candidate subunits for the D2-activated, G protein-gated channel in pituitary lactotropes, where they play a critical role in excitation-secretion coupling.

Original languageEnglish (US)
Pages (from-to)2820-2832
Number of pages13
JournalEndocrinology
Volume142
Issue number7
DOIs
StatePublished - Jan 1 2001
Externally publishedYes

ASJC Scopus subject areas

  • Endocrinology

Fingerprint Dive into the research topics of 'Identification of G protein-coupled, inward rectifier potassium channel gene products from the rat anterior pituitary gland'. Together they form a unique fingerprint.

  • Cite this