Identification of G protein-coupled, inward rectifier potassium channel gene products from the rat anterior pituitary gland.
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 Ca(2+)-dependent action potentials. Both the activation of this effector K(+) channel and the inhibition of PRL release are mediated by D(2)-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 (beta(1)gamma(2)) 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 coexpressed with the D(2) receptor in Xenopus oocytes. Dose-response data fit to a Michaelis-Menten equation gave an apparent K(d) 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 D(2)-activated, G protein-gated channel in pituitary lactotropes, where they play a critical role in excitation-secretion coupling.
Gregerson
K A
KA
Department of Obstetrics, University of Maryland, Baltimore, Maryland 21201, USA. kregers@umaryland.edu
Flagg
T P
TP
O'Neill
T J
TJ
Anderson
M
M
Lauring
O
O
Horel
J S
JS
Welling
P A
PA
D000818:Animals; D015152:Blotting, Northern; D004553:Electric Conductivity; D005260:Female; D051676:G Protein-Coupled Inwardly-Rectifying Potassium Channels; D007700:Kinetics; D009865:Oocytes; D010903:Pituitary Gland, Anterior / Q000378:metabolism*; D015221:Potassium Channels / Q000378:metabolism* / Q000502:physiology; D024661:Potassium Channels, Inwardly Rectifying; D011233:Precipitin Tests; D051381:Rats; D017207:Rats, Sprague-Dawley; D017448:Receptors, Dopamine D2 / Q000502:physiology; D020133:Reverse Transcriptase Polymerase Chain Reaction; D014982:Xenopus laevis
D051676:G Protein-Coupled Inwardly-Rectifying Potassium Channels; D015221:Potassium Channels; D024661:Potassium Channels, Inwardly Rectifying; D017448:Receptors, Dopamine D2