G protein-gated inwardly rectifying K⁺ (GIRK) channels are predominantly expressed in neurones and atrial myocytes. A characteristic feature of these channels is their activation in response to stimulation of G_i/o-coupled receptors. For example, in neurones they are activated by adenosine and GABA and as such they play an important role in neurotransmitter-mediated regulation of membrane excitability. In response to stimulation of heptahelical receptors coupled to pertussis toxin (PTx)-sensitive G_i/o proteins, the G_i/o heterotrimer dissociates into its component G_i/o α and Gβλ subunits. The Gβλ dimer binds directly to the GIRK channel complex activating it. The channels are tetrameric assemblies of Kir3.x subunits (Kir3.1-3.4 and splice variants). Atrial myocytes express Kir3.1 and 3.4 and neurones express Kir3.1, 3.2 and 3.3 subunits. It is therefore possible that greater channel diversity exists in the brain and that neuronal GIRK channel composition may differ in different brain regions.

In this study I have characterised a neuronal GIRK current in primary cultured hippocampal neurones and compared this with cloned channel subunits heterologously expressed in HEK293 cells. RT-PCR and immunocytochemistry were used to assess expression of Kir3.x subunits in hippocampus. Kir3.1, 3.2A, 3.2C (but not 3.2B or 3.2D) and 3.3 were detected. Cultured neurones were obtained using a protocol modified from Shah & Haylett (2000). Neonatal (P3-P5) Sprague-Dawley rats were humanely killed by cervical dislocation. Hippocampi were dissected out, cut into pieces and incubated in trypsin (1 h, 37 °C). Enzymatic action was terminated by the addition of serum and bovine serum albumin and individual cells obtained by trituration using fire-polished Pasteur pipettes. Cells were plated in Neurobasal medium (+2 % B27 supplement, 0.5 mM L-glutamine, 5 % serum) onto poly-D-lysine-coated glass coverslips. After 24 h the medium was replaced with serum-free medium plus 1 µM cytosine arabinoside. Cells were maintained at 37 °C with humidified 92.5 % O₂-7.5 % CO₂.

Whole-cell patch clamp recordings were made from neurones after 7-11 days in culture. The GIRK current was identified on the basis of its inward rectification, inhibition by tertiapin, enhancement by a number of G_i/o-coupled receptor agonists (e.g. baclofen, adenosine, somatostatin) and sensitivity to PTx. The properties of the neuronal GIRK current were compared to the cloned Kir3.1+3.2A channel currents activated by stimulation of either the GABA_B1a+2 or the GABA_B1b+2 receptors, also heterologously and stably expressed in HEK293 cells. Striking similarities between the native hippocampal GIRK current and the cloned channel current were observed in terms of channel kinetics, activation by baclofen and inhibition by tertiapin.

This research was funded by the Royal Society and the British Heart Foundation. J.L.L. is a Royal Society Dorothy Hodgkin Fellow.