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Cerebellar granule (CG) neurons express a G protein-gated K+ current (GIRK) that is involved in the neurotransmitter regulation of the excitatory input to the Purkinje fibres of the cerebellum. Here, we characterized the single-channel behaviour of GIRK in CG neurons, and examined the effects of several known modulators of GIRK and their putative physiological roles. Whole-cell GIRKs were activated by baclofen, a GABAB receptor agonist. In cell-attached patches, baclofen activated GIRK with a single-channel conductance of 34 pS and a mean open time of 0.5 ms. In inside-out patches, application of GTPgammaS to the cytoplasmic side activated GIRK with similar kinetic properties. Addition of 2 mM ATP resulted in a marked increase in GIRK activity and induced longer-lived openings with a mean open time of 2.3 ms (ATP-dependent gating). Brain cytosolic fraction or free fatty acids inhibited this effect of ATP, and this was reversed by addition of purified recombinant brain fatty acid binding protein. Applying phosphatidylinositol 4,5-bisphosphate (PIP2) to inside-out patches in place of ATP also increased GIRK activity; however, only an increase in the frequency of opening was observed. The stimulatory effect of PIP2 on GIRK activity was not inhibited by the cytosolic fraction. Following maximal activation by PIP2, ATP caused an additional 2.2-fold increase in GIRK activity. These results show that GIRKs in CG neurons are regulated by positive and negative modulators that affect frequency as well as open time duration. The net effect is that the ligand-activated GIRK is in the 'low activity' state associated with short-lived openings, mainly due to strong action of the cytosolic inhibitor of ATP-dependent gating. Our results also show that intracellular ATP modulates GIRK via pathways different from that of PIP2 in CG neurons.

Abstract: Carotid body glomus (CB) cells express different types of K+ channels such as TASK, BK, and Kv channels, and hypoxia has been shown to inhibit these channels. Here we report the presence of a ∼72-pS channel that has not been described previously in CB cells. In cell-attached patches with 150mM K+ in the pipette and bath solutions, TASK-like channels were present (∼15 and ∼36-pS). After formation of inside-out patches, a 72-pS channel became transiently active in ∼18% of patches. The 72-pS channel was K+-selective, inhibited by 2–4mM ATP and 10–100μM glybenclamide. The 72-pS channel was observed in CB cells isolated from newborn, 2–3 week and 10–12 week-old rats. Reverse transcriptase-PCR and immunocytochemistry showed that Kir6.1, Kir6.2, SUR1 and SUR2 were expressed in CB glomus cells as well as in non-glomus cells. Acute hypoxia (∼15mmHg O2) inhibited TASK-like channels but failed to activate the 72-pS channel in cell-attached CB cells. K+ channel openers (diazoxide, pinacidil, levcromakalim), sodium cyanide and removal of extracellular glucose also did not activate the 72-pS channel in the cell-attached state. The hypoxia-induced elevation of intracellular [Ca2+] was unchanged by glybenclamide or diazoxide. NaCN-induced increase in [Ca2+] was not affected by 10μM glybenclamide but inhibited by 100μM glybenclamide. Acute glucose deprivation did not elevate [Ca2+] in the presence or absence of glybenclamide. These results show that an ATP-sensitive K+ channel is expressed in the plasma membrane of CB cells, but is not activated by short-term metabolic inhibition. The functional relevance of the 72-pS channel remains to be determined. [Copyright &y& Elsevier]

Examines adenosine triphosphate (ATP)-dependent regulation of a G protein-coupled potassium channel expressed in oocytes. Effect of the antagonistic regulation of G protein-gated potassium channels by ATP; Reversal of effects to ATP by cytosolic extract from atria; Inward rectification of potassium channels by polyamines.