G protein activated K+ channels (GIRK, Kir3) are switched on by direct binding of Gβγ following activation of G i/o proteins via G protein-coupled receptors (GPCRs). Although Gα i subunits do not activate GIRKs, they interact with the channels and regulate the gating pattern of the neuronal heterotetrameric GIRK1/2 channel (composed of GIRK1 and GIRK2 subunits) expressed in Xenopus oocytes. Coexpressed Gα i3 decreases the basal activity (I basal ) and increases the extent of activation by purified or coexpressed Gβγ. Here we show that this regulation is exerted by the 'inactive' GDP-bound Gα i3 GDP and involves the formation of Gα i3 βγ heterotrimers, by a mechanism distinct from mere sequestration of Gβγ 'away' from the channel. The regulation of basal and Gβγ-evoked current was produced by the 'constitutively inactive' mutant of Gα i3 , Gα i3 G203A, which strongly binds Gβγ, but not by the 'constitutively active' mutant, Gα i3 Q204L, or by Gβγ-scavenging proteins. Furthermore, regulation by Gα i3 G203A was unique to the GIRK1 subunit; it was not observed in homomeric GIRK2 channels. In vitro protein interaction experiments showed that purified Gβγ enhanced the binding of Gα i3 GDP to the cytosolic domain of GIRK1, but not GIRK2. Homomeric GIRK2 channels behaved as a 'classical' Gβγ effector, showing low I basal and strong Gβγ-dependent activation. Expression of Gα i3 G203A did not affect either I basal or Gβγ-induced activation. In contrast, homomeric GIRK1 * (a pore mutant able to form functional homomeric channels) exhibited large I basal and was poorly activated by Gβγ. Expression of Gα i3 GDP reduced I basal and restored the ability of Gβγ to activate GIRK1 * , like in GIRK1/2. Transferring the unique distal segment of the C terminus of GIRK1 to GIRK2 rendered the latter functionally similar to GIRK1 * . These results demonstrate that GIRK1 containing channels are regulated by both Gα i3 GDP and Gβγ, while GIRK2 is a Gβγ-effector insensitive to Gα i3 GDP .

Background-The hyperpolarization-activated nucleotide-gated channel-HCN4 plays a major role in the diastolic depolarization of sinus atrial node cells. Mutant HCN4 channels have been found to be associated with inherited sinus bradycardia. Methods and Results-Sixteen members of a family with sinus bradycardia were evaluated. Evaluation included a clinical questionnaire, 12-lead ECGs, Holter monitoring, echocardiography, and treadmill exercise testing. Eight family members (5 males) were classified as affected. All affected family members were asymptomatic with normal exercise capacity during long-term follow-up. Electrophysiological testing performed on 2 affected family members confirmed significant isolated sinus node dysfunction. Segregation analysis suggested autosomal-dominant inheritance. Direct sequencing of the exons encoding HCN4 revealed a missense mutation, G480R, in the ion channel pore domain in all affected family members. Function analysis, including expression of HCN4 wild-type and G480R in Xenopus oocytes and human embryonic kidney 293 cells, revealed that mutant channels were activated at more negative voltages compared with wild-type channels. Synthesis and expression of the wild-type and mutant HCN4 channel on the plasma membrane tested in human embryonic kidney 293 cells using biotinylation and Western blot analysis demonstrated a reduction in synthesis and a trafficking defect in mutant compared with wild-type channels. Conclusions-We describe an inherited, autosomal-dominant form of sinus node dysfunction caused by a missense mutation in the HCN4 ion channel pore. Despite its critical location, this mutation carries a favorable prognosis without the need for pacemaker implantation during long-term follow-up.

G protein-activated K + channels (GIRK) mediate postsynaptic inhibitory effects of neurotransmitters in the atrium and in the brain by coupling to G protein-coupled receptors (GPCRs). In neurotransmitter-dependent GIRK signalling, Gβγ is released from the heterotrimeric Gαβγ complex upon GPCR activation, activating the channel and attenuating its rectification. Now it becomes clear that Gα is more than a mere Gβγ donor. We have proposed that Gα i3 -GDP regulates GIRK gating, keeping its basal activity low but priming (predisposing) the channel for activation by agonist in intact cells, and by Gβγ in excised patches. Here we have further investigated GIRK priming by Gα i3 using a model in which the channel was activated by coexpression of Gβγ, and the currents were measured in intact Xenopus oocytes using the two-electrode voltage clamp technique. This method enables the bypass of GPCR activation during examination of the regulation of the channel in intact cells. Using this method, we further characterize the priming phenomenon. We tested and excluded the possibility that our estimates of priming are affected by artifacts caused by series resistance or large K + fluxes. We demonstrate that both Gα i3 and membrane-attached Gβγ scavenger protein, m-phosducin, reduce the basal channel activity. However, Gα i3 allows robust channel activation by coexpressed Gβγ, in sharp contrast to m-phosducin, which causes a substantial reduction in the total Gβγ-induced current. Furthermore, Gα i3 also does not impair the Gβγ-dependent attenuation of the channel rectification, in contrast to m-phosducin, which prevents this Gβγ-induced modulation. The Gα i3 -induced enhancement of direct activation of GIRK by Gβγ, demonstrated here for the first time in intact cells, strongly supports the hypothesis that Gα i regulates GIRK gating under physiological conditions.

ObjectivesA key clinical paradox in osteoarthritis (OA), a prevalent age-related joint disorder characterised by cartilage degeneration and debilitating pain, is that the severity of joint pain does not strictly correlate with radiographic and histological defects in joint tissues. Here, we determined whether protein kinase Cδ (PKCδ), a key mediator of cartilage degeneration, is critical to the mechanism by which OA develops from an asymptomatic joint-degenerative condition to a painful disease.MethodsOA was induced in 10-week-old PKCδ null (PKCδ−/−) and wild-type mice by destabilisation of the medial meniscus (DMM) followed by comprehensive examination of the histology, molecular pathways and knee-pain-related-behaviours in mice, and comparisons with human biopsies.ResultsIn the DMM model, the loss of PKCδ expression prevented cartilage degeneration but exacerbated OA-associated hyperalgesia. Cartilage preservation corresponded with reduced levels of inflammatory cytokines and of cartilage-degrading enzymes in the joints of PKCδ-deficient DMM mice. Hyperalgesia was associated with stimulation of nerve growth factor (NGF) by fibroblast-like synovial cells and with increased synovial angiogenesis. Results from tissue specimens of patients with symptomatic OA strikingly resembled our findings from the OA animal model. In PKCδ null mice, increases in sensory neuron distribution in knee OA synovium and activation of the NGF-tropomyosin receptor kinase (TrkA) axis in innervating dorsal root ganglia were highly correlated with knee OA hyperalgesia.ConclusionsIncreased distribution of synovial sensory neurons in the joints, and augmentation of NGF/TrkA signalling, causes OA hyperalgesia independently of cartilage preservation.

Objectives-To conduct a clinical, genetic and functional analysis of three unrelated families with familial sinus bradycardia (FSB).Background-Mutations in the hyperpolarization-activated nucleotide-gated channel (HCN4) are known to be associated with FSB.