A kinetic study of receptor activation of the G-protein gated K+ channel

The cloned G-protein gated inwardly rectifying K+ channel (a tetramer composed of Kir3.1-3.4 subunits) is activated by direct binding of Gβγ dimers, liberated by receptor activation of the Gi/o subfamily of heterotrimeric guanine nucleotide binding (G)-proteins. The interaction of these three membrane-associated components, G-protein coupled receptor (GPCR), heterotrimeric G-protein and channel, is rapid in native cells, with full channel activation via the GABA-B receptor occurring within a few hundred milliseconds (Sodickson & Bean, 1996 and 1998), and current deactivation occurring with a time constant of 1-2 seconds. Recent discovery of the Regulators of G-protein signalling (RGS) protein family has solved a major discrepancy between the slow deactivation of purified G-proteins and the fast deactivation of G-protein mediated signalling pathways. Their discovery has generated considerable interest in the kinetics of G-protein signalling and the organisation of these signalling components in the cell membrane. For these studies, the GIRK signalling system was reconstituted in mammalian HEK-293 cell lines, stably expressing the cloned neuronal channel subunits (Kir3.1 and Kir3.2A) plus a Gi/o-coupled GPCR (α2A adrenergic, A1 adenosine, D2 dopamine, M4 muscarinic and the heterodimeric GABA-B1b/2 receptors). Chapter 1 provides a general introduction to G-protein signalling and reviews our current understanding of the factors involved in the regulation of GERK channels. In Chapter 2, the methods and experimental protocols used in the study are described. In Chapter 3, I present a systematic analysis of the factors that contribute to the rapid activation of the channel complex, and in Chapter 4 the characteristic fast desensitisation of receptor-activated currents is examined. Factors influencing channel deactivation upon removal of agonist are explored in Chapter 5, and in Chapter 6 I describe the effects of the novel RGS protein family in these cell lines. Conclusions and future directions for this work are presented in Chapter 7

HL-1 cells express an inwardly rectifying K+ current activated via muscarinic receptors comparable to that in mouse atrial myocytes

An inwardly rectifying K^+ current is present in atrial cardiac myocytes that is activated by acetylcholine (I_{KACh}). Physiologically, activation of the current in the SA node is important in slowing the heart rate with increased parasympathetic tone. It is a paradigm for the direct regulation of signaling effectors by the Gβγ G-protein subunit. Many questions have been addressed in heterologous expression systems with less focus on the behaviour in native myocytes partly because of the technical difficulties in undertaking comparable studies in native cells. In this study, we characterise a potassium current in the atrial-derived cell line HL-1. Using an electrophysiological approach, we compare the characteristics of the potassium current with those in native atrial cells and in a HEK cell line expressing the cloned Kir3.1/3.4 channel. The potassium current recorded in HL-1 is inwardly rectifying and activated by the muscarinic agonist carbachol. Carbachol-activated currents were inhibited by pertussis toxin and tertiapin-Q. The basal current was time-dependently increased when GTP was substituted in the patch-clamp pipette by the non-hydrolysable analogue GTPγS. We compared the kinetics of current modulation in HL-1 with those of freshly isolated atrial mouse cardiomyocytes. The current activation and deactivation kinetics in HL-1 cells are comparable to those measured in atrial cardiomyocytes. Using immunofluorescence, we found GIRK4 at the membrane in HL-1 cells. Real-time RT-PCR confirms the presence of mRNA for the main G-protein subunits, as well as for M2 muscarinic and A1 adenosine receptors. The data suggest HL-1 cells are a good model to study IKAch

Regulators of G protein signalling proteins in the human myometrium

The contractile state of the human myometrium is controlled by extracellular signals that promote relaxation or contraction. Many of these signals function through G proteincoupled receptors at the cell surface, stimulating heterotrimeric G proteins and leading to changes in the activity of effector proteins responsible for bringing about the response. G proteins can interact with multiple receptors and many different effectors and are key players in the response. Regulators of G protein signalling (RGS) proteins are GTPase activating proteins for heterotrimeric G proteins and help terminate the signal. Little is known about the function of RGS proteins in human myometrium and we have therefore analysed transcript levels for RGS proteins at various stages of pregnancy (non-pregnant, preterm, term non-labouring, term labouring). RGS2 and RGS5 were the most abundantly expressed isolates in each of the patient groups. The levels of RGS4 and RGS16 (and to a lesser extent RGS2 and RGS14) increased in term labouring samples relative to the other groups. Yeast two-hybrid analysis and co-immunoprecipitation in myometrial cells revealed that both RGS2 and RGS5 interact directly with the cytoplasmic tail of the oxytocin receptor, suggesting they might help regulate signalling through this receptor. Key words: G protein-coupled receptors; labour; myometrium; RGS protein

A kinetic study of receptor activation of the G-protein gated K+ channel

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