Abstract
Numerous heptahelical receptors use activation of heterotrimeric G proteins to convey a multitude of extracellular signals to appropriate effector molecules in the cell. Both high specificity and correct integration of these signals are required for reliable cell function. Yet the molecular machineries that allow each cell to merge information flowing across different receptors are not well understood. Here we demonstrate that G protein-regulated inwardly rectifying K(+) (GIRK) channels can operate as dynamic integrators of alpha-adrenergic and cholinergic signals in atrial myocytes. Acting at the last step of the cholinergic signaling cascade, these channels are activated by direct interactions with betagamma subunits of the inhibitory G proteins (G betagamma), and efficiently translate M(2) muscarinic acetylcholine receptor (M2R) activation into membrane hyperpolarization. The parallel activation of alpha-adrenergic receptors imposed a distinctive "signature" on the function of M2R-activated GIRK1/4 channels, affecting both the probability of G betagamma binding to the channel and its desensitization. This modulation of channel function was correlated with a parallel depletion of G beta and protein phosphatase 1 from the oligomeric GIRK1 complexes. Such plasticity of the immediate GIRK signaling environment suggests that multireceptor integration involves large protein networks undergoing dynamic changes upon receptor activation.
Highlights
The heart functions in a continuously changing environment responding to excitatory and inhibitory signals from the sympathetic and parasympathetic branches of autonomic nervous system
We demonstrate that G protein-regulated inwardly rectifying K؉ (GIRK) channels can operate as dynamic integrators of ␣-adrenergic and cholinergic signals in atrial myocytes
Effects of ␣-AR Stimulation on GIRK1/4 Channel Function— Numerous studies, involving different cell types, have indicated that activation of ␣1-adrenergic or other Gq-coupled receptors leads to inhibition of whole-cell GIRK channel currents [15,16,17,18,19,20,21,22,23,24,25]
Summary
Materials—Mouse monoclonal antibodies against G, PP1, phosphoinositide-specific phospholipase C (PLC) 1, phosphoinositide 3-kinase (PI 3-kinase), -arrestin, G␣i, G␣s, and G␣q were purchased from BD Transduction Laboratories. The atrial tissue homogenate was first centrifuged at 10,000 ϫ g for 15 min at 4 °C to remove the nuclei and mitochondria, and plasma membranes were obtained by centrifugation (100,000 ϫ g for 65 min) of the resulting supernatant on a cushion of HME-PIPhI buffer with 1.3 M sucrose [28]. The membranes at the interface were collected, washed twice, resuspended in HME-PI-PhI buffer containing 0.32 M sucrose and frozen as above. The atrial membranes were solubilized in MEB buffer (20 mM HEPES, pH 8.0, 2 mM MgCl2, 1 mM EDTA, 100 mM NaCl, 10 mM -mercaptoethanol, 1% sodium deoxycholate, and 0.45% IGEPAL CA-630) with PI and PhI as described previously [4, 10]. To verify that the protein bands of interest were properly quantified, in each experiment, a range of known amounts of atrial membrane proteins was loaded on the same gel, and the relative protein levels were examined to confirm that pixel intensity of the immunoreactive bands grows linearly with increasing protein amount
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