Abstract
Inward rectifier K+ channels are important for maintaining normal electrical function in many cell types. The proper function of these channels requires the presence of membrane phosphoinositide 4,5-bisphosphate (PIP2). Stimulation of the Ca2+-sensing receptor CaR, a pleiotropic G protein-coupled receptor, activates both Gq/11, which decreases PIP2, and phosphatidylinositol 4-kinase (PI-4-K), which, conversely, increases PIP2. How membrane PIP2 levels are regulated by CaR activation and whether these changes modulate inward rectifier K+ are unknown. In this study, we found that activation of CaR by the allosteric agonist, NPSR568, increased inward rectifier K+ current (I K1) in guinea pig ventricular myocytes and currents mediated by Kir2.1 channels exogenously expressed in HEK293T cells with a similar sensitivity. Moreover, using the fluorescent PIP2 reporter tubby-R332H-cYFP to monitor PIP2 levels, we found that CaR activation in HEK293T cells increased membrane PIP2 concentrations. Pharmacological studies showed that both phospholipase C (PLC) and PI-4-K are activated by CaR stimulation with the latter played a dominant role in regulating membrane PIP2 and, thus, Kir currents. These results provide the first direct evidence that CaR activation upregulates currents through inward rectifier K+ channels by accelerating PIP2 synthesis. The regulation of I K1 plays a critical role in the stability of the electrical properties of many excitable cells, including cardiac myocytes and neurons. Further, synthetic allosteric modulators that increase CaR activity have been used to treat hyperparathyroidism, and negative CaR modulators are of potential importance in the treatment of osteoporosis. Thus, our results provide further insight into the roles played by CaR in the cardiovascular system and are potentially valuable for heart disease treatment and drug safety.
Highlights
Kir2.x channels are inward rectifier K+ channels that play an important role in maintaining stable resting membrane potentials, controlling excitability, and shaping the initial depolarization and final repolarization of ventricular myocytes [13, 19, 21, 27]
We previously demonstrated that extracellular spermine inhibits the outward current through Kir2.1 channels expressed in oocytes and the outward IK1 of myocytes, but the effect was much greater in oocytes than in cardiac myocytes [3]
It is noted that the time course of effect was slow with and without CaR expression (Fig. 1e, f), suggesting that the direct effect of spermine on the Kir2.1 channel is as slow as the indirect effect (i.e., accumulation of PIP2 against the phospholipase C (PLC)-dependent hydrolysis via CaR activation)
Summary
Kir2.x channels are inward rectifier K+ channels that play an important role in maintaining stable resting membrane potentials, controlling excitability, and shaping the initial depolarization and final repolarization of ventricular myocytes [13, 19, 21, 27]. Gain and loss of function of Kir2.x channels, which mediate cardiac inwardly rectifying currents (IK1), can cause reentry and arrhythmia, respectively [19]. We previously demonstrated that extracellular spermine inhibits the outward current through Kir2.1 channels expressed in oocytes and the outward IK1 of myocytes, but the effect was much greater in oocytes than in cardiac myocytes [3]. The actions of extracellular spermine may be more diverse in complex cell types such as Pflugers Arch - Eur J Physiol (2016) 468:1931–1943 cardiac myocytes. Extracellular spermine can activate the calcium-sensing receptor, CaR [34]. How membrane PIP2 levels are regulated by CaR activation and whether the resulting changes regulate inward rectifier K+ channels are unknown
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