Adrenergic Regulation of the Human Ether-A-Go-Go-Related Gene Channel Protein Abundance Occurs at the Surface of the Endoplasmic Reticulum

Abstract

Long QT syndrome (LQTS) is a disorder of ion channels that can lead to the potentially lethal ventricular arrhythmias. Human ether-a-go-go-related gene product (HERG), a potassium channel responsible for the rapidly inactivating delayed rectifier current, is a genetic target in hereditary LQTS (specifically, LQTS2). Furthermore, pharmacological compounds of multiple classes can result in abnormal synthesis and/or function of HERG. Because arrhythmic events are often triggered in patients with LQTS2 following various stressors, it has been postulated that adrenergic regulation plays a key role in acute HERG regulation and LQTS pathogenesis. Recently, we have shown that chronic cAMP treatment (a surrogate for β-adrenergic signaling) significantly augments HERG protein abundance in HEK293 cells. Here, using velocity gradient centrifugations, we show that upon elevation of cAMP, the channel accumulates in the endoplasmic reticulum (ER) disproportionately more than at the plasma membrane. We also show that localized inhibition of protein kinase A (PKA) signaling at the surface of the ER by a targeted PKA inhibitory peptide (PKI) completely abolishes the effect; channels on the plasma membrane are unaffected by this inhibitor, as shown by patch-clamp experiments. Next, we targeted specific cAMP and PKA activity FRET-based biosensors (ICUE2 and AKAR3, respectively) to the ER surface; cell-permeable cAMP as well as the β-agonist isoproterenol elicited major FRET signal in the case of either biosensor, indicating that χAMΠ/ΠKA σιγναλινγ ισ ινταχτ ιν τηισ χoμπαρτμeντ. Tηe χAMΠ-δeπeνδeντ αυγμeντατιoν ρeμαινσ πρeσeντ eϖeν αϕτeρ τηe HEPΓ χoδινγ σeνeνχe ισ χoδoν-oπτιμιζeδ (38% διϕϕeρeντ ϕρoμ τηe oριγιναλ ατ τηe πριμαρψ σeνeνχe λeϖeλ), ινδιχατινγ τηατ χAMΠ-δeπeνδeντ αυγμeντατιoν ισ νoτ μPNA δeπeνδeντ. Tηeρeϕoρe, ωe χoνχλυδe τηατ β-adrenergic regulation of HERG occurs during the early stages of protein synthesis or folding at the level of the ER.