Although the modulation of ion channel gating by hormones and drugs has been extensively studied, much less is known about how cell surface ion channel expression levels are regulated. Here, we demonstrate that the cell surface density of both the heterologously expressed K + channel encoded by the human ether-a-gogo-related gene (HERG) and its native counterpart, the rapidly activating delayed rectifier K + channel (I Kr IntroductionThe amplitude of ion channel currents is determined by a combination of channel gating (open versus closed times) and channel density in the plasma membrane. Whereas ion channel gating is known to be modulated by various means, such as hormones and drugs, much less is known about how the density of WT ion channels in the plasma membrane is regulated.The human ether-a-go-go-related gene (HERG) encodes the pore-forming subunits of the rapidly activating delayed rectifier K + channel (I Kr ) in the heart (1, 2). Mutations in HERG reduce I Kr and cause type 2 long QT syndrome (LQT2), a disorder that predisposes individuals to life-threatening arrhythmias (3). In addition, the HERG channel is a common target for diverse classes of drugs that induce acquired long QT syndrome (LQTS; ref. 4). Whereas drugs usually reduce HERG channel current (I HERG ) by blocking the channel, LQTS-causing HERG mutations often decrease I HERG by disrupting forward trafficking of the channel, thereby reducing expression levels of HERG at the plasma membrane (5). However, little is presently known about how the plasma membrane density of WT HERG channels is regulated under either physiological or pathophysiological conditions. Disorders of extracellular K + concentration ([K + ] o ) are the most common electrolyte abnormality found in clinical practice and can be life threatening. For example, abrupt, insulin-induced shifts of K + from the extracellular compartment into cells, abnormal K + losses caused by digestive or kidney disorders, and the use of certain diuretics are common causes of low [K + ] o (hypokalemia). It is also

The human ether-a-go-go-related gene (hERG) encodes a channel that conducts the rapidly activating delayed rectifier K ϩ current (I Kr ), which is important for cardiac repolarization. Mutations in hERG reduce I Kr and cause congenital long QT syndrome (LQTS). More frequently, common medications can reduce I Kr and cause LQTS as a side effect. Protein trafficking abnormalities are responsible for most hERG mutation-related LQTS and are recently recognized as a mechanism for druginduced LQTS. Whereas hERG trafficking has been studied in recombinant expression systems, there has been no reported study on cardiac I Kr trafficking at the protein level. In the present study, we identified that I Kr is present in cultured neonatal rat ventricular myocytes and can be robustly recorded using Cs ϩ as the charge carrier. We further discovered that 4,4Ј-(isopropylidenedithio)-bis-(2,6-di-t-butylphenol) (probucol), a cholesterol-lowering drug that induces LQTS, disrupted I Kr trafficking and prolonged the cardiac action potential duration. Probucol did not directly block I Kr . Probucol also disrupted hERG trafficking and did not block hERG channels expressed in human embryonic kidney 293 cells. We conclude that probucol induces LQTS by disrupting ether-a-go-go-related gene trafficking, and that primary culture of neonatal rat cardiomyocytes represents a useful system for studying native I Kr trafficking.

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Rationale: The human ether-a-go-go-related gene (HERG) encodes the pore-forming subunits of the rapidly activating delayed rectifier potassium channel (I Kr ) that is important for cardiac repolarization. Key Words: arrhythmia Ⅲ cardiac electrophysiology Ⅲ ion channels Ⅲ potassium channels Ⅲ voltage-gated potassium channels P otassium channels play important roles in vital cellular signaling processes such as cardiac rhythm. The function of ion channels is determined by a combination of channel gating (opening versus closing) and channel numbers (channel density) in the plasma membrane. Although the function and modulation of preexisting ion channels have been extensively studied, information about the stability of ion channels in the plasma membrane is limited. In particular, it is not known whether and how the gating states of a channel affect the channel's stability in the plasma membrane.The rapidly activating delayed rectifier K ϩ channel (I Kr ) is important for repolarization of cardiac action potentials and is encoded by the human ether-a-go-go-related gene (HERG). 1-3 Reduction of I Kr , attributable to mutations in HERG or drug block, slows repolarization and causes long QT syndrome (LQTS), a cardiac electric disorder that predisposes affected individuals to the potentially lethal arrhythmia, Torsades de Pointes. 4

Objective-Elevated apolipoprotein D (apoD) levels are associated with reduced proliferation of cancer cells. We therefore investigated whether apoD, which occurs free or associated with HDL, suppresses vascular smooth muscle cell (VSMC) proliferation, which is related to the pathobiology of disease. Methods and Results-Intense immunoreactivity for apoD was observed in human atherosclerotic plaque but not in normal coronary artery. However, an increase in apoD mRNA was seen in quiescent relative to proliferating fetal lamb aortic VSMCs, and in the rat aortic VSMC line (A10), we demonstrated uptake of apoD from serum. Stable transfection of apoD in A10 cells in the absence of serum did not influence VSMC proliferation assessed by [ 3 H]-thymidine incorporation. ApoD, administered at a dose of 100 ng/mL, completely inhibited basal as well as platelet-derived growth factor (PDGF)-BB-induced VSMC proliferation (PϽ0.01) but had no effect on fibroblast growth factor-induced VSMC proliferation. ApoD did not suppress PDGF-BB or fibroblast growth factor-2-induced phosphorylation of extracellular signal regulated kinase (ERK) 1/2 but selectively inhibited PDGF-BB-mediated ERK1/2 nuclear translocation. Conclusions-Our data suggest that apoD selectively modulates the proliferative response of VSMC to growth factors by a mechanism related to nuclear translocation of ERK1/2. Key Words: apolipoprotein D Ⅲ vascular smooth muscle cells Ⅲ platelet-derived growth factor-BB Ⅲ fibroblast growth factor-2 Ⅲ extracellular signal-regulated kinase phosphorylation and nuclear translocation Ⅲ proliferation A bnormal proliferation of vascular smooth muscle cells (VSMCs) is a critical component of atherosclerosis and arterial restenosis after angioplasty. 1 The mechanism has been related to a response to injury in which growth factors such as basic fibroblast growth factor (FGF-2) and plateletderived growth factor (PDGF) are released, stimulating proliferation and migration of VSMCs, leading to the formation of a neointima. Binding of PDGF to its receptor leads to the activation of several cell-signaling pathways associated with both VSMC proliferation and migration, such as those related to mitogen-activated protein kinase (MAPK), extracellular signal-regulated kinase (ERK) 1/2, phosphatidylinositol 3-kinase (PI3-kinase), and phospholipase C-␥ (PLC-␥). 2 Many epidemiological studies have demonstrated that elevated levels of LDL and reduced levels of HDL are risk factors in the development of atherosclerosis. 3 The apolipoprotein (apo) portion of HDL consists mainly of apoA-I (70%) and A-II (20%), in addition to A-IV, C, E, J, and D. There is an inverse relationship, both in human subjects and in experimental animals, between apoA-I, apoE, and atherosclerosis. 3,4 ApoE is induced by growth arrest in human and mouse fibroblasts, 5 and in rat VSMC, apoE significantly inhibits PDGF-BB-induced VSMC proliferation by partially suppressing MAPK activity and by preventing the increase in cyclin D1, which is necessary for cells to enter the G1 phase o...