Kv1.5 Surface Expression Is Modulated by Retrograde Trafficking of Newly Endocytosed Channels by the Dynein Motor

Abstract

Abstract-In this article we have investigated the mechanisms by which retrograde trafficking regulates the surface expression of the voltage-gated potassium channel, Kv1.5. Overexpression of p50/dynamitin, known to disrupt the dynein-dynactin complex responsible for carrying vesicle cargo, substantially increased outward K ϩ currents in HEK293 cells stably expressing Kv1.5 (0.57Ϯ0.07 nA/pF, nϭ12; to 1.18Ϯ0.2 nA/pF, nϭ12, PϽ0.01), as did treatment of the cells with a dynamin inhibitory peptide, which blocks endocytosis. Nocodazole pretreatment, which depolymerizes the microtubule cytoskeleton along which dynein tracks, also doubled Kv1.5 currents in HEK cells and sustained K ϩ currents in isolated rat atrial myocytes. These increased currents were blocked by 1 mmol/L 4-aminopyridine, and the specific Kv1.5 antagonist, DMM (100 nM). Confocal imaging of both HEK cells and myocytes, as well as experiments testing the sensitivity of the channel in living cells to external Proteinase K, showed that this increase of K ϩ current density was caused by a redistribution of channels toward the plasma membrane. Coimmunoprecipitation experiments demonstrated a direct interaction between Kv1.5 and the dynein motor complex in both heterologous cells and rat cardiac myocytes, supporting the role of this complex in Kv1.5 trafficking, which required an intact SH3-binding domain in the Kv1.5 N terminus to occur. These experiments highlight a pathway for Kv1.5 internalization from the cell surface involving early endosomes, followed by later trafficking by the dynein motor along microtubules. This work has significant implications for understanding the way Kv channel surface expression is regulated. (Circ Res. 2005;97:363-371.)Key Words: atrial myocyte Ⅲ cardiomyocytes Ⅲ intracellular protein transport Ⅲ ion channels Ⅲ potassium channels V oltage-gated K ϩ channels (Kv channels) are intimately involved in the cellular regulation of excitation in all cardiovascular cells, and their activity depends on the presence of active channel subunits at the plasmalemma. Surface expression is regulated by changes in gene expression, 1-3 interactions with accessory subunits, by phosphorylation, and by cellular components that regulate their trafficking to the cell surface. Trafficking can also provide an explanation for the mechanisms by which drugs may act to achieve their therapeutic actions. 4 Although several groups have investigated motifs within K ϩ channels that affect trafficking, [5][6][7][8] little is known about the molecules and machinery involved in these processes in the heart. Surface expression requires movement from the endoplasmic reticulum through the Golgi apparatus to the plasma membrane, and several studies have investigated channel determinants that affect this trafficking process. Motifs in the C termini and pore domains, 6,7 of Kv channels have been implicated in their differential surface expression presumably through effects on forward trafficking. 5 Functional expression of channels can also be regulated by the rem...

Dynamic of Ion Channel Expression at the Plasma Membrane of Cardiomyocytes

“…Manipulation of microtubule integrity with microtubule depolymerizing agents such as colchicine and nocodazole has been shown to affect the surface expression of both potassium and sodium channels in cardiomyocytes. In atrial myocytes, nocodazole treatment increases the surface expression of Kv1.5 channels and the amplitude of the corresponding current I Kur (72). Similar results were reported for Kv4.2 and hERG channels, respectively mediating I to and I Kr (241).…”

“…However, despite, for example, the fact that dynamin-dependent endocytosis has been shown to be operative for various ion channels such as Kv1.2 (299), Kv1.5 (72,267,384), Kv7.1 (459), and Cav1. 2 (174), the specificity of the pathway(s) has not been characterized.…”

Potassium channels in pulmonary arterial hypertension

“…More importantly, COGOLLUDO et al [31], demonstrated that stimulation with serotonin leads to caveolin-dependent internalisation of K V 1.5, ensuring dynamic cell surface channel expression [31,34]. Likewise, endocytosis of K V 2.1 and K V 1.5 channels has been reported to be a key mechanism in the control of K V channel activity, which is regulated by tyrosine phosphorylation mediated by Src kinase [35][36][37]. Thus, serotonin signalling appears to modulate K V current in PASMCs through direct alterations in K V 1.5 trafficking and surface expression.…”

Impaired endocytosis of the ion channel TRPM4 is associated with human progressive familial heart block type I

“…Because mutations mostly obstruct protein function (22)(23)(24)(25)(26)(27), we considered a default in retrograde (i.e., endocytotic) TRPM4 trafficking as a likely explanation for the PFHBI mutational effect. Dynein-based endocytotic trafficking, a common pathway in ion channel endocytosis (28)(29)(30), is effectively inhibited in cells that overexpress dynamitin (31,32). Overexpression of dynamitin is an established method to disrupt the dynein motor system and to interfere with ion channel endocytosis.…”