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...

requirement for dynein function and intact microtubule cytoskeleton for normal surface expression of cardiac potassium channels. Am J Physiol Heart Circ Physiol 296: H71-H83, 2009. First published October 31, 2008 doi:10.1152/ajpheart.00260.2008-Potassium channels at the cardiomyocyte surface must eventually be internalized and degraded, and changes in cardiac potassium channel expression are known to occur during myocardial disease. It is not known which trafficking pathways are involved in the control of cardiac potassium channel surface expression, and it is not clear whether all cardiac potassium channels follow a common pathway or many pathways. In the present study we have surveyed the role of retrograde microtubule-dependent transport in modulating the surface expression of several cardiac potassium channels in ventricular myocytes and heterologous cells. The disruption of microtubule transport in rat ventricular myocytes with nocodazole resulted in significant changes in potassium currents. A-type currents were enhanced 1.6-fold at ϩ90 mV, rising from control densities of 20.9 Ϯ 2.8 to 34.0 Ϯ 5.4 pA/pF in the nocodazole-treated cells, whereas inward rectifier currents were reduced by one-third, perhaps due to a higher nocodazole sensitivity of Kir channel forward trafficking. These changes in potassium currents were associated with a significant decrease in action potential duration. When expressed in heterologous human embryonic kidney (HEK-293) cells, surface expression of Kv4.2, known to substantially underlie A-type currents in rat myocytes, was increased by nocodazole, by the dynein inhibitor erythro-9-(2-hydroxy-3-nonyl) adenine hydrochloride, and by p50 overexpression, which specifically interferes with dynein motor function. Peak current density was 360 Ϯ 61.0 pA/pF in control cells and 658 Ϯ 94.5 pA/pF in cells overexpressing p50. The expression levels of Kv2.1, Kv3.1, human ether-a-go-go-related gene, and Kir2.1 were similarly increased by p50 overexpression in this system. Thus the regulation of potassium channel expression involves a common dynein-dependent process operating similarly on the various channels.voltage-gated potassium channel; inward rectifier; microtubules; p50/ dynamitin THE PLATEAU PHASE of the action potential in the heart involves numerous K ϩ currents, and the delicate balance of their timeand voltage-dependent properties gives the cardiac action potential its characteristic shape and time course (23). Each of these repolarizing currents is underlain by one or more specific potassium channels: in humans, transient outward current (I to ) is underlain by Kv4.2 and Kv4.3, ultrarapid delayed rectifier current (I Kur ) by Kv1.5, and rapid delayed rectifier current (I Kr ) by the human ether-a-go-go-related gene product (hERG) (24). Because they are responsible for determining the durations of the action potential and refractory period (13, 24), minor differences in K ϩ currents can have dramatic effects on cellular electrophysiology. The functioning of these channels at the c...

Background: ATP-sensitive potassium (K ATP ) channels translate cellular metabolism (generation of ATP) in an electrical signal. Results: Mutual repulsion between specific substituted titratable residues in the bundle crossing forces K ATP channels to open and changes their apparent ATP sensitivity. Conclusion: ATP-dependent gating involves conformational changes in the bundle crossing region of K ATP channels. Significance: This reflects an engineered method for control of ion channel activity by a non-natural mechanism.

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