The cardiac potassium channel hERG encodes the α-subunit of the rapid delayed rectifier current I_Kr in the heart which contributes to terminal repolarization in human cardiomyocytes. The crucial role played by hERG/I_Kr in cardiac repolarization was uncovered first in patients with inherited long QT syndrome (LQTS2) with the identification of mutations in the hERG gene that reduced hERG/I_Kr currents. In symptomatic patients, LQTS2 is characterized by a prolongation of the QT interval on the electrocardiogram and associated with an increased propensity to develop arrhythmias. More recently, trafficking deficient LQTS2 mutations have been recognized as an important disease mechanism. Although hERG currents have been studied extensively, only few proteins have been identified so far that are involved in hERG trafficking to the cell surface. We have isolated two cytosolic chaperones, Hsp70 and Hsp90 that interacted with newly synthesized hERG. HERG-chaperone interactions were transient with wildtype protein, but prolonged with trafficking deficient LQTS2 mutants. Inhibition of Hsp90 function with geldanamycin prevented the association of Hsp90 with hERG, inhibited hERG maturation and reduced expression of hERG/IKr currents in cardiomyocytes. These data suggested that Hsp90 is a crucial player in the maturation of the cardiac potassium channel hERG/I_Kr and that disruption of Hsp90 function will lead to a reduction in the number of functional channels at the cell surface with acquired long QT syndrome as a possible consequence. Based on our observations with geldanamycin, a specific inhibitor of Hsp90, we speculated that proteins in the processing pathway are not only crucial for the maturation of wildtype channels but at the same time provide targets for therapeutic compounds that have been linked in the past to acquired long QT syndrome with no indication of direct channel block as the underlying mechanism. In subsequent studies we have identified for the first time acquired trafficking inhibition of hERG as a novel mechanism causing acquired long QT syndrome. For example, arsenic trioxide (As₂O₃) produces dramatic remissions in patients with acute promyelocytic leukemia. However, its therapeutic use is burdened by toxicity including QT prolongation and torsade de pointes arrhythmias. Using electrophysiological recordings, surface expression assays and Western blotting we have shown that As₂O₃ inhibits maturation and surface expression of the cardiac potassium channel hERG at clinically relevant concentrations. As₂O₃ interfered with the processing and maturation of hERG by inhibition of hERG-Hsp90 complexes. At the same time, direct hERG block was not detected. While arsenic trioxide as a metalloid is an unusual therapeutic compound, pentamidine represents a more conventional small molecule compound that is clinically used for treatment of leishmaniasis, trypanosomiasis and Pneumocystis carinii pneumonia. Once again, pentamidine use was accompanied by electrographic abnormalities with no clear indication of direct hERG block. We were able to confirm that pentamidine -just like arsenic trioxide- inhibited maturation and surface expression of hERG. HERG currents and the fully-glycosylated cell surface form of hERG were suppressed after overnight exposure to clinically relevant concentrations while other membrane currents were not affected. Thus, pentamidine represents another example of a therapeutic compound that produces QT prolongation by suppression of hERG trafficking. To survey therapeutic compounds in a more systematic manner for effects on hERG trafficking, we have developed a novel chemiluminesence-based surface expression assay performed in 96-well format. We have used this assay to identify novel compounds that interfere with hERG trafficking and found that cardiac glycosides represent a large class of potent inhibitors of hERG cell surface expression. Cardiac glycosides reduced expression of the fully-glycosylated cell surface form of hERG on Western blots indicating that channel exit from the endoplasmic reticulum was blocked. Similarly, hERG currents were reduced with nanomolar affinity on long-term exposure. Importantly, hERG trafficking inhibition was initiated by cardiac glycosides through direct block of Na/K pumps and not via off-target interactions with hERG or another closely associated protein in the processing or export pathway. Taken together, we have identified several of a growing number of hERG trafficking inhibitors which poses a novel problem for the identification of compounds that impair hERG channel function in pre-clinical safety studies that are geared towards detection of acute channel block.