Background: Cardiac potassium channels are implicated in a range of inherited and acquired ion channelopathies. Consequently, the regulation of potassium channels is a critical process; malfunction of which results in changes to membrane surface expression, current density, and channel kinetics. Decreases or increases in current can arise from defects in the channels themselves or from dysregulation by chaperone, trafficking, degradation, or accessory proteins, in addition to intracellular and extracellular factors (e.g. Ca2+ concentrations, second messenger signaling molecules, hormonal influences, ion concentrations, and pH). Here, we investigated the effects of regulatory mechanisms on potassium channel function and cardiac arrhythmogenesis. We evaluated the roles of interacting proteins on atrial and ventricular K+ channel function. Using heterologous expression systems (HEK 293 cells), mouse models, and human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs), we evaluated the impacts of quality control mechanisms on the human ether-à-go-go related gene (hERG) K+ channel; interacting proteins on small-conductance Ca2+-activated K+ channels (SK); and anion exchanger Slc26a6 on cardiac excitability and action potential. Specifically, we tested the hypotheses that E3 ubiquitin ligase, RNF207, regulates hERG channel expression and function through endoplasmic reticulum (ER)-associated degradation (chapter 2); calmodulin (CaM) mutants decrease SK2 current density through regulation of channel activation kinetics (chapter 3); [alpha]-actinin and filamin A regulate SK2 channel trafficking and surface expression (chapter 4); and Slc26a6 regulates cardiac excitability and action potential via its effects on intracellular pH and membrane potential (chapter 5). Methods and Results: To further evaluate these effects, we utilized a combination of electrophysiology, molecular biology, and imaging techniques. Whole-cell voltage-clamp, perforated patch action potential recordings, co-immunoprecipitation, degradation and ubiquitinylation assays, and immunofluorescence through confocal microscopy and stimulated emission depletion (STED) high-resolution microscopy were used in this study. We found that wild-type RNF207 is able to ubiquitinylate mutant hERG subunits (T613M; hERG[subscript T613M]), whereas mutant RNF207 (G603fs; RNF207[subscript G603fs]) fails to tag mutant hERG for degradation, allowing significant reduction in current density. SK2 channel surface expression was shown to be significantly enhanced by alpha-actinin and filamin a coexpression. SK2 current density was regulated by Ca2+-CaM activation, which was altered in the presence of CaM mutants. Lastly, we found that knockdown of Slc26a6 resulted in significantly prolonged action potential duration, decreased calcium transients, and intracellular pH irregularities. Conclusions Regulatory mechanisms play a key role in K+ channel expression and function. Potentially detrimental effects of heterozygously inherited channel mutations, which would otherwise be masked, may be revealed in the case of simultaneously inherited mutations in quality control mechanisms. Consequently, the effects of mutations in interacting proteins, cardiac ion channels or exchangers, and intracellular messengers may be widespread and warrants further study.