Book Description

Proper mechanical activity of the heart is delicately interrelated with its timely electrical activation. It is the underlying ionic currents in each cell that lead to electrical activity, manifested as the cardiac action potential. In order to have a normal cardiac action potential, a specific number of functional ion channels need to be present on the plasma membrane. Defects in channel assembly or trafficking may lead to abnormalities in the corresponding ionic currents and shape of the action potential, which may lead to cardiac arrhythmias. The most prominent type of arrhythmia is atrial fibrillation (AF), which is associated with a significant increase in the risk of stroke. The incidence of AF is projected to increase three-fold by 2050 and therapeutic options for AF remain suboptimal. Current pharmacological therapies are associated with extracardiac toxicity while catheter ablation is invasive and can be associated with serious adverse events.Our group, as well as others, have previously documented the expression of several isoforms of small-conductance Ca2+ -activated K+ (SK) channels in human and mouse atrial myocytes. SK channels mediate the repolarization phase of the atrial action potential, with little effect on ventricular excitation. Indeed, we have previously documented that SK2 channel knock-out mice are prone to the development of AF. On the other hand, a recent study by Diness JG, et al. suggests that inhibition of Ca2+ activated K+ current may prevent AF. Taken together, these studies underpin the importance of these channels in atria and their potential to serve as a future therapeutic target for AF. Three isoforms of SK channel subunits (SK1, SK2 and SK3) are found to be expressed with SK2 as the most predominant isotype. In the first portion (CHAPTER 2) of this dissertation, I investigate the heteromultimeric formation and the domain necessary for the assembly of three SK channel subunits (SK1-SK3). My biochemical and functional data provides evidence for the formation of heteromultimeric complexes among different SK channel subunits in atrial myocytes. Using an innovative patch-clamp technique, applied here for the first time in cardiac myocytes, I show reduction of I[K,Ca] via inhibition of heteromultimerization. Since SK channels are predominantly expressed in atrial myocytes, specific ligands of the different isoforms of SK channel subunits may offer a unique therapeutic opportunity to directly modify atrial cells without interfering with ventricular myocytes. In addition to having proper subunit assembly and channel formation, there need to be a precise number of channels at specific locations on the plasma membrane. This means that there must be highly regulated sorting and trafficking pathways for ion channels. The importance of these processes is underscored by a number of disease conditions that involve mishandled trafficking of membrane proteins. It is important to note that the complete intracellular trafficking pathways are not known for any single channel. In the subsequent chapters, I identify [alpha]-actinin2 and Filamin A molecules, as important regulators of SK2 channel trafficking. Using various functional methods, including total internal reflection fluorescence microscopy (TIRF-M), I show SK2 channel interacts with FLNA to increase number of channels on the sarcolemma through increasing rate of recycling via recycling endosomes. I also show increased forward trafficking of SK2 as a result of interaction with [alpha]-actinin2 via an early endosomal-mediated trafficking pathway. Insight into the trafficking of SK2 channels may serve as a novel approach to modify SK2 current and atrial excitability without interfering with ventricular activity. The work done advances a new frontier towards understanding membrane localization of ion channels in cardiac muscle and other excitable cells. The demonstration of the mechanisms for targeting, anchoring and cell surface expression of the channel would help further understanding the ion channel function.