Abstract
The small- and intermediate-conductance Ca2+-activated K+ channels KCa2.3 and KCa3.1 regulate a plethora of physiological processes: including water and electrolyte transport in polarised epithelia, neuronal firing, and vascular tone. Critical for KCa2.3 and KCa3.1 function is the regulation of the number of channels at the plasma membrane; however, the mechanisms that regulate the trafficking of KCa2.3 and KCa3.1 to and from the plasma membrane are still poorly understood. There are several marked differences in the trafficking pathways of these two channels, for example, KCa2.3 has previously been established to recycle back to the plasma membrane after endocytosis; however, there is conflicting evidence in the literature if KCa3.1 recycles back to the plasma membrane.
This research project has focused on the regulation of KCa2.3 and KCa3.1 by two multiprotein complexes: exocyst and retromer. Exocyst is an eight-protein complex that tethers post-secretory vesicles to the plasma membrane. Additionally, exocyst has previously been established to regulate the trafficking of several proteins to the basolateral membrane of polarised epithelia. whereas the retromer complex is associated with the endosomal compartment and been demonstrated to regulate the retrieval and recycling of many membrane-bound proteins back to the plasma membrane, including ion channels. By using a combination of cell surface biotinylation, immunoblotting, and Ussing chamber electrophysiology, this project aimed to determine if exocyst and retromer regulated the trafficking of KCa2.3 and KCa3.1.
Knockdown of the exocyst subunit Sec3 significantly reduced KCa3.1-specific current (n = 5, P < 0.01) and significantly reduced the basolateral membrane population of KCa3.1 in FRT epithelia (n = 4, P < 0.05). These data suggest that the exocyst complex is required for the delivery of KCa3.1 to the basolateral membrane of polarised epithelia.
Stabilisation of the retromer complex with the pharmacological chaperone R55 increased the KCa2.3 population at the cell surface (n = 3, P < 0.05). Additionally, siRNA-induced knockdown of the retromer subunit VPS35 or the retromer-associated protein SNX3 decreased KCa2.3 levels at the cell surface (n = 3 each, P < 0.05). These data suggest the retromer regulates recycling of KCa2.3 back to the plasma membrane. Furthermore, cell surface levels of a mutated KCa2.3 channel, with a short deletion in the N-terminal domain, was not affected by R55. Surprisingly, stabilising retromer decreased the basolateral population of KCa3.1 (n = 4, P < 0.01) and KCa3.1 specific current; suggesting, that retromer does not regulate the trafficking or recycling of KCa3.1. Cumulatively, these data suggest, for the first time, that retromer is involved in the recycling of KCa2.3, but not the genetically related KCa3.1 channel.