Control of the movement of ions and water across epithelia is essential for homeostasis. Changing the number or activity of ion channels at the plasma membrane is a significant regulator of epithelial transport. In polarized epithelia, the intermediate-conductance calcium-activated potassium channel, KCa3.1 is delivered to the basolateral membrane where it generates and maintains the electrochemical gradients required for epithelial transport. The mechanisms that control the delivery of KCa3.1 to the basolateral membrane are still emerging. Herein we investigated the role of the highly conserved tethering complex exocyst. In epithelia, exocyst is involved in the tethering of post-Golgi secretory vesicles with the basolateral membrane, which is required before membrane fusion. In our Fisher rat thyroid cell line that stably expresses KCa3.1, siRNA knockdown of either of the exocyst subunits Sec3, Sec6, or Sec8 significantly decreased KCa3.1-specific current. Additionally, knockdown of exocyst complex subunits significantly reduced the basolateral membrane protein level of KCa3.1. Finally, co-immunoprecipitation experiments, suggest associations between Sec6 and KCa3.1, but not between Sec8 and KCa3.1. Collectively, based on these data and our previous studies, we suggest that components of exocyst complex are crucially important in the tethering of KCa3.1 to the basolateral membrane. After which, SNARE proteins aid in the insertion of KCa3.1-containing vesicles into the basolateral membrane of polarized epithelia.
The small‐ and intermediate‐conductance Ca2+‐activated K+ channels KCa2.3 (KCNN3) and KCa3.1 (KCNN4) 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, e.g. KCa2.3 has previously been established to recycle back to the plasma membrane after endocytosis; however, there are differences in opinion if KCa3.1 recycles in the literature. The highly conserved multi‐protein complex retromer has been demonstrated to regulate the retrieval and recycling of many membrane‐bound proteins, including ion channels. We hypothesised that retromer was required for the recycling of both KCa2.3 and KCa3.1. To test this hypothesis, we utilised a combination of biochemical and electrophysiological methods. Stabilisation of retromer with the pharmacological chaperone R55 increased the KCa2.3 population at the cell surface. Additionally, siRNA‐induced knockdown of the retromer subunit VPS35 decreased KCa2.3 levels at the cell surface. Surprisingly, even though KCa2.3 and KCa3.1 are in the same gene family, similar biochemical and Ussing chamber electrophysiological experiments demonstrated that R55 did not have an effect on KCa3.1 cell surface levels or 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 KCa3.1, a member of the same gene family.
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