Retromer- and Retriever-Associated Sorting Nexins Regulate Traffcking of KCa2.3 Channels

Abstract

Correct function of the Ca2+-activated K+ channel KCa2.3 (SK3) is essential for several physiological processes. Activity of KCa2.3 in neurons modulates the rate of action potential firing; in endothelial cells activation of KCa2.3 channels induces vasodilation, and accordingly, a decrease in blood pressure. KCa2.3 activity (i.e. currents) can be modulated by two main variables: the open probability ( Po) and the number of channels inserted into the plasma membrane ( N). Numerous pharmacological compounds have been identified and developed to alter KCa2.3 Po. In contrast, the mechanisms that control delivery of KCa2.3 to and from the plasma membrane are still poorly understood. After endocytosis, plasma membrane proteins are targeted to the endosomal network which is a key area for sorting membrane-bound proteins. Several endosomal-associated coat complexes have been identified that ‘rescue’ proteins from degradation and sequester proteins into recycling tubules for transport back to the plasma membrane. These complexes include Retromer, Retriever, and Commander. Associated with these complexes are members of the Sorting Nexin (SNX) family. SNX proteins bind to specific amino acid motifs to sort proteins for Retromer- or Retriever-mediated recycling. Interestingly, the Devor laboratory has demonstrated KCa2.3 can be rapidly recycled back into the plasma membrane after endocytosis. Based on those data, we hypothesised that the Retromer and Retriever complexes regulate KCa2.3 recycling. To investigate this, we are using biochemical [e.g. cell surface biotinylation, co-immunoprecipitation (co-IP)] and electrophysiological (e.g. patch-clamp) techniques. First, we determined the role of SNXs in KCa2.3 traffcking by using siRNA to knock down SNX protein expression in Fisher rat thyroid (FRT) cells expressing heterologous KCa2.3. Knockdown of Retromer-associated SNX3 or Retriever-associated SNX17 significantly decreased the cell surface population of KCa2.3 ( P<0.01, n=4, each). With protein-protein interaction experiments, KCa2.3 co-IPed with SNX17 and SNX3, but not Retromer-associated SNX27 ( n=3, each). Electrophysiology experiments are underway to determine if the knockdown of SNXs alters the functional expression of KCa2.3. and We are also investigating if KCa2.3 traffcking in primary endothelial cells requires SNX3 and SNX17. These results suggest that SNX3 and SNX17 are novel regulators of KCa2.3 traffcking. This work was supported by grants from NIH (HL083060, HL092157) and the Cystic Fibrosis Foundation (DEVOR20GO) to D.C.D.; Lottery Health New Zealand (R-LHR-2019-101706) to F.J.M.; and Aim Fund grant to K.L.H., and the Department of Physiology, University of Otago. M.J.E.L. was supported by a doctoral scholarship from the University of Otago. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.