Abstract
BackgroundPotassium channels play a fundamental role in resetting the resting membrane potential of excitable cells. Determining the intracellular trafficking and localization mechanisms of potassium channels provides a platform to fully characterize their maturation and functionality. Previous investigations have discovered residues or motifs that exist in their primary structure, which directly promote anterograde trafficking of nascent potassium channels. Recently, a non-conical di-acidic motif (E483/484) has been discovered in the C-terminus of the mammalian homologue of the Shaker voltage-gated potassium channel subfamily member 3 (Kv1.3), and was shown to disrupt the anterograde trafficking of Kv1.3.ResultsWe have further investigated the intracellular trafficking requirements of Kv1.3 both in vivo and in vitro. First, three alternative C-terminal acidic residues, E443, E445, E447 were probed for their involvement within the early secretory pathway of Kv1.3. Single point (E443A, E445A, and E447A) and double point (E443A-E445A, E445A-E447A) mutations exhibited no significant changes in their endoplasmic reticulum (ER) retention. The triple point mutant E443A-E445A-E447A displayed a modest ER retention while deletion of the C-terminus showed dramatic ER retention. Second, we demonstrate in vivo the requirement for the Sec24a isoform to confer anterograde trafficking using a siRNA knockdown assay. Third, we show in vitro the association of recombinantly expressed Kv1.3 and Sec24a proteins.ConclusionThese results expand upon previous studies aimed at deciphering the Kv1.3 secretory trafficking mechanisms and further show in vitro evidence of the association between Kv1.3 and the COPII cargo adaptor subunit isoform Sec24a.Electronic supplementary materialThe online version of this article (doi:10.1186/s12858-015-0045-6) contains supplementary material, which is available to authorized users.
Highlights
Potassium channels play a fundamental role in resetting the resting membrane potential of excitable cells
Fluorescent tag of Kv1.3 channel does not perturb biophysical properties it has been previously examined in other expression systems, we confirmed that our N-terminal enhanced green fluorescent protein insertion did not alter Kv1.3 channel function
Addition of the N-terminal enhanced green fluorescent protein (eGFP) did not significantly alter the magnitude of the peak or sustained current, the current-voltage relationship, the voltage at half activation (V1/2), or the kinetics of inactivation or deactivation (Additional file 2). These results suggest that there are no deleterious alterations of the Kv1.3 channel properties when fused with the eGFP reporter
Summary
Potassium channels play a fundamental role in resetting the resting membrane potential of excitable cells. One-third of all eukaryotic proteins undergo transport from the endoplasmic reticulum (ER) to the ER-Golgi intermediate complex (ERGIC). This pathway is termed the anterograde early secretory pathway and is facilitated by the coat protein complex II (COPII). It has been shown that the N-terminus contains the T1 tetramerization domain, which promotes the formation of properly assembled channels [28, 29], while the C-terminus is recognized to harbor the ER export motif that facilitates efficient processing and localization of Kv channels to the plasma membrane [5, 30,31,32,33]
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