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. 2015 Jul 10:16:16.
doi: 10.1186/s12858-015-0045-6.

Kv1.3 contains an alternative C-terminal ER exit motif and is recruited into COPII vesicles by Sec24a

John M Spear  1 , Dolly Al Koborssy  2 , Austin B Schwartz  3 , Adam J Johnson  4 , Anjon Audhya  5 , Debra A Fadool  6   7   8 , Scott M Stagg  9   10
Affiliations

Kv1.3 contains an alternative C-terminal ER exit motif and is recruited into COPII vesicles by Sec24a

John M Spear et al. BMC Biochem. .

Abstract

Background: Potassium 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.

Results: We 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.

Conclusion: These 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.

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Figures

Fig. 1
Fig. 1
Biophysical properties of Kv1.3 channels following mutations of the acidic ER export motif. a Bar graph of the mean peak (left) or sustained (middle) current (± s.e.m.) for various voltage-clamped Kv1.3-eGFP or mutant channels as recorded in cell-attached patches using a single step depolarization of +40 mV (Vc) from a holding potential (Vh) of -80 mV. Representative current traces comparing Kv1.3-eGFP with that of Kv1.3-eGFP ∆C (right). b Same as in (A) but comparing inactivation (left) or deactivation (middle) kinetics of Kv1.3-eGFP. Significantly different by one-way ANOVA, Bonferoni’s post-hoc test, * = 0.001. c Line graph of the normalized tail currents is fit with a Boltzmann relation to calculate voltage at half-activation (V1/2). Significantly different V1/2 by one-way ANOVA, Bonferoni’s post-hoc test, *** = 0.0001, * = 0.001
Fig. 2
Fig. 2
Localization of Kv1.3 channels after sequential mutation of the acidic ER export motif. The Kv1.3-eGFP and each of the mutated Kv1.3-eGFP proteins (see methods) were transiently expressed in BSC40 cells and the fluorescence was observed using confocal microscopy (a-h) in the presence of the membranous ER resident protein Sec61β and the Golgi resident protein Golgin97. The Kv1.3-eGFP co-localization with these organelle specific resident proteins and the resulting fluorescent trafficking profile (a) is similar in appearance with the Kv1.3-eGFP E443A (b), Kv1.3-eGFP E445A (c), Kv1.3-eGFP E443A-E445A (e), and Kv1.3-eGFP E445A-E447A (f) profiles indicating that there is no significant ER retention of any of these mutated proteins. A noticeable difference in the co-localization of Kv1.3-eGFP and Sec61β is observed in the Kv1.3-eGFP E447A (d), Kv1.3-eGFP E443A-E445A-E447A (g), and Kv1.3-eGFP ∆C (h) trafficking profiles where there is either a modest (d and g) or severe (h) retention within the ER network (d, g, and h; white arrows). Line scans of each fluorescent channel are shown. The white arrow in the merged image represents the placement and direction of the line scan. Scale bar = 10 μm
Fig. 3
Fig. 3
Retention of Kv1.3-eGFP in the ER upon sequential mutation of the acidic motif. Bar graph depicting the amount of Kv1.3-eGFP or mutant proteins retained in the ER. Ratio of relative percent intensity is equal to the amount of protein retained in the ER microsome fractions divided by the total amount of protein from whole cell homogenates. Resulting values were plotted as the mean ± standard error of the mean of three replicates (n = 3). The only statistically different mutant was the Kv1.3-eGFP E443A-E445A-E447A by one-way ANOVA, Bonferoni correction applied for Type-1 errors (p > 0.008)
Fig. 4
Fig. 4
Kv1.3-eGFP trafficking after siRNA mediated knockdown of Sec24. Kv1.3-eGFP trafficking was examined after the knockdown of Sec24 isoforms (as indicated) in the presences of the membranous ER resident protein Sec61β tagged with the mCherry fluorophore (Sec61β-mCherry). Cellular nuclei were stained with DAPI. The wild-type (wt) trafficking profile is similar to the trafficking profile of Sec24c and Sec24cd knockdown conditions. An altered trafficking profile is seen in Sec24a, Sec24b, Sec24ab, and Sec24abcd conditions. Interestingly, there is also an altered trafficking profile in the Sec24d condition, but the Kv1.3-eGFP signal does not overlap well with the Sec61β protein. Line scans of each fluorescent channel are shown. The white arrow in the merged image represents the placement and direction of the line scan. Scale bar = 5 μm
Fig. 5
Fig. 5
In vitro Kv1.3-Sec24a membrane floatation assay. Membrane floatation assay used to test for the association between Kv1.3 and Sec24a341. (a) Kv1.3 proteins reconstituted into synthetic lipid vesicles (proteoliposomes) and (b) control lipid vesicles (liposomes). (c) Schematic of the floatation assay. Proteoliposomes, drawn as small black circles, migrate through the three-step sucrose gradient (0 %, 25 %, and 30 % w/v sucrose; top (1), middle (2) and bottom (3), respectively) after incubation and centrifugation. (d) Kv1.3 proteoliposomes were found in the top fraction after centrifugation. (e) When Kv1.3 proteoliposomes (~65 kDa as a monomer) were incubated with Sec24a341 (~80 kDa), both Kv1.3 and Sec24a341 were detected in the top fraction. (f) Sec24a341 alone was not detected in the top fraction. (g) When Kv1.3 proteins in detergent micelles were mixed with Sec24a341 in the presence of control liposomes, both Kv1.3 and Sec24a341 were found in the top fraction. (h) Sec24a341 incubated with control liposomes was not found in the top fraction. (i) Kv1.3 micelles and Sec24a341 do not float in the absence of membranes. (j) Kv1.3 was detected in the top fraction when Kv1.3 in detergent micelles were incubated with control liposomes. Scale bar = 100 nm
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