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. 2020 Dec 4;295(49):16487-16496.
doi: 10.1074/jbc.RA120.015349. Epub 2020 Sep 10.

Site-specific deacylation by ABHD17a controls BK channel splice variant activity

Heather McClafferty  1 , Hamish Runciman  1 , Michael J Shipston  2
Affiliations

Site-specific deacylation by ABHD17a controls BK channel splice variant activity

Heather McClafferty et al. J Biol Chem. .

Abstract

S-Acylation, the reversible post-translational lipid modification of proteins, is an important mechanism to control the properties and function of ion channels and other polytopic transmembrane proteins. However, although increasing evidence reveals the role of diverse acyl protein transferases (zDHHC) in controlling ion channel S-acylation, the acyl protein thioesterases that control ion channel deacylation are very poorly defined. Here we show that ABHD17a (α/β-hydrolase domain-containing protein 17a) deacylates the stress-regulated exon domain of large conductance voltage- and calcium-activated potassium (BK) channels inhibiting channel activity independently of effects on channel surface expression. Importantly, ABHD17a deacylates BK channels in a site-specific manner because it has no effect on the S-acylated S0-S1 domain conserved in all BK channels that controls membrane trafficking and is deacylated by the acyl protein thioesterase Lypla1. Thus, distinct S-acylated domains in the same polytopic transmembrane protein can be regulated by different acyl protein thioesterases revealing mechanisms for generating both specificity and diversity for these important enzymes to control the properties and functions of ion channels.

Keywords: Kcnma1; Kcnmb1; S-acylation; acyl protein thioesterase; acyl thioesterase; ion channel; lipid; lipid modification; membrane trafficking; palmitoylation; post-translational modification (PTM); potassium channel; protein trafficking.

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Conflict of interest statement

Conflict of interest—The authors declare that they have no conflicts of interest with the contents of this article.

Figures

Figure 1
Figure 1
ABHD17a controls plasma membrane association of BK channel STREX domain.A, schematic of pore-forming subunit of the STREX BK channel splice variant indicating the two S-acylated domains of the channel: the S0–S1 loop present in all channel variants and the alternatively spliced STREX insert in the intracellular C terminus of the channel located between the two regulator of potassium conductance (RCK) domains. B, the isolated S-acylated STREX–CRD fusion protein is associated with the plasma membrane when expressed in HEK293 cells and remains cytosolic when deacylated. C, representative confocal sections of HEK293 cells expressing the STREX–CRD (as a GFP fusion protein) and upon co-expression with ABHD17a. Scale bars, 2 µm. D, immunostaining of populations of HEK293 cells in 96 wells for overexpressed FLAG-tagged putative acyl thioesterases from the ABHD and Lypla families. E, quantification of membrane expression of STREX–CRD, as a GFP fusion protein, in HEK293 upon co-expression with ABHD and Lypla acyl thioesterase family members. The data are expressed as the fraction of cells expressing STREX–CRD at the plasma membrane compared with cells expressing STREX–CRD alone. The data are means ± S.D. (n = 23–54 independent experiments/group). **, p < 0.01; *, p < 0.05 compared with STREX–CRD alone using a nonparametric Kruskal–Wallis test with post hoc Dunn's test.
Figure 2
Figure 2
ABHD17a deacylates the BK channel STREX, but not S0–S1 loop, domain.A and B, representative Western blots from an acyl-RAC assay of ZERO BK channel variant that lacks the STREX domain (A) or the STREX-C53A/C54A/C56A mutant that cannot be S-acylated in the S0–S1 loop (B), expressed in HEK293 cells alone or with ABHD17a or ABHD17c. Cell lysate input to the acyl-RAC assay is shown together with pulldowns following cleavage of thioester bond with hydroxylamine (+NH2OH) or in salt control (−NH2OH). C and D, quantification of S-acylation of ZERO (C) or STREX-C53A/C54A/C56A (D) in the presence of ABHD17a or ABHD17c. The data are expressed as percentages of S-acylation of corresponding ZERO or STREX-C53A/C54A/C56A channel alone (control) as means ± S.D. (n = 4 indepen-dent experiments/group). **, p < 0.01; *, p < 0.05 compared with STREX-C53A/C54A/C56A alone using nonparametric Kruskal–Wallis test with post hoc Dunn's test.
Figure 3
Figure 3
ABHD17a–dependent deacylation does not control BK channel surface expression.A, representative on-cell Western assay of cell surface expression of epitope-tagged BK ZERO, ZERO-C53A/C54A/C56A, and STREX channels alone or co-expressed with WT β1 or co-expressed with WT ABHD17a, the catalytically inactive ABHD17a mutant (ABHD17a-mut) or the N-terminally truncated, targeting-deficient ABHD17a (Δ19-ABHD17a). Surface BK channel was quantified using an extracellular FLAG tag (green), whereas total BK expression was measured using an intracellular HA tag (red) following cell permeabilization. Four replicates from an individual experiment are shown. B–E, quantification of corresponding BK channel surface expression in the presence of acyl thioesterases and their related mutants as in A and expressed as a fraction of the corresponding BK channel subunit expressed alone. The data are means ± S.D. (n = 5–17 independent experiments/group). **, p < 0.01; *, p < 0.05 compared with corresponding BK channel group alone using nonparametric Kruskal–Wallis test with post hoc Dunn's test.
Figure 4
Figure 4
ABHD17a does not control BK channel internalization.A, schematic of internalization assay. B, representative images from an internalization assay of epitope-tagged ZERO BK channels alone or co-expressed with ABHD17a and its mutants in HEK293 cells. Internalized BK α-subunit was quantified, after 60 min at 37 °C, using the extracellular FLAG tag (green) following acid-strip of surface staining in nonpermeabilized cells and normalized to surface expression at time 0. Four replicates from an individual experiment are shown. C, quantification of BK channel α-subunit internalization as a percentage of initial total surface expression. The data are means ± S.D. (n = 4 independent experiments/group).
Figure 5
Figure 5
ABHD17a-mediated deacylation of the STREX domain inhibits STREX channel function.A, representative experimental time courses of ionomycin (1 μm started at 16 s, horizontal gray bar)–induced changes in membrane potential of HEK293 cells transfected with STREX channel variants and ABHD17a using a 96-well format cell population Flexstation membrane potential assy. Positive changes in RFU denote membrane depolarization, whereas negative changes in RFU reflect membrane hyperpolarization. The assays were performed in physiological ion gradients with 2 mm extracellular calcium. The data are means ± S.D. from a single typical independent experiment with eight experimental replicates. B, quantification of peak hyperpolarization response to ionomycin in membrane potential assays expressed as a fraction of the peak hyperpolarization in cells expressing STREX channels alone. The data are means ± S.D. (n = 5–20/group). **, p < 0.01 compared with STREX using nonparametric Kruskal–Wallis test with post hoc Dunn's test.
Figure 6
Figure 6
Distinct acyl protein thioesterases control deacylation at distinct sites in BK channels.A, schematic of the STREX variant of the BK channel pore-forming subunit (Kcnma1) and sites of S-acylation. The STREX domain is deacylated by Abhd17a and the S0–S1 domain by Lypla1. B, amino acid sequence of other targets for ABHD17 acyl thioesterases showing the 10 most vicinal residues surrounding S-acylated cysteines. Mouse sequences and numbering are used throughout and include proteins with S-acylation at the (i) N terminus including Psd-95 (11, 12), Gap-43 (12), and Map6 (13); (ii) internal/intracellular loop cysteines such as Mpp6 (14) and the STREX variant of Kcnma1 shown here; and (iii) cysteines toward the C terminus of proteins including H-Ras and N-Ras (11, 12).
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