Distinct acyl protein transferases and thioesterases control surface expression of calcium-activated potassium channels

Abstract

Protein palmitoylation is rapidly emerging as an important determinant in the regulation of ion channels, including large conductance calcium-activated potassium (BK) channels. However, the enzymes that control channel palmitoylation are largely unknown. Indeed, although palmitoylation is the only reversible lipid modification of proteins, acyl thioesterases that control ion channel depalmitoylation have not been identified. Here, we demonstrate that palmitoylation of the intracellular S0-S1 loop of BK channels is controlled by two of the 23 mammalian palmitoyl-transferases, zDHHC22 and zDHHC23. Palmitoylation by these acyl transferases is essential for efficient cell surface expression of BK channels. In contrast, depalmitoylation is controlled by the cytosolic thioesterase APT1 (LYPLA1), but not APT2 (LYPLA2). In addition, we identify a splice variant of LYPLAL1, a homolog with ∼30% identity to APT1, that also controls BK channel depalmitoylation. Thus, both palmitoyl acyltransferases and acyl thioesterases display discrete substrate specificity for BK channels. Because depalmitoylated BK channels are retarded in the trans-Golgi network, reversible protein palmitoylation provides a critical checkpoint to regulate exit from the trans-Golgi network and thus control BK channel cell surface expression.

FIGURE 1.

siRNA-based imaging screen reveals zDHHC22 and zDHHC23 as acyl transferases that palmitoylate the S0–S1 loop of BK channels. A, schematic of the pore-forming α-subunit indicating the intracellular S0–S1 loop and the previously identified palmitoylated cysteine residues Cys-53, Cys-54, and Cys-56. B, representative single confocal images of a -YFP fusion protein of the S0–S1 loop in HEK293 cells treated with a scrambled siRNA (scr-siRNA) or siRNAs targeting zDHHC22 or zDHHC23. Scale bars are 5 μm. C, bar graph of membrane expression of the S0–S1-YFP fusion protein, normalized to the scr-siRNA control, after zDHHC knockdown by siRNA. Data are means ± S.E. N >4, n >200. **, p < 0.01 decrease compared with scr-siRNA group, ANOVA with post-hoc Dunnetts test.

FIGURE 2.

zDHHC22 and -23 control BK channel palmitoylation. A, representative fluorograph (top panel) of [³H]palmitate incorporation and corresponding Western blot (anti-HA) of the full-length BK channel ZERO variant immunoprecipitated from HEK293 cells following knockdown of the respective zDHHC. B, quantification of [³H]palmitate incorporation from experiments (n = 3) as in A. Data are means ± S.E. **, p < 0.01 decrease compared with scrambled (scr) group, ANOVA with post-hoc Dunnett's test.

FIGURE 3.

zDHHC22 and -23 control cell surface expression of BK channels. A, representative single confocal images of ZERO channel surface (non-permeabilized, Flag-) and total (permeabilized, -HA) labeling following knockdown of the respective zDHHC. Scale bars are 5 μm. B, quantification of the ZERO channel surface (Flag-) to total (-HA) ratio as in A (N >4, n >200) following knockdown of the respective zDHHC or using the palmitoylation-deficient BK channel mutant C53:54:56. Data are means ± S.E. **, p < 0.01 decrease compared with scrambled (scr) group, ANOVA with post-hoc Dunnett's test.

FIGURE 4.

Overexpression of acyl thioesterases LYPLA1 and LYPLAL1, but not LYPLA2, reduces BK channel surface expression. A, mRNA expression of acyl thioesterases LYPLA1 and LYAPL2 and the homolog LYPLAL1 in HEK293 cells expressed as a fraction of LYPLA1 mRNA. B, representative single confocal images of full-length ZERO variant BK channels in HEK293 cells co-expressed with CFP- or -CFP-tagged LYPLA1 or LYPLAL1 and the palmitoylation-deficient BK channel mutant C53:54:56A. Surface (Flag-) and total (-HA) channel labeling was determined as in Fig. 2. Scale bars are 5 μm. C, quantitative immunofluorescence analysis of BK channel surface expression under different conditions (N > 5, n > 200/group) expressed as a percentage of the FLAG/HA ratio of ZERO. PPT1 is the lysosomal palmitoyl protein thioesterase 1. LYPLA1_D169A:H203 and LYPLAL1_D173A:H211A contain mutations in the respective catalytic triad required for thioesterase activity. Data are means ± S.E. **, p < 0.01 compared with ZERO, ANOVA with post-hoc Dunnett's test.

FIGURE 5.

Acyl thioesterases LYPLA1 and LYPLAL1, but not LYPLA2, depalmitoylate BK channels. A, representative fluorograph (top panel) of [³H]palmitate incorporation and corresponding Western blot (anti-HA) of BK channels co-expressed with LYPLA1, LYPLAL1, or their catalytic triad mutants. B, quantification of [³H]palmitate incorporation from experiments (n = 3) as in A. Data are means ± S.E. **, p < 0.01 compared with ZERO, ANOVA with post-hoc Dunnett's test.

FIGURE 6.

LYPLA1 and LYPLAL1 have no effect of GABA_BR1a(ASRR) cell surface trafficking. A, representative single confocal images of the HA-tagged trafficking competent GABA_BR1a(ASRR) receptor subunit in HEK293 cells co-expressed with -CFP-tagged LYPLA1, LYPLAL1, or their corresponding catalytic triad mutants LYPLA1_D169A:H203 and LYPLAL1_D173A:H211A. The extracellular HA tag was labeled under non-permeabilized conditions (surface) and following permeabilization (Total) in the same cells as described under “Experimental Procedures.” Scale bars are 5 μm. B, quantitative immunofluorescence analysis of receptor surface expression under different conditions (N > 3, n > 48/group) expressed as a percentage of the surface expression of GABA_BR1a(ASRR). Data are means ± S.E.

FIGURE 7.

Acyl thioesterases retard BK channels in the TGN. Quantitative co-localization analysis of ZERO variant BK channels co-expressed with LYPLA1 or LYPLAL1 and the C53:54:56A palmitoylation-deficient BK channel with markers of A, the endoplasmic reticulum (ER - pdsRed-ER); B, trans-golgi network (TGN - TGN38); or C, recycling endosomes (RE - rab11). Inset images are representative confocal images indicating distribution of the respective compartment marker, the outline indicates the plasma membrane in the cells stained for the TGN and RE, respectively. Scale bars are 5 μm. Data are expressed as the respective Pearson's correlation coefficient (R) from deconvolved data, where a value of +1 indicates 100% co-localization. Data are means ± S.E., N > 4, n > 48/group. *, p < 0.05; **, p < 0.01 compared with ZERO group, ANOVA with post-hoc Dunnett's test.