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. 2013 May 3;288(18):13136-44.
doi: 10.1074/jbc.M113.461830. Epub 2013 Mar 16.

Palmitoylation of the β4-subunit regulates surface expression of large conductance calcium-activated potassium channel splice variants

Lie Chen  1 , Danlei Bi, Lijun Tian, Heather McClafferty, Franziska Steeb, Peter Ruth, Hans Guenther Knaus, Michael J Shipston
Affiliations

Palmitoylation of the β4-subunit regulates surface expression of large conductance calcium-activated potassium channel splice variants

Lie Chen et al. J Biol Chem. .

Abstract

Regulatory β-subunits of large conductance calcium- and voltage-activated potassium (BK) channels play an important role in generating functional diversity and control of cell surface expression of the pore forming α-subunits. However, in contrast to α-subunits, the role of reversible post-translational modification of intracellular residues on β-subunit function is largely unknown. Here we demonstrate that the human β4-subunit is S-acylated (palmitoylated) on a juxtamembrane cysteine residue (Cys-193) in the intracellular C terminus of the regulatory β-subunit. β4-Subunit palmitoylation is important for cell surface expression and endoplasmic reticulum (ER) exit of the β4-subunit alone. Importantly, palmitoylated β4-subunits promote the ER exit and surface expression of the pore-forming α-subunit, whereas β4-subunits that cannot be palmitoylated do not increase ER exit or surface expression of α-subunits. Strikingly, however, this palmitoylation- and β4-dependent enhancement of α-subunit surface expression was only observed in α-subunits that contain a putative trafficking motif (… REVEDEC) at the very C terminus of the α-subunit. Engineering this trafficking motif to other C-terminal α-subunit splice variants results in α-subunits with reduced surface expression that can be rescued by palmitoylated, but not depalmitoylated, β4-subunits. Our data reveal a novel mechanism by which palmitoylated β4-subunit controls surface expression of BK channels through masking of a trafficking motif in the C terminus of the α-subunit. As palmitoylation is dynamic, this mechanism would allow precise control of specific splice variants to the cell surface. Our data provide new insights into how complex interplay between the repertoire of post-transcriptional and post-translational mechanisms controls cell surface expression of BK channels.

Keywords: Ion Channels; Membrane Transport; Potassium Channels; Protein Palmitoylation; S-Acylation; Signal Transduction.

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Figures

FIGURE 1.
FIGURE 1.
Palmitoylation controls exit of the β4-subunit from the endoplasmic reticulum. A, schematic of the β4 regulatory subunit of large conductance calcium- and voltage-activated potassium (BK) channels indicating the palmitoylated cysteine residue (Cys-193) juxtaposed to the intracellular C terminus of the second transmembrane domain. B representative fluorographs of [3H]palmitate (3H-palm) incorporation and corresponding Western blot (anti-Myc) of the wild-type β4-subunit and the alanine mutant C193A. C, acyl-RAC of murine cerebellum with Western blot probed with anti-b4. D, representative single confocal images of the β4 and C193A mutant expressed in HEK293 cells and co-labeled for the ER. Scale bars are 2 μm. E and F, bar graphs of membrane expression (expressed as a percentage of wild-type β4) (E) and co-localization with the ER (expressed as Pearson's correlation coefficient, R) (F) of the wild-type β4 and C193A mutant. Data are means ± S.E. N >5, n >200. **, p < 0.01 when compared with wild-type β4 group, ANOVA with post hoc Dunnett's test.
FIGURE 2.
FIGURE 2.
β4-Subunit palmitoylation controls surface expression and ER retention of the pore-forming α-subunit ZERO variant of BK channels. A, representative single confocal images of the ZERO variant of the pore-forming α-subunit of BK channels expressed in HEK293 cells in the absence and presence of the wild-type β4-subunit and the palmitoylation-deficient C193A subunit. Total α-subunit expression and co-labeling for the ER with merged images are shown. Scale bars are 2 μm. B and C, quantification of the effect of β4, or its C193A mutant, on ER co-localization (ER coloc.) (B) and cell surface expression of the ZERO α-subunit (C). D, β4, but not C193A, also increases cell surface expression of the ZERO α-subunit palmitoylation-deficient mutant C53:54:56A. E, recapitulation of cell surface enhancement of ZERO variant expression by β4 in the neuronal cell line N2a. For cell surface expression of ZERO α-subunit channel, protein was probed under nonpermeabilized (surface) and permeabilized (total) conditions, and the surface/total ratio was expressed as a percentage of the α-subunit in the absence of regulatory subunit as indicated. Data are means ± S.E., N > 7. **, p < 0.01 when compared with respective α-subunit expressed alone, ANOVA with post hoc Dunnett's test.
FIGURE 3.
FIGURE 3.
β4-Subunit palmitoylation modifies channel deactivation kinetics. A, representative macropatch recordings from isolated inside-out patches of HEK293 cells expressing the ZERO α-subunit variant (□) with and without WT β4 (○ or palmitoylation-deficient C193A β4-subunits (●) in the presence of 10 μm intracellular free calcium. B, corresponding normalized G/V relationships with Boltzmann fits determined from tail currents recorded as above. C and D, activation time (Act. time) constants determined at +60 mV (C) and deactivation time (Deact. time) constants determined at −60 mV (D). Data are means ± S.E., n = 10–17 per group. **, p < 0.01 when compared with ZERO expressed alone, #, p < 0.05 when compared with β4-subunit, ANOVA with post hoc Dunnett's test.
FIGURE 4.
FIGURE 4.
β4-mediated enhancement of channel surface expression is α-subunit splice variant-dependent. A, schematic of three distinct α-subunit splice variants that differ only in their very N or C termini, analyzed with the first and last 3 amino acids shown. The ZERO variant used in Fig. 2 is MDA-DEC with the variant with the same start methionine but shorter C terminus (MDA-ERL) and variant with upstream methionine with truncated C terminus (MAN-ERL) indicated. B, representative single confocal images of the surface (nonpermeabilized), total (permeabilized), and merged images of the three α-subunit splice variants. Scale bars are 2 μm. C, quantification of the surface expression of the three variants expressed as a percentage of ZERO (MDA-DEC) variant BK channels in HEK293. D and E, quantification of the effect of β4 or its C193A mutant on the MDA-ERL (D) and MAN-ERL (E) splice variants expressed as a percentage of the surface expression of the respective α-subunit alone. Data are means ± S.E., N > 5, n > 200. **, p < 0.01 when compared with respective α-subunit alone, ANOVA with post hoc Dunnett's test.
FIGURE 5.
FIGURE 5.
The heptapeptide … REVEDEC is sufficient to confer β4-mediated enhancement of BK channel cell surface expression. A, representative confocal images of the MAN-ERL α-subunit variant and the chimera in which the last 7 amino acids of MAN-ERL have been replaced by the heptapeptide REVEDEC (MAN-(R … DEC) expressed in N2a cells with or without the WT β4-subunit. B and C, quantification of surface expression of MAN-ERL (B) and MAN-(R … DEC) (C) in N2a cells expressed in the absence and presence of WT β4-subunit or the C193A mutant. Data are expressed as a percentage of MAN-ERL surface expression. D, cell surface expression of MAN-(R … DEC) in the presence or absence of WT β4-subunit or the C193A mutant expressed in HEK293 cells. Data are means ± S.E., N > 4, n > 96/group. *, p < 0.05, **, p < 0.01 when compared with MAN-ERL in panels B and C or MAN-(R … DEC) in panel D variant surface expression, ANOVA with post hoc Dunnett's test.
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