Palmitoylation gates phosphorylation-dependent regulation of BK potassium channels

Abstract

Large conductance calcium- and voltage-gated potassium (BK) channels are important regulators of physiological homeostasis and their function is potently modulated by protein kinase A (PKA) phosphorylation. PKA regulates the channel through phosphorylation of residues within the intracellular C terminus of the pore-forming alpha-subunits. However, the molecular mechanism(s) by which phosphorylation of the alpha-subunit effects changes in channel activity are unknown. Inhibition of BK channels by PKA depends on phosphorylation of only a single alpha-subunit in the channel tetramer containing an alternatively spliced insert (STREX) suggesting that phosphorylation results in major conformational rearrangements of the C terminus. Here, we define the mechanism of PKA inhibition of BK channels and demonstrate that this regulation is conditional on the palmitoylation status of the channel. We show that the cytosolic C terminus of the STREX BK channel uniquely interacts with the plasma membrane via palmitoylation of evolutionarily conserved cysteine residues in the STREX insert. PKA phosphorylation of the serine residue immediately upstream of the conserved palmitoylated cysteine residues within STREX dissociates the C terminus from the plasma membrane, inhibiting STREX channel activity. Abolition of STREX palmitoylation by site-directed mutagenesis or pharmacological inhibition of palmitoyl transferases prevents PKA-mediated inhibition of BK channels. Thus, palmitoylation gates BK channel regulation by PKA phosphorylation. Palmitoylation and phosphorylation are both dynamically regulated; thus, cross-talk between these 2 major posttranslational signaling cascades provides a mechanism for conditional regulation of BK channels. Interplay of these distinct signaling cascades has important implications for the dynamic regulation of BK channels and physiological homeostasis.

Fig. 1.

STREX targets BK channel C terminus to the plasma membrane. (A) Schematic illustrating the topology of the BK channel pore forming α-subunit. The STREX insert is located in the linker between the 2 predicted regulator of K⁺ conductance (RCK) domains in the intracellular C terminus. Inclusion of STREX generates a CRD encompassing the heme-binding domain (hbd) and STREX. Sequence alignment indicates evolutionarily conserved cysteine residues in the STREX insert predicted to be palmitoylated by the CSS-palm algorithm (shaded) and the PKA phosphorylation site serine S3 (indicated by the asterisk). Cysteine residues are numbered in the CRD as follows: STREX residues numbered from the first STREX residue (K) and upstream cysteines labeled by letters. (B and C) Schematic of C-terminal GFP fusion (B) and CRD domain (C) fused between CFP and YFP constructs and representative single confocal section images of STREX, STREX cysteine mutants C12:13A and C23:25A, and the ZERO variant (STREX insert excluded) expression in HEK293 cells. In B Lower Right, the C-terminal ZERO–GFP fusion construct was cotransfected with a modified C-terminal STREX construct (STREX*) in which the −GFP tag of STREX was replaced with an −HA epitope. (Scale bars: 5 μm.) (D) Summary bar chart of the respective C-terminal (■) or CRD (□) construct localization at the plasma membrane expressed as a percentage of the respective STREX expression. Data are means ± SEM, N > 12, n > 350 for each construct. **, P < 0.01 compared with respective STREX construct (ANOVA with Student–Neuman–Keuls post hoc test).

Fig. 2.

Palmitoylation of STREX required for membrane localization. (A) Representative fluorographs (Upper) and Western blots (Lower) of full-length STREX-HA and ZERO-HA channels (Left) and the wild-type STREX and mutant C12:13A CRD-YFP constructs (Right) expressed in HEK293 cells. Constructs were labeled with ³H-palmitate for 4 h and the respective constructs immunoprecipitated (IP) by using α-HA or α-GFP magnetic microbeads respectively and detected by fluorography. (B) Representative blots from a cysteine-accessibility assay after IP and treatment of STREX channels with 1 M neutral hydroxylamine to cleave endogenous palmitate thioester bonds to cysteine residues. Accessible cysteines were probed by using biotin-BMCC (Upper) with total protein probed by α-HA (Lower). (C) Summary imaging data of STREX and C12:13A C terminus (■) or CRD (□) as in Fig. 1 B–D and the effect of 24-h pretreatment of cells with the palmitoyltransferase inhibitor 2-bromopalmitate (2-BP, 100 μM) or the myristoylation inhibitor 2-hydroxymyristate (2-HM, 0.1–1 mM). Data are means ± SEM, N > 14, n > 950 for each construct/treatment. **, P < 0.01 compared with respective STREX construct (ANOVA with Student–Neuman–Keuls post hoc test).

Fig. 3.

PKA phosphorylation of STREX dissociates STREX from plasma membrane. (A) Representative single confocal sections from HEK293 cells expressing wild-type STREX C-terminal constructs and the corresponding STREX and C-terminal PKA phosphorylation site phosphomimetic constructs S3E and S899E. (Scale bars: 5 μm.) (B) Effect of cell-permeable cAMP analogue 8-CPT-cAMP (0.1 mM, 10–30 min) on STREX C terminus (■) or CRD (□) membrane localization in the presence or absence of 10 nM okadaic acid or the PKA inhibitor H89 (1 μM). (C) Summary of S3E and S899E construct expression at the plasma membrane (na, S899 site not present in CRD construct). (D) Effect of acute hypoxia (<3% O₂), PKG activation with the cell-permeable cGMP analogue 8-CPT-cGMP (0.1 mM in the presence of 10 nM okadaic acid) or PKC activation with the phorbol ester PMA (100 nM in the presence of 10 nM okadaic acid) on construct localization at the plasma membrane. Data are means ± SEM, N > 4, n > 350. (E and F) Representative single-channel traces and diary plots of single-channel mean open probability (Po) from isolated inside-out patches of HEK293 cells expressing full-length STREX (E) or C12:13A (F) channels before and 10 min after exposure to cAMP. Single channels were assayed in physiological K⁺ gradients exposed to 0.2 μM free calcium and 2 mM Mg-ATP. (G and H) Inhibition of STREX (G) or C12:13A (H) channel Po by cAMP (0.1–1.0 mM) in the presence or absence of the PKA inhibitor PKI_5-24 (0.45 μM) or 24-h cell pretreatment with 100 μM 2-BP; application of catalytic subunit of PKAc (300 nM) or exposure to acute hypoxia (<3%O₂). Data are means ± SEM, n = 5–14 for each treatment. **, P < 0.01 compared with respective control (ANOVA with Student–Neuman–Keuls post hoc test).

Fig. 4.

Palmitoylation gates PKA inhibition of BK channels in native cells. (A and B) Representative whole-cell outward current traces from mouse anterior pituitary corticotrope (AtT20) cells pretreated with vehicle (0.01% DMSO) (A) or 2-BP (24 h, 100 μM) (B) before (control) or 10 min after intracellular dialysis with 0.1 mM cAMP or extracellular application of a 1 μM concentration of the BK channel inhibitor paxilline. Cells were voltage clamped at −50 mV in physiological potassium gradients and traces shown recorded during voltage steps to +40 mV for 100 ms. (C) Mean current density (pA/pF) of paxilline-sensitive current from vehicle- and 2-BP-treated AtT20 cells determined 90 ms into the pulse at +40 mV. (D) Percentage inhibition of paxilline-sensitive current (Ipax) by cAMP in vehicle- and 2-BP-treated cells. Data are means ± SEM, n = 9 for each treatment. **, P < 0.01 compared with cAMP inhibition in vehicle control (ANOVA with Student–Neuman–Keuls post hoc test).