Palmitoylation of the β4-Subunit Regulates Surface Expression of Large Conductance Calcium-activated Potassium Channel Splice Variants

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

Palmitoylation of large conductance voltage- and calcium-dependent potassium (bk) channels

S-palmitoylation is a reversible post-translational lipid modification of proteins by adding a 16-carbon palmitate onto a cysteine residue. Palmitoylation has been shown to control the trafficking and function of many signalling proteins including ion channels. In this Thesis, palmitoylation is shown to control both the plasma membrane expression and gating properties of large conductance calcium- and voltage- dependent potassium (BK) channels. The BK channel is assembled from four pore-forming α-subunits. Each α-subunit contains seven transmembrane domains (S0-S6), with an extracellular N-terminus and a large intracellular C-terminus. BK channel α-subunit is encoded by a single gene Kcnma1 that undergoes extensive pre mRNA splicing at various splice sites, thus there are a number of alternatively spliced variants of α-subunits. Using quantitative imaging assays, palmitoylation of the intracellular S0-S1 loop controlled trafficking of full length ZERO variant BK channels to the plasma membrane in HEK293 cells as well as neuronal N2a cells. Importantly, all four α-subunits need to be palmitoylated for robust surface expression. Thus, palmitoylation of the S0-S1 loop of the α-subunit is important for surface expression of BK channels. The BK channel may also assemble with auxiliary β-subunits (β1-4) that regulate surface expression and gating properties of BK channels. The N-terminus of the β1- subunit and the C-terminus of the β4-subunit were shown to be palmitoylated using [3H]-palmitate incorporation, respectively. However, mutation of the palmitoylated cysteine (C18 in β1 and C193 in β4) to alanine to generate depalmitoylated β- subunits had no significant effects on the electrophysiological properties resulting from co-expression with the ZERO variant of the BK channel. However, although palmitoylation of the S0-S1 loop does not affect the electrophysiological properties of the ZERO channels alone, it is important for the shift in the V0.5max of ZERO channel when co-expressed with the β1-subunit, but not β4-subunit. These data suggest that palmitoylation of the S0-S1 loop controls the functional coupling between the ZERO α-subunit and β1-subunit. Although palmitoylation of C18 in the N-terminus of the β1-subunit was not required for functional coupling to α-subunits, we identified other critical residues within the short intracellular N-terminus of the β1-subunit that are essential. The functional coupling between BK α- and β1-subunit was predicted to be controlled by the interaction between a non-classic amphipathic α-helix in the β1 subunit N-terminus and the plasma membrane. Deletion, or mutations predicted to disrupt the interaction significantly decreased the β1-subunit induced left shift in the BK channel V0.5max. This suggests that the amphipathic in-plane anchor is critical for functional coupling of β1-subunits with BK channel α-subunits. In this Thesis, we demonstrated: i) palmitoylation of the α-subunit S0-S1 loop controls surface membrane expression of BK channels, and also controls functional regulation by β1, but not β4-subunits; and ii) a potential non-classical amphipathic in-plane anchor in the β1 N-terminus is essential for functional coupling with α- subunits. These studies help us further understand the regulation of BK channels and suggest potential therapeutic targets for various diseases related to dysfunctional BK channels, such as hypertension

The complement inhibitor CD59 is required for GABAergic synaptic transmission in the dentate gyrus

Summary: Complement-dependent microglia pruning of excitatory synapses has been widely reported in physiological and pathological conditions, with few reports concerning pruning of inhibitory synapses or direct regulation of synaptic transmission by complement components. Here, we report that loss of CD59, an important endogenous inhibitor of the complement system, leads to compromised spatial memory performance. Furthermore, CD59 deficiency impairs GABAergic synaptic transmission in the hippocampal dentate gyrus (DG). This depends on regulation of GABA release triggered by Ca2+ influx through voltage-gated calcium channels (VGCCs) rather than inhibitory synaptic pruning by microglia. Notably, CD59 colocalizes with inhibitory pre-synaptic terminals and regulates SNARE complex assembly. Together, these results demonstrate that the complement regulator CD59 plays an important role in normal hippocampal function