Identification by mass spectrometry and functional characterization of two phosphorylation sites of KCNQ2/KCNQ3 channels

Abstract

Neuronal potassium channel subunits of the KCNQ (Kv7) family underlie M-current (I(M)), and may also underlie the slow potassium current at the node of Ranvier, I(Ks). I(M) and I(Ks) are outwardly rectifying currents that regulate excitability of neurons and myelinated axons, respectively. Studies of native I(M) and heterologously expressed Kv7 subunits suggest that, in vivo, KCNQ channels exist within heterogeneous, multicomponent protein complexes. KCNQ channel properties are regulated by protein phosphorylation, protein-protein interactions, and protein-lipid interactions within such complexes. To better understand the regulation of neuronal KCNQ channels, we searched directly for posttranslational modifications on KCNQ2/KCNQ3 channels in vivo by using mass spectrometry. Here we describe two sites of phosphorylation. One site, specific for KCNQ3, appears functionally silent in electrophysiological assays but is located in a domain previously shown to be important for subunit tetramerization. Mutagenesis and electrophysiological studies of the second site, located in the S4-S5 intracellular loop of all KCNQ subunits, reveal a mechanism of channel inhibition.

Fig. 1.

Topology of KCNQ2 and KCNQ3 channels. KCNQ2 (843 aa) and KCNQ3 (872 aa) polypeptides are predicted to have six membrane-spanning helices and intracellular N and C termini, like other voltage-gated potassium channels. Stars indicate the positions of the phosphorylation sites identified in this study.

Fig. 2.

Affinity purification of KCNQ2/KCNQ3 channels and identification and electrophysiological characterization of a site of phosphorylation in the C-terminal region of KCNQ3. (A) Protein stained gel showing heterologously expressed, affinity-purified KCNQ2/KCNQ3 channel bands excised for mass spectrometric analysis. Other protein bands identified by Maldi TOF MS are KCNQ2/KCNQ3 oligomers (≈400 kDa) (1), HSP70 (67 kDa) (2), and actin (≈47 kDa) (3). (B) MS/MS spectrum of phosphopeptide representing residues 568-581 of KCNQ3. As shown, collision-induced dissociation spectrum contains doubly charged parent ion (, 798.89) and many b and y series fragment ions (b and y ions are, respectively, the N- or C-terminal fragments produced when the parent peptide is fragmented at peptide bonds). Ions marked exhibit masses of x_n -98 Da, where x_n is a b_n or y_n ion, reflecting the loss of H₃PO₄, a characteristic signature of phosphopeptides. (C) Schematic of fragment ion coverage in MS/MS spectrum from B. The presence of y_6, y_4, y₇^*, and y₈^* ions localizes the site of phosphorylation to S578 or T579. (D) Wild-type and S578/T579 mutant channels behave similarly. Recordings shown are representative of >15 oocytes from three different batches.

Fig. 3.

Analysis of a potential phosphorylation site in the S4-S5 loop. (A) A set of overlapping peptide mass matches implicates a conserved threonine in the S4-S5 loop as a site of phosphorylation. The observed and calculated monoisotopic (MH⁺) masses of the KCNQ subunit peptides, the error δ (in parts per million), the peptide sequence, and position in each subunit are indicated. (B) MS spectrum showing low intensity triply charged ion with m/z (mass normalized by charge) of 534.91 atomic mass units (amu), detected in tryptic digest of KCNQ2. This corresponds to monoisotopic mass of 1602.714 amu, a close match for the residues 208-219 of KCNQ2 if Thr-217 is phosphorylated. (C) The KCNQ S4-S5 loop sequence is highly conserved. All family members contain a threonine corresponding to the phosphorylated threonines, Thr-217 of KCNQ2 and Thr-246 of KCNQ3, and surrounding basic residues.

Fig. 4.

Physiological characterization of S4-S5 loop mutants. (A) Both mutant and wild-type channels generate large currents upon depolarization, with similar activation kinetics. (B) Current-voltage relation for KCNQ2_T217A/KCNQ3_T246A, KCNQ2_T217D/KCNQ3_T246D, wild-type KCNQ, and endogenous channels. KCNQ2_T217D/KCNQ3_T246D channels are nonfunctional. Results shown are representative of more than five batches of oocytes, with n ≥ 5 oocytes per condition per batch. Error bars represent SEM.

Fig. 5.

The loss of channel activity exhibited by S4-S5 loop aspartate mutants is not caused by reduced subunit protein expression or impaired surface trafficking. (A) Quantification of surface channels using a whole-oocyte chemiluminescence assay (see Materials and Methods). Mean surface expression levels are shown in absolute luminescence units (LU, Ai) or relative LU (RLU), where values have been normalized to KCNQ2/HA-KCNQ3 (Aii) or KCNQ2_T217D/HA-KCNQ3_T246D (Aiii) levels. Error bars represent standard error of the mean. (Ai) Surface expression levels of wild-type and mutant HA-KCNQ2 are comparable, regardless of whether KCNQ3 or KCNQ3_T246D subunits are coexpressed. (A ii and iii) Compared to the background level of surface expression of HA-KCNQ3 or HA-KCNQ3_T246D when expressed alone, KCNQ2 coexpression with HA-KCNQ3 greatly increased the surface luminescence (fold-increase = 60.7 ± 16.4; Aii) as did coexpression of KCNQ2_T217D with HA-KCNQ3_T246D (fold-increase = 59.7 ± 11.5) and coexpression of wild-type KCNQ2 with HA-KCNQ3_T246D (fold-increase = 70.6 ± 12.8). (B) The KCNQ3_T246D mutant forms functional heteromeric channels with KCNQ2. Although expression of KCNQ3 or KCNQ3_T246D (data not shown) alone did not result in current above endogenous levels, coexpression of KCNQ2 and wild-type KCNQ3 (n = 8) or KCNQ3_T246D (n = 5) resulted in larger average currents than expression of KCNQ2 alone (n = 5).