Protein-protein interaction between cPLA2 and splice variants of alpha-subunit of BK channels

Abstract

Altering the splice variant composition of large-conductance Ca(2+)-activated potassium (BK) channels can alter their activity and apparent sensitivity to Ca(2+) and other regulators of activity. We hypothesized that differences in the responsiveness to arachidonic acid of GH3 and GH4 cells was due to a difference in two splice variants, one present in GH3 cells and the other in GH4 cells. The sequences of the two splice variants differ from one another in several ways, but the largest difference is the presence or absence of 27 amino acids in the COOH terminus of the BK alpha-subunit. Open probability of the variant containing the 27 amino acids is significantly increased by arachidonic acid, while the variant lacking the 27 amino acids is insensitive to arachidonic acid. In addition, sensitivity of BK channels to arachidonic acid depends on cytosolic phospholipase A(2) (cPLA(2)). Here we used the Mammalian Matchmaker two-hybrid assay and two BK alpha-subunit constructs with [rSlo(27)] and without [rSlo(0)] the 27-amino acid motif to determine whether cPLA(2) associates with one construct [rSlo(27)] and not the other. We hypothesized that differential association of cPLA(2) might explain the differing responsiveness of the two constructs and GH3 and GH4 cells to arachidonic acid. We found that cPLA(2) is strongly associated with the COOH terminus of rSlo(27) and only very weakly associated with rSlo(0). We also found that arachidonic acid has a lower affinity for rSlo(0) than for rSlo(27). We conclude that the lack of response of BK channels in GH4 cells to arachidonic acid can be explained, in part, by the poor binding of cPLA(2) to the COOH terminus of the rSlo(0) alpha-subunit, which is very similar to the splice variant found in the arachidonic acid-insensitive GH4 cells.

Fig. 1.

Arachidonic acid differentially affects splice variants in the α-subunit of large-conductance Ca²⁺-activated potassium (BK) channels from GH3 and GH4 cells. Experiments were conducted with inside-out patches depolarized to +20 mV and exposed to 0.1 μM Ca²⁺ Open probability (P_o) values for individual patches are plotted along with means ± SD, and significant changes in P_o are indicated by an asterisk. One micromolar arachidonic acid caused P_o to increase from 0.2 ± 0.05 to 0.57 ± 0.13 (P < 0.0001) in GH3 cells (n = 8) (A), while P_o in GH4 cells was unaffected by arachidonic acid (0.16 ± 0.09 vs. 0.16 ± 0.10) (n = 7) (B). C and D: representative single-channel records from BK channels in GH3 (C) and GH4 (D) cells. The “o” and “c” denote the open and closed state of the channel(s), respectively.

Fig. 2.

Quantitative PCR (qPCR) reveals a large difference in the relative amounts of 2 different splice variants in GH3 and GH4 cells. Using primers that were based on the clones we had isolated to select different splice variants, we amplified 100- to 200-base regions by qPCR and examined the amount of the 2 splice variants that differed between GH3 and GH4 cells. Relative amounts of the 2 splice variants in GH3 (A) and GH4 (B) cells are shown (note logarithmic scale). In GH3 cells there is 86 ± 12 times (mean ± SD; n = 3) as much of a rat splice variant, rnBKα1, as that of a splice variant previously only observed in mouse, muBKα1. In contrast, in GH4 cells there is 549 ± 80 times (mean ± SD; n = 3) as much muBKα1 as rnBKα1. The larger difference in the GH4 cells may reflect the fact that they are a clonal line derived from GH3 cells.

Fig. 3.

Alignment of cytosolic domains of α-subunit splice variants from GH3 cells and GH4 cells. BK channels in GH3 cells are sensitive to cytosolic arachidonic acid, while BK channels from GH4 cells are not. We hypothesized that the difference could arise because the 2 clonal lines express different splice variants of the α- or β-subunits. Using PCR with degenerate primers as described in materials and methods, we isolated β-subunits and the cytosolic domains of α-subunits. BK β-subunits were identical in both types of cells. Examination of the α-subunits revealed 2 different splice variants from the 2 cell types. The α-subunit of GH3 cells is identical to a splice variant previously identified in rat brain and rat adrenal chromaffin cells (accession nos.: nucleotide AF135265, protein AAD34786). The GH4 α splice variant has not been previously described in rat but is homologous to a mouse variant (muBKα; accession nos.: nucleotide L16912, protein AAA39746).

Fig. 4.

GH3 and GH4 cells contain the β₁- but not the β₂-subunit of BK channels. A: 1.5% agarose gel containing the PCR products obtained from total RNA from GH3 and GH4 cells and PCR primers specific for rat β₁- and β₂-subunits of BK channels as described in materials and methods. Gel shows the presence of the β₁-subunit in both GH3 and GH4 cells. It is also apparent that the β₂-subunit is not present in either GH3 or GH4 cells. HEK cells were used as a positive control for the β₁-subunit, and mouse brain was used as a positive control for the β₂-subunit. B: GH3 and GH4 cell lysates were probed with a polyclonal antibody to the β₁-subunit of BK channels. First 2 lanes show the presence of the β₁-subunit in both cell lines. Second 2 lanes are identical to first 2 lanes except that the lysates were probed with the polyclonal antibody to the β₁-subunit in the presence of the antigenic peptide to show the successful competition of the β₁-subunit band. C: GH3 and GH4 cell lysates (1st 2 lanes) and rat kidney lysate (3rd lane) were probed with a polyclonal antibody to the β₂-subunit of BK channels. First and second lanes show the absence of the β₂-subunit in both cell lines. On the other hand, 3rd lane shows that the antibody is functional since the β₂-subunit was readily visualized in rat kidney. Fourth lane is identical to 3rd lane and 5th and 6th lanes are identical to 1st and 2nd lanes except that the lysates were probed with the polyclonal antibody to the β₂-subunit in the presence of the corresponding antigenic peptide, showing the successful competition of the β₂-subunit band.

Fig. 5.

Primary structure and proposed membrane topology of BK channel splice variants. BK channel α-subunits contain 7 putative transmembrane domains, S0–S6, a conserved K⁺-selective pore region between S5 and S6, and a long COOH-terminal cytosolic tail with 4 additional hydrophobic segments, S7–S10. The splice site that is different in GH3 and GH4 cells near the Ca²⁺ bowl (blue) is highlighted with a red asterisk. Primary sequence of the COOH terminus is shown at bottom. Sequence of the subunit prior to S9 (pink) is invariant in both constructs [rSlo(27) and rSlo(0)]. The 27 amino acids that are present in GH3 cells and rSlo(27) but missing in GH4 cells and rSlo(0) are shown in red, and the Ca²⁺ bowl is shown in blue.

Fig. 6.

Arachidonic acid differentially affects splice variants rSlo(27) and rSlo(0) isolated from rat brain. P_o values for individual patches are plotted along with means ± SD, and significant changes in P_o are indicated by an asterisk. When the α-subunit [rSlo(27)], similar to the splice variant from GH3 cells, was transfected into HEK-293 cells, arachidonic acid treatment significantly increased P_o from 0.07 ± 0.03 to 0.19 ± 0.06 (P < 0.005) (n = 6) (A). On the other hand, when the α-subunit [rSlo(0)], similar to the splice variant from GH4 cells, was transfected into HEK-293 cells, arachidonic acid treatment had no significant effect on P_o (0.05 ± 0.03 to 0.05 ± 0.03) (n = 8) (B). C and D: representative single-channel records from BK channels in HEK-293 cells expressing rSlo(27) (C) and rSlo(0) (D). The “o” and “c” denote the open and closed state of the channel(s), respectively. These data suggest that the effect of arachidonic acid requires the presence of the 27-amino acid sequence contained in rSlo(27), since when it is absent, either in rSlo(0) or in GH4 cells, arachidonic acid has no effect on channel P_o.

Fig. 7.

Coexpression of either cytosolic phospholipase A₂ (cPLA₂) or the β₁-subunit with muBKα results in an increase in Ca²⁺ sensitivity. Groups of 6–8 cells from each treatment were studied at each Ca²⁺ concentration. For these experiments, inside-out patches depolarized to +20 mV were used. Compared with wild-type GH3 cells, the Ca²⁺ sensitivity of BK channels derived from stable HEK cell line expressing only the muBKα was greatly diminished. Coexpression with the β₁-subunit, cPLA₂, or both the β₁-subunit and cPLA₂ resulted in a sequential marked increase in Ca²⁺ responsiveness of the channels.

Fig. 8.

cPLA₂ is equally expressed in both GH3 and GH4 cells but associates more strongly with the α-subunit of BK channels from GH3 cells. A: cPLA₂ activities for GH3 and GH4 cells were not different [67.3 ± 7.4 and 62.4 ± 6.9 pmol palmitic acid·min⁻¹·mg protein⁻¹, respectively, by assay of PLA₂ function in whole cell lysates from GH3 and GH4 cells as described previously (21, 22)]. B: typical Western blot in which whole cell lysates from both GH3 and GH4 cells were probed with a commercially available antibody (Millipore) to cPLA₂. This blot shows that cPLA₂ is expressed to the same extent in both cell lines (as expected from A). C: we immunoprecipitated BK channel α-subunits, resolved the immunoprecipitate on SDS gels, and probed with a commercially available antibody (Millipore) to cPLA₂. cPLA₂ was associated with BK channels in both GH3 and GH4 cells but was much more strongly associated in GH3 cells. These data suggest that BK channel α-subunits in both GH3 and GH4 cells are associated with cPLA₂, albeit to different extents.

Fig. 9.

Protein-protein interactions between cPLA₂ and splice variants of α-BK channels. A: lane 1 shows result of coexpressing Matchmaker binding domain + full-length cPLA₂ and Matchmaker activation domain + NH₂-terminal region that rSlo(27) and rSlo(0) have in common. Lane 2 shows result of expressing Matchmaker-cPLA₂ and Matchmaker activation domain + COOH-terminal region of rSlo(0). Lane 3 shows result of expressing Matchmaker-cPLA₂ and Matchmaker activation domain + COOH-terminal region of rSlo(27). Lane 4 is a negative control resulting from expression of unaltered Matchmaker binding domain and unaltered Matchmaker activation domain, lane 5 is a positive control supplied in the Matchmaker kit, and lane 6 is the unreacted chloramphenicol acetyltransferase (CAT) standard. This blot shows that cPLA₂ binds weakly to the NH₂ terminus of the α-subunit [which rSlo(27) and rSlo(0) have in common] and binds strongly to the COOH terminus of rSlo(27) but not to the COOH terminus of rSlo(0). B: intensities of expressed CAT resulting from physical association between cPLA₂ and the COOH termini of rSlo(27) and rSlo(0) were compared with intensities arising from physical association between cPLA₂ and NH₂ terminus of rSlo(27) [NH₂ terminus is identical for rSlo(27) and rSlo(0)]. All data are presented as means ± SD. In all experiments, the association was significantly greater for the COOH terminus of rSlo(27) compared with the NH₂ terminus (*P < 0.04) and significantly weaker for the COOH terminus of rSlo(0) compared with the NH₂ terminus (^#P < 0.02). C, top: in the absence of cPLA₂, BK-β interacts equally well with the NH₂ terminus of rSlo, the COOH terminus of rSlo(0), or the COOH terminus of rSlo(27). Lane 1 contains results of expressing Matchmaker binding domain plasmid + BK-β and Matchmaker activation domain + NH₂-terminal region that rSlo(27) and rSlo(0) have in common, lane 2 results of expressing Matchmaker BK-β and Matchmaker rSlo(0), and lane 3 results of expressing Matchmaker BK-β and Matchmaker rSlo(27). Since all lanes show equivalent amounts of CAT, activity binding interaction is the same for all constructs. Bottom: all lanes are identical to those at top except that cells were also transfected with cPLA₂ in PCDNA3. Top row shows that β-subunit can physically associate with both the NH₂ and either of the splice variant COOH termini of the α-subunit. However, when cPLA₂ is present, we know that cPLA₂ preferentially binds to the COOH terminus of rSlo(27), and this figure shows that when cPLA₂ binds to rSlo(27) it also prevents the β-subunit from strongly associating with the COOH terminus of the BK α-subunit to which cPLA₂ binds [rSlo(27)].

Fig. 10.

rSlo(27) has a higher affinity for arachidonic acid than rSlo(0). We bound anti-BK-α to A/G beads and then immunoprecipitated rSlo(0) or rSlo(27) from transfected Chinese hamster ovary cells. We used the immobilized proteins as targets for [³H]arachidonic acid binding. Since under these conditions there might be significant nonspecific binding of arachidonic acid to the A/G beads and antibody + protein complexes, we also prepared beads with anti-GAPDH antibody and GAPDH protein. The rate of release of arachidonic acid from the 3 different sets of beads was determined. The half-lives of arachidonic acid binding for binding of arachidonic acid to rSlo(27), rSlo(0), and GAPDH (control) were 0.89 ± 0.15, 0.55 ± 0.09, and 0.52 ± 0.11 h, respectively (means ± SE, n = 5) (see inset for example of typical rate of arachidonic acid loss). It should be noted that the rates of arachidonic acid loss were biexponential. All of the initial rates of loss (1st exponential) were identical and presumably represent nonspecific binding of arachidonic acid to the beads. Only the slower (2nd exponential) rates of loss were significantly different. The unbinding half-life for rSlo(27) was significantly longer (P < 0.05) than either rSlo(0) or GAPDH, while the unbinding half-life for rSlo(0) was not significantly different from the GAPDH control.