Similar to other tetrameric potassium channels BK_Ca channels are potently regulated by a variety of serine/threonine protein kinases (16), including cAMP-dependent protein kinase (PKA) (10, 17–20). The functional response of BK_Ca channels to PKA phosphorylation depends on the splice-variant α-subunit composition of the tetramer (10, 19). For example, PKA activates homotetramers of mammalian ZERO splice variants (10, 17, 19), whereas PKA inhibits homotetramers of STREX variants (10). This differential regulation of BK_Ca channels by PKA depends on functional consensus of PKA phosphorylation sites within the C terminus of the α-subunit. PKA activation of ZERO variants requires a functional conserved C-terminal PKA site (S899) (10, 17, 19), whereas PKA inhibition of STREX requires a functional PKA site (S4_STREX) within the STREX insert (10).

However, it is unknown how many α-subunits within the BK_Ca channel tetramer must be phosphorylated for functional regulation. An important step toward understanding how phosphorylation controls BK_Ca channel activity would be to determine the contribution of phosphorylating individual or multiple α-subunits within the tetramer. For example, do all four S899 sites within ZERO tetramers have to be phosphorylated for activation by PKA? Is phosphorylation of a single S4_STREX site in STREX tetramers sufficient for PKA inhibition? Moreover, as the BK_Ca channel tetramerization domain is conserved between splice variants (8), does heterotetramerization of splice-variant α-subunits, such as ZERO and STREX, result in channels with a null or “smoothed” response to PKA phosphorylation or do mechanisms exist to allow either the activation or inhibition phenotype in response to PKA phosphorylation to be retained?

In Vitro PKA Phosphorylation Assays. Immunoprecipitates (IPs) of -HA-tagged channels transiently expressed in HEK293 cells were subject to PKA-phosphorylation assay. In brief, IPs were incubated with 1 mM cold ATP, 10 μCi (1 Ci = 37 GBq) of [γ-³²P]ATP (Amersham Pharmacia), and 40 units of PKA catalytic subunit (PKAc; Promega) in 2 mM MgCl₂/4 mM Tris·HCl (pH 7.4) in a 30-μl reaction. Additionally, 100 nM protein phosphatase inhibitor, okadaic acid, was added to inhibit endogenous serine/threonine protein phosphatase activity. Negative control reactions were performed either in the absence of PKAc or by pretreating PKAc with 10 μM H-89 10 min before and during the reaction; kemptide was used as a positive control for PKAc activity (data not shown). Reactions were incubated (30°C for 10 min), then quenched by being placed on ice and the addition of an equal volume of 2× gel-loading buffer (lithium dodecyl sulfate) before analysis by autoradiography.

Previous site-directed mutagenesis studies have revealed a role for S899 (Figs. 1 A and 2A) in PKA regulation of mammalian ZERO homotetramers (10, 17, 19), and in vitro phosphorylation studies demonstrate that the equivalent site in Drosophila (S942) is phosphorylated by PKA (20). To confirm biochemically that S899 is required for phosphorylation of full-length murine ZERO BK_Ca channels in HEK293 cells, homotetramers of ZERO channels immunoprecipitated from HEK293 cells were subjected to in vitro PKA phosphorylation (Fig. 2B). Full-length ZERO channels were phosphorylated by PKA in vitro, and this phosphorylation was completely abolished in the S899A mutant.

To investigate the number of α-subunits within the tetramer that must be phosphorylated by PKA for activation of homotetrameric ZERO channels, depending on the conserved S899 PKA consensus motif, we examined channels formed from mixtures of ZERO α-subunits containing the TEA_o mutation (Y334V, which retains the functional S899 PKA consensus site) coexpressed with TEA-sensitive ZERO subunits in which the S899 site has been mutated to alanine (S899A, Fig. 2 A). The TEA_o sensitivity of S899A homotetramers was not significantly different from ZERO channels (not shown). Activation of endogenous PKA in isolated inside-out patches with cAMP resulted in a robust activation of homotetrameric Y334V channels to an extent similar to that of wild-type ZERO channels (mean activation, 62.6 ± 4.5%, n = 5 vs. 60.3 ± 7.4, n = 6), demonstrating that the TEA mutation per se has no effect on channel activation by PKA (Fig. 2 C and E). The effect of cAMP was completely blocked by preincubating patches with the PKA inhibitor, PKI_5–24 (10). In the presence of 0.45 μM PKI_5–24, the mean percentage change in channel activity after addition of cAMP to the intracellular face of the patch was -4.3 ± 6.7.8% (n = 6), confirming that the effect of cAMP is mediated by activation of endogenous PKA activity associated with the isolated patch. PKI_5–24 alone applied after patch excision (as for cAMP in Fig. 2C) had no significant effect on channel activity, suggesting that “basal” PKA activity in isolated patches is low under our recording conditions (the mean percentage change in activity was -2.7 ± 4.1%, n = 4). Homotetrameric S899A channels were unresponsive to endogenous PKA (Fig. 2E), supporting the biochemical evidence (Fig. 2B) that S899 is required for channel activation by PKA. In channels containing a mixture of Y334V and S899A α-subunits no significant channel activation (mean change in activity, 5.3 ± 9.8%, n = 11) was observed upon application of cAMP (Fig. 2 D and E). Because cAMP failed to activate any channel in which fewer than four intact S899 sites are present, it is most unlikely that the lack of channel activation is due to the channel already being phosphorylated at all of its remaining S899 sites before cAMP application. The lack of PKA activation was observed even in channels that expressed a single S899A α-subunit (the mean change in activity in four patches was 1.6 ± 9.2%, Fig. 2 D and E). Altogether, these findings suggest that channel activation requires the coordinate phosphorylation of the S899 site on each of the four α-subunits of the tetramer and thus obeys an all-or-nothing rule.

PKA Phosphorylation of a Single α-Subunit at a Splice-Insert-Specific Consensus PKA Site Is Sufficient for Inhibition of STREX Homotetramer Channels. We sought to investigate whether phosphorylation of all four subunits within the tetramer is crucial for channels assembled from other BK_Ca channel α-subunit splice variants. The STREX variant is potently inhibited by endogenous PKA associated with the channel complex, and this regulation depends on a conserved serine residue (S4_STREX) within the 59-aa STREX insert (Figs. 1 A and 3A and ref. 10). Full-length STREX channels were phosphorylated by PKA, and phosphorylation was abolished in the PKA consensus site double-mutant S4_STREXA-S899A (Fig. 3B). Mutation of either the S4_STREX or S899 sites alone in STREX channels resulted in a reduced, but not abolished, phosphate incorporation, suggesting that either site is available for PKA phosphorylation in STREX homotetramer channels (Fig. 3B).

To directly examine whether each STREX α-subunit in the tetramer must be phosphorylated at the STREX-insert-specific PKA site, S4_STREX, we generated STREX channels with (STREX-Y334V) or without (STREX) the TEA_o mutation and STREX channels in which the S4_STREX site is mutated to alanine (S4_STREXA, Fig. 3A). In all three constructs, the conserved C-terminal S899 site is intact, and phosphorylation assays indicate it is available for phosphorylation (Fig. 3B). Both STREX and STREX-Y334V homotetramers were inhibited to the same extent by activation of endogenous PKA by cAMP in isolated inside-out patches (mean inhibition, -56.5 ± 6.8%, n = 12, and -60.6 ± 10.3%, n = 5, respectively), demonstrating that the TEA_o mutation had no effect on the ability of endogenous PKA to inhibit the channel (Fig. 3 C and E). The effect of cAMP was completely prevented upon prior application of the PKA inhibitor peptide PKI_5–24 to the patch (the mean percentage change in channel activity was 1.8 ± 4.5%, n = 8). PKI_5–24 alone had no significant effect on channel activity (not shown). Furthermore, the TEA_o sensitivity of STREX and STREX-Y334V was not significantly different from ZERO or Y334V channels, respectively. (The IC₅₀ for TEA_o inhibition of STREX was 0.19 ± 0.09 mM, and for STREX-Y334V it was 83.5 ± 10.5 mM.) Homotetrameric S4_STREXA channels are activated under the same conditions depending on a functional S899 site (10) (Fig. 3E). However, channels formed from mixtures of STREX-Y334V and S4_STREXA α-subunits were inhibited by endogenous PKA to the same extent as homotetramers of STREX or STREX-Y334V subunits (Fig. 3 D and E). Similar inhibition of STREX-Y334V and S4_STREXA heterotetramers was observed irrespective of the presence of 1, 2, or 3 S4_STREXA α-subunits in the heterotetramer, suggesting that even a single S4_STREX site is sufficient to exert inhibition in channels containing four STREX inserts. [In all three patches containing channels with predicted 1:3 STREX-Y334V/S4_STREXA α-subunit stoichiometry, channel activity was significantly inhibited; mean inhibition was -67.5 ± 9.3%, not significantly different from STREX-Y334V homotetramers alone (Fig. 3 D and E); P > 0.05, Mann–Whitney U test).] This finding suggests that, under our recording conditions, the S4_STREX site is constitutively dephosphorylated before cAMP application. In addition, these data, in conjunction with the phosphorylation assays in Fig. 3B, suggest that a single S4_STREX site in a tetramer composed of four STREX α-subunits may override four functional S899 sites. Thus, in STREX homotetramers, phosphorylation of a single S4_STREX site is sufficient for channel inhibition and thus obeys a single-subunit rule.

ZERO/STREX Heterotetramers Are Activated by PKA Through Phosphorylation of All Four Subunits at S899. Because BK_Ca channel activation depends on the presence of a functional S899 site in each α-subunit, whereas channel inhibition only requires phosphorylation of a single S4_STREX site, we sought to address the functional consequence of channel regulation by PKA in heterotetramers formed from ZERO and STREX α-subunits in which multiple PKA phosphorylation sites are available (Fig. 4A). For example, does STREX act as a dominant α-subunit to confer inhibition in all heterotetramers, as might be predicted from the single-subunit rule in STREX homotetramers? Does regulation depend on a titration of α-subunit stoichiometry? Or, alternatively, is the activation by PKA through phosphorylation of all four α-subunits at the conserved S899 site the default phenotype of heterotetramers?

Coexpression of epitope-tagged ZERO and STREX α-subunits in HEK293 cells resulted in robust coimmunoprecipitation of the respective channel α-subunit, confirming biochemically that heterotetramers may exist in vivo (Fig. 4B). Furthermore, coexpression of Y334V α-subunits with STREX α-subunits (or ZERO with STREX-Y334V α-subunits) resulted in functional expression of heterotetramers as determined by single-channel TEA_o sensitivity (Fig. 4 C and D). However, in contrast to the dominant effect of PKA inhibition determined by the S4_STREX site in STREX homotetramers, in heterotetrameric channels containing ZERO and STREX α-subunits, channels were always activated by endogenous PKA (Fig. 4 C and E). For ZERO/STREX heterotetramers in which all S899 and all S4_STREX sites are available for phosphorylation, mean activation was 72.3 ± 10.0% (n = 26) (Fig. 4E). Activation was independent of whether the TEA_o mutation for determination of subunit stoichiometry was in the ZERO or STREX α-subunit (for ZERO + STREX-Y334V, activation was 64.6 ± 15.0%, n = 12; for Y334V + STREX, mean activation was 79.4 ± 15.5%, n = 14) or the number of respective TEA-mutant subunits in the tetramer (not shown). These heterotetrameric channels contain fewer than four α-subunits expressing the STREX insert (and, thus, fewer than four S4_STREX sites), but they contain four α-subunits each with a functional S899 site. Thus, to examine whether this dominant activation requires functional S899 sites in each of the four α-subunits, as would be predicted from the all-or-nothing rule of ZERO homotetramers, we coexpressed STREX-Y334V α-subunits with S899A α-subunits. Under these conditions (i.e., functional conserved S899 sites are only present in STREX subunits), no significant change of channel activity was observed (% change in activity was -1.7 ± 6.8%, n = 12; Fig. 4C). Furthermore, if intact S899 sites were in the ZERO but not STREX α-subunit of the tetramer, no significant effect of PKA was observed (Fig. 4E). In heterotetramers of ZERO α-subunits and STREX α-subunits that contain both the TEA_o mutation and the S899A mutation (STREX-Y334V-S899A α-subunits), no significant effect of endogenous PKA was observed (mean change in activity, 3.9 ± 6.3%, n = 8). These data are consistent with the hypothesis that the default mode of PKA regulation of ZERO/STREX heterotetramers is activation-dependent on phosphorylation of all four α-subunits at S899 by the all-or-nothing rule.

PKA activation of BK_Ca channels depends on all four α-subunits being phosphorylated at the conserved C-terminal PKA consensus site, S899. This four-subunit all-or-nothing rule applies for channels assembled as homotetramers of the ZERO α-subunit splice variant, which contains a single PKA consensus site (S899), and for heterotetrameric channels assembled from ZERO and STREX α-subunits.

In contrast, PKA-dependent inhibition of BK_Ca channel activity requires only a single α-subunit to be phosphorylated at a splice-insert-specific (STREX) PKA consensus motif (S4_STREX) and thus follows a single-subunit rule. STREX α-subunits contain two PKA consensus motifs: the conserved C-terminal S899 site and the STREX-insert-specific site, S4_STREX. In channels assembled as STREX homotetramers, phosphorylation of a single S4_STREX site can override the effect of phosphorylating the S899 sites within the STREX α-subunits. Although the single-subunit rule for PKA inhibition can apply to ZERO/STREX heterotetramers, inhibition mediated by PKA phosphorylation of a S4_STREX site is conditional upon all four S899 sites within the tetramer being dephosphorylated. The rules of phosphorylation regulation are distinct from those of BK_Ca channel tetramer regulation by intracellular free Ca²⁺ through the C-terminal “calcium bowl”; each α-subunit calcium bowl provides a stepwise contribution to the cooperative activation and apparent Ca²⁺ affinity of BK_Ca channel tetramers (23). These inter- and intra-subunit phosphorylation rules have important implications for the coordinated regulation of BK_Ca channels by reversible protein phosphorylation.

Because PKA-dependent activation follows the four-subunit all-or-nothing phosphorylation rule, but PKA-dependent inhibition follows the single-subunit rule, a functional consequence of our data is that PKA-dependent activation and inhibition may be subject to differential combinatorial control by protein phosphatases. For example, for PKA-dependent activation dephosphorylation of any one S899 site within the tetramer would prevent PKA activation and could be accomplished by distinct serine/threonine protein phosphatases targeting separate α-subunits, i.e., multiple phosphatases may converge at a single BK_Ca tetramer to regulate PKA activation. Conversely, antagonism of PKA inhibition would require all available S4_STREX PKA consensus sites to be dephosphorylated. Although BK_Ca channels are potently regulated by dephosphorylation by multiple protein phosphatases (18, 24, 25), how or if these enzymes are targeted to BK_Ca channel α-subunits is currently unknown.

Our data also suggest an additional level of conditional regulation by reversible protein phosphorylation (19, 25, 26). In ZERO/STREX heterotetramers, PKA inhibition is conditional upon the phosphorylation status of the S899 sites in the tetramer, and this effect can be exerted across α-subunits within the heterotetramer (i.e., the phosphorylated S899 site does not need to reside on the same subunit as the S4_STREX site). Although it is unknown whether differential, site-specific dephosphorylation/phosphorylation can occur in vivo, our data imply that cross talk between α-subunits within the tetramer is an important determinant of BK_Ca channel regulation by phosphorylation.

These data suggest that alternative pre-mRNA splicing and posttranslational PKA phosphorylation of BK_Ca channel α-subunits act in concert to enable two distinct BK_Ca behaviors to be expressed from tetramers assembled from distinct BK_Ca channel α-subunit splice variants. The default action of BK_Ca channels is to facilitate cell repolarization, and this function can be enhanced through PKA activation of the tetramer by means of the four-subunit all-or-nothing rule (ZERO or default heterotetramer phenotype). In contrast, the single-subunit PKA-inhibition rule allows a subset of BK_Ca channels to facilitate depolarization upon PKA phosphorylation (STREX homotetramers or ZERO/STREX heterotetramers in which all S899 sites are dephosphorylated). Considerable phenotypic diversity in the regulation of native BK_Ca channels by PKA-dependent phosphorylation has been reported. In many neurons and smooth muscle cells, PKA activates BK_Ca channels (13, 16, 27–29), whereas in endocrine cells of the anterior pituitary, BK_Ca channels are inhibited (6, 24, 30). Whether such physiological diversity results from homo- and/or heterotetramer assembly of α-subunit splice variants in native tissues and/or involves accessory subunits remains to be determined.