From the Membrane Biology Group & Medical Research
Council Membrane and Adapter Protein Co-operative
Group, University of Edinburgh Medical School, Teviot Place,
Edinburgh EH8 9AG, United Kingdom and the ¶ Department of
Neuroscience, University of Pennsylvania School of Medicine,
Philadelphia, Pennsylvania 19104-6074
Received for publication, October 19, 2000, and in revised form, December 22, 2000
![]() |
ABSTRACT |
---|
![]() ![]() ![]() ![]() ![]() ![]() |
---|
Alternative exon splicing and reversible protein
phosphorylation of large conductance calcium-activated potassium (BK)
channels represent fundamental control mechanisms for the regulation of cellular excitability. BK channels are encoded by a single gene that
undergoes extensive, hormonally regulated exon splicing. In native
tissues BK channels display considerable diversity and plasticity in
their regulation by cAMP-dependent protein kinase (PKA).
Differential regulation of alternatively spliced BK channels by PKA may
provide a molecular basis for the diversity and plasticity of BK
channel sensitivities to PKA. Here we demonstrate that PKA activates BK
channels lacking splice inserts (ZERO) but inhibits channels expressing
a 59-amino acid exon at splice site 2 (STREX-1). Channel activation is
dependent upon a conserved C-terminal PKA consensus motif (S869),
whereas inhibition is mediated via a STREX-1 exon-specific PKA
consensus site. Thus, alternative splicing acts as a molecular switch
to determine the sensitivity of potassium channels to protein phosphorylation.
Large conductance calcium- and voltage-activated potassium
(BK)1 channels link
intracellular chemical signaling events with the electrical properties
of excitable cells in the endocrine, nervous, and vascular systems
(1-3). BK channels are further potently modulated by reversible
protein phosphorylation (4-7). In native tissues BK channels display
considerable diversity and plasticity in their regulation by reversible
protein phosphorylation. For example, cAMP-dependent
protein kinase (PKA) phosphorylation activates BK channels in smooth
muscle cells and many neurones but inhibits channel activity in
endocrine cells of the anterior pituitary (5, 7-11). Furthermore, the
direction of channel regulation by PKA can be modified during
challenges to homeostasis (9-11).
The pore-forming To address whether BK channel alternative splice variants are
differentially regulated by PKA-mediated protein phosphorylation, we
have examined the regulation of three mouse (mslo) BK channel variants
(20-22) expressed in HEK293 cells. BK channels are regulated by
multiple protein kinase signaling pathways (5, 19, 23, 24). We have
thus assayed the functional regulation of BK channel splice variants by
directly activating PKA that remains closely associated with the
channels in excised inside-out patches.
Molecular and Cell Biology--
cDNAs encoding mouse BK
channel variants were subcloned into the mammalian expression vector
pcDNA3 or pcDNA3.1+ (Invitrogen BV, Leek, The Netherlands) and
transfected into HEK293 cells using lipofectAMINE as previously
described (22). The cloning and subcloning of the three splice variants
have been described previously (ZERO and STREX-1 (22); and ZERO1/4 Electrophysiology--
All experiments were performed in the
inside-out configuration of the patch clamp technique at room
temperature (20-24 °C) using physiological potassium gradients
essentially as described previously (22). The pipette solution
(extracellular) contained (in mM): 140 NaCl, 5 KCl, 0.1 CaCl2, 2 MgCl2, 20 glucose, 10 HEPES, pH 7.4. The bath solution (intracellular) contained (in mM): 140 KCl, 5 NaCl, 2 MgCl2, 1 or 5 BAPTA, 30 glucose, 10 HEPES, 1 mM ATP, pH 7.3, with free calcium
[Ca2+]i buffered to 0.2 µM, unless
indicated otherwise. Data acquisition and voltage protocols were
controlled by an Axopatch 200 A or B amplifier and pCLAMP6 software
(Axon Instruments Inc., Foster City, CA). All recordings were sampled
at 10 kHz and filtered at 2 kHz. Following patch excision channel
activity was allowed to stabilize for at least 10 min (typically 10-15
min after excision) before application of cAMP or other reagents to the
intracellular face of patches by gravity-driven perfusion or direct
application to the bath. In all experiments 1 mM cAMP was
used to activate endogenous PKA, similar results were also observed
using 0.1 mM cAMP (not shown). Patches were held at 0 mV,
and channel activity was determined during voltage steps to +40 mV. To
determine mean percent change in channel activity after a treatment, in
patches with low to moderate levels of channel expression, mean
NPo (number of functional channels × open
probability of channel) was averaged from 60 s of recording at +40
mV immediately before and 10 min after the respective drug treatment.
Mean change in activity was expressed as a percentage of the
pretreatment control ± S.E. with analysis undertaken using
pCLAMP6 and IGOR Pro3.1 (Wavemetrics). In the respective figure legends
and text a positive percentage change in activity reflects activation,
whereas a negative percentage change reflects channel inhibition.
Statistical significance was defined at p < 0.05 using
nonparametric Kruskal-Wallis or Mann-Whiney U test as appropriate.
Five major alternative splice sites and a conserved weak PKA
consensus motif (RQPS869) have been identified in
the C-terminal region of mammalian BK channels (14, 20-22, 26, 27)
(Fig. 1a).
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES
-subunits of BK channels are derived from a single
gene (Slo) that undergoes extensive alternative splicing to
produce channels with distinct phenotypes (12-15). Importantly, alternative splicing of the
-subunit is dynamically regulated in
adults, for example during stress or pregnancy (15, 16). Thus the
diversity and plasticity of responses to PKA-dependent protein phosphorylation observed between BK channels in native tissues
may result either from differential modulation of alternatively spliced
BK channel
-subunits (12-15) or through their interaction with
different signaling complexes and
-subunits (17-19).
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES
C,
also referred to as mB2 (21, 25)). ZERO lacks inserts at mammalian
splice sites 1-5 and STREX-1 channels are identical except for a
59-amino acid cysteine-rich insert at mammalian splice site 2. ZERO1/4
C contains inserts at splice sites 1 and 4 and has modified N
and C termini compared with ZERO constructs. Site-directed mutagenesis
was performed using QuikChange (Stratagene) and verified by DNA
sequencing. Site-directed mutants displayed no significant change in
single channel kinetics or conductance, although S869A channels
displayed a significant shift in V0.5 (10-12 mV
in the absence of intracellular ATP) compared with wild type ZERO
channels. GST fusion proteins of the STREX-1 splice insert were
constructed in pGEX-5x-1 by polymerase chain reaction and recombinant
protein purified and assayed in a PKA phosphorylation reaction under
standard conditions. Coimmunoprecipitation of channel variants with the
catalytic subunit of PKA (PKAc) was performed essentially as described
previously (24).
RESULTS AND DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES
View larger version (20K):
[in a new window]
Fig. 1.
Activation of endogenous PKA differentially
regulates BK channel splice variants. a, diagrammatic
representation (not to scale) of BK channel -subunit domain
structure. Numbered triangles indicate the five alternative
mammalian splice sites in the C-terminal tail of BK channels downstream
of the transmembrane domains (black boxes), including the
pore region and voltage sensor common to other voltage gated potassium
channels. ZERO represents channels lacking inserts at any splice site,
STREX-1 represents channels identical to ZERO except for an additional
59-amino acid exon at splice site 2 (stippled box). Serine
residue 869 (S869) is conserved in all mammalian BK channel
splice variants. The indicated serine residue in the STREX-1 exon
(S4STREX) is conserved in all mammalian STREX-1
splice variants. b, representative single channel recordings
and diary plots of mean single channel open probability
(Po) of STREX-1 and ZERO channels in inside-out
patches from HEK293 cells before (Control) and 10 min after
application of 1 mM cAMP to the intracellular face of the
patch. All currents were recorded in physiological potassium gradients
at +40 mV in the presence of 0.2 µM intracellular free
calcium and 2 mM MgCl2. c, summary
of the mean percent change in activity for each splice variant ± S.E. (shown on the left) after activation of endogenous PKA
with 1 mM cAMP. cAMP was applied in the presence
(+cAMP) and absence (
ATP) of 1 mM
ATP or after pretreatment with 0.45 µM
concentration of the specific PKA inhibitor PKI5-24
in the presence of ATP. A positive percent change in activity indicates
activation, a negative percent change indicates inhibition.
Application of cAMP to the intracellular face of excised inside-out
patches containing channels that lack inserts at any splice site (ZERO
construct channels (Fig. 1a) (20, 22)) resulted in
significant (p < 0.01) activation of mean channel
activity in 10 out of 16 patches (Fig. 1, b and
c). The mean percentage activation in response to cAMP was
128.6 ± 45.2%. cAMP-mediated inhibition of channel activity was
never observed in patches containing ZERO channels. This activation was
dependent upon closely associated cAMP-dependent protein
kinase activity as cAMP had no effect in any patch in the absence of
ATP (mean percent change in activity was: 2.9 ± 15.6%,
n = 4) or in the presence of the specific PKA inhibitor
peptide PKI5-24 (9.3 ± 8.2%, n = 6).
In contrast, under identical recording conditions, application of cAMP
to the intracellular face of patches containing channels expressing the
59-amino acid cysteine-rich exon at splice site 2 (STREX-1, Fig.
1a (22, 26)) resulted in significant inhibition of channel
activity in eight out of nine patches (Fig. 1, b and c; mean percent change in activity: 56.5 ± 6.8%).
cAMP had no significant effect in the absence of ATP (18.6 ± 11.9%, n = 4) or in the presence of
PKI5-24 (7.6 ± 11.8%, n = 6). The STREX-1 exon is widely expressed in neuroendocrine tissues and confers
enhanced apparent calcium sensitivity on native BK channels compared
with the ZERO variant that is highly expressed in brain (22, 26). The
inhibitory effect of cAMP was not a result of the enhanced single
channel Po observed in STREX-1 compared with ZERO channels under identical recording conditions as cAMP also inhibited STREX-1 channels when recording conditions were adjusted to
give a Po similar to that of ZERO (not shown).
The data suggest that an endogenous cAMP-dependent protein
kinase A activity (PKA) remains closely associated with STREX-1 or ZERO
channels in excised patches from HEK293 cells. Indeed, the catalytic
subunit of PKA (PKAc) binds directly to the C-terminal tail of dSlo
(24) and coimmunoprecipitates with both mouse ZERO and STREX-1 channel
splice variants expressed in HEK293 cells (Fig.
2a). This interaction is
specific as PKAc did not coimmunoprecipitate with KCNQ3 potassium
channel subunits under the same
conditions.2 It is
interesting that, in the case of one splice variant of dSlo, PKA
regulatory subunit competes with the channel for PKAc binding, and
hence the holoenzyme does not bind to the
channel.3
|
In contrast to the robust regulation of ZERO or STREX-1 channels, cAMP
had no significant effect on channels containing inserts at splice
sites 1 and 4 with different N and C termini (Fig. 1c; ZERO1/4C variant (21), mean percent change in activity was 7.8 ± 5.2%, n = 8). As PKAc also coimmunoprecipitates
with the ZERO1/4
C variant, the lack of regulation by cAMP is
unlikely to be a result of the inability of PKAc to interact with this variant. Thus, the functional effect of PKA-mediated phosphorylation on
BK channel activity is determined by alternative exon splicing of the
BK channel pore-forming subunit.
The differential regulation of the ZERO and STREX-1 splice variants may
result from (i) the STREX-1 exon modifying the functional effect of PKA
protein phosphorylation at conserved sites within the -subunit or,
alternatively, (ii) the STREX-1 exon itself introducing additional PKA
consensus motifs. To address these issues we undertook site-directed
mutagenesis of putative conserved C-terminal and STREX-1
insert-specific PKA consensus motifs (Fig. 1a). Although
mammalian BK channel
-subunits do not contain strong PKA consensus
sites, sequence analysis reveals more than 10 putative weaker PKA sites
in the C terminus (20, 27). The conserved mammalian C-terminal serine
residue at position 869 (Ser869) has been reported
as the target for PKA-mediated activation of the hSlo ZERO variant
expressed in Xenopus oocytes (27). Furthermore, the
equivalent site in dSlo (Ser942) is phosphorylated by PKA
(24).
Mutation of serine 869 to alanine (S869A) completely abolished
cAMP-mediated activation of the ZERO variant (S869A construct, mean
percent change activity was 3.2 ± 1.5%, n = 8)
but had no significant effect on cAMP-mediated inhibition of the
site-directed STREX-1 variant (STREX-S869A, mean percent change was
58.1 ± 12.1%, n = 11) in excised patches (Fig.
3, a and b).
Importantly, the inhibitory effect of cAMP on STREX-S869A channels is
dependent upon endogenous PKA activity as cAMP had no effect on the
activity of STREX-S869A channels in the absence of ATP
(n = 5, not shown) or in the presence of
PKI5-24 (n = 6, Fig. 3b). Thus
the effect of cAMP in the STREX-S869A variant is specific to activation of endogenous cAMP-dependent protein kinase and not a
result of nonspecific actions or activation of protein kinase G under
the recording conditions used. These data suggest that
Ser869 is essential for PKA-mediated activation of the ZERO
variant but is not required for PKA-mediated inhibition of STREX-1.
This implies that the presence of the STREX-1 exon per se
does not modify the functional effect of PKA-mediated phosphorylation
of Ser869. The presence of the STREX-1 exon may expose or
introduce additional protein kinase A consensus sites within the
-subunit.
|
To address whether the STREX-1 exon is itself a target for PKA phosphorylation, we analyzed whether a STREX-1 insert GST fusion protein is a substrate for PKA-dependent phosphorylation in vitro. Sequence analysis of mammalian STREX-1 exons reveals putative weak PKA consensus sites with serine residue 4 (S4STREX, Fig. 1a) conserved in all mammalian STREX-1 inserts identified to date (22, 26). PKA phosphorylates a GST-STREX-1 fusion protein in vitro (Fig. 2b), and this phosphorylation is completely eliminated when S4STREX is mutated to alanine (GST-S4STREXA; Fig. 2b). We thus addressed whether S4STREX plays a functional role in mediating PKA inhibition of STREX-1 using the full-length site-directed S4STREXA mutant expressed in HEK293 cells. In contrast to the robust cAMP-mediated inhibition of channel activity observed with the STREX-1 or STREX-S869A channels cAMP-mediated protein phosphorylation resulted in significant stimulation (mean percent increase in activity was 45.8 ± 5.9%, n = 7) of the S4STREXA site-directed mutant (Fig. 3, a and b). This suggests that S4STREX plays a dominant inhibitory role in the regulation of BK channels expressing the STREX-1 exon as the activation of S4STREXA channels dependent upon the conserved C-terminal Ser869 site is overridden in the presence of a functional S4STREX site. cAMP-dependent protein kinase-mediated regulation was abolished when both the conserved C-terminal Ser869 and exon-specific S4STREX serine residues were mutated to alanine (double mutant, S4STREXA-S869A, percent change was 6.5 ± 6.8%, n = 8, Fig. 3b).
Several mechanisms contribute to the diversity and plasticity of the properties and regulation of potassium ion channels, including multiple gene families, heteromultimerization of subunits, association with regulatory or accessory subunits, and post-translational modifications such as reversible protein phosphorylation. As BK channels are derived from a single gene alternative, exon splicing plays a major role in determining the functional diversity of BK channels.
Our data reveal that alternative exon splicing of BK channels also provides a fundamental context-sensitive mechanism for switching BK channel sensitivity to protein phosphorylation that has significant implications for both the long and short term control of cellular excitability. As BK channel splice variant expression can be dynamically regulated, for example during stress or pregnancy (15, 16), the functional consequence of PKA-mediated regulation of BK channel activity may be switched in the long term by altering the splice variant expressed (e.g. from ZERO to STREX-1). In contrast, short term switching of BK channel sensitivity to PKA could be achieved by specific dephosphorylation of residues important for PKA regulation within a splice insert. For example, in STREX-1 containing channels that are normally inhibited by PKA, dephosphorylation of the PKA site specifically within the STREX-1 insert would allow PKA-dependent activation of the channel through conserved sites in the C terminus (as for S4STREXA construct). Further diversity and plasticity may be achieved through splice variant heteromultimerization and/or association with regulatory subunits (17-19).
In conclusion, alternative exon splicing acts as a powerful long and
short term molecular switch to define the sensitivity of BK channels to
protein phosphorylation and provides a fundamental mechanism for
specifying the diversity, plasticity, and functional role of such
modular coincidence detectors of excitable cells.
![]() |
ACKNOWLEDGEMENTS |
---|
We thank Hannah Florance and Janet Philp for expert technical assistance and other members of the Membrane Biology Group and Medical Research Council Membrane and Adapter Protein Co-operative Group for useful discussion. Mouse brain BK channel clones used to construct expression plasmids were generous gifts from L. Salkoff and L. Pallanck, respectively.
![]() |
FOOTNOTES |
---|
* This work was supported by grants from The Wellcome Trust (046787) and the Biotechnology and Biological Sciences Research Council (15/C11774) (to M. J. S.) and a grant (to I. B. L.) from the National Institutes of Health.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
§ University of Edinburgh Faculty of Medicine postgraduate Crichton Scholar.
Current addresses: Dept. of Physiology and Biophysics, Mt.
Sinai Medical Center, New York, NY 10029.
** Current addresses: Organon Laboratories Ltd., Newhouse, Lanarkshire ML1 5SH, United Kingdom.
To whom all correspondence should be addressed: Membrane
Biology Group, University of Edinburgh Medical School, Teviot Place, Edinburgh EH8 9AG, United Kingdom. Tel.: 44-131-650-3253; Fax: 44-131-650-6527; E-mail: mike.shipston@ed.ac.uk.
Published, JBC Papers in Press, January 19, 2001, DOI 10.1074/jbc.C000741200
2 H. Wen and I. B. Levitan, unpublished data.
3 Y. Zhou and I. B. Levitan, unpublished data.
![]() |
ABBREVIATIONS |
---|
The abbreviations used are: BK, large conductance calcium-activated potassium; PKA, cAMP-dependent protein kinase; GST, glutathione S-transferase; BAPTA, 1,2-bis(aminophenoxy)ethane-N,N,N',N'-tetraacetic acid.
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() |
---|
1. | Brenner, R., Perez, G. J., Bonev, A. D., Eckman, D. M., Kosek, J. C., Wiler, S. W., Patterson, A. J., Nelson, M. T., and Aldrich, R. (2000) Nature 407, 870-876[CrossRef][Medline] [Order article via Infotrieve] |
2. | Robitaille, R., and Charlton, M. P. (1992) J. Neurosci. 12, 297-305[Abstract] |
3. | Vergara, C., Latorre, R., Marrion, N., and Adelman, J. (1998) Curr. Opin. Neurobiol. 8, 321-329[CrossRef][Medline] [Order article via Infotrieve] |
4. | Chung, S., Reinhart, P., Martin, B., Brautigan, D., and Levitan, I. (1991) Science 253, 560-562[Medline] [Order article via Infotrieve] |
5. | Levitan, I. B. (1994) Annu. Rev. Physiol. 56, 193-212[CrossRef][Medline] [Order article via Infotrieve] |
6. | Reinhart, P., Chung, S., Martin, B., Brautigan, D., and Levitan, I. (1991) J. Neurosci. 11, 1627-1635[Abstract] |
7. | White, R. E., Schonbrunn, A., and Armstrong, D. L. (1991) Nature 351, 570-573[CrossRef][Medline] [Order article via Infotrieve] |
8. | Lee, K., Rowe, I., and Ashford, M. (1995) J. Physiol. (Lond.) 488, 319-337[Abstract] |
9. |
Perez, G.,
and Toro, L.
(1994)
Am. J. Physiol.
266,
C1459-C1463 |
10. |
Shipston, M.,
Kelly, J.,
and Antoni, F.
(1996)
J. Biol. Chem.
271,
9197-9200 |
11. |
Zhou, X.-B.,
Wang, G.-X.,
Huneke, B.,
Wieland, T.,
and Korth, M.
(2000)
J. Physiol. (Lond.)
524,
339-352 |
12. | Adelman, J., Shen, K., Kavanaugh, M., Warren, R., Wu, Y., Lagrutta, A., Bond, C., and North, R. (1992) Neuron 9, 209-216[Medline] [Order article via Infotrieve] |
13. |
Lagrutta, A.,
Shen, K.-Z.,
North, R. A.,
and Adelman, J. P.
(1994)
J. Biol. Chem.
269,
20347-20351 |
14. | Tseng-Crank, J., Foster, C., Krause, J., Mertz, R., Godinot, N., DiChiara, T., and Reinhart, P. (1994) Neuron 13, 1315-1330[Medline] [Order article via Infotrieve] |
15. |
Xie, J.,
and McCobb, D. P.
(1998)
Science
280,
443-446 |
16. |
Benkusky, N.,
Fergus, D.,
Zucchero, T.,
and England, S.
(2000)
J. Biol. Chem.
275,
27712-27719 |
17. |
Dworetzky, S.,
Boissard, C.,
Lum-ragan, J.,
McKay, M.,
Post-Munson, D.,
Trojnacki, J.,
Chang, C.,
and Gribkoff, V.
(1996)
J. Neurosci.
16,
4543-4550 |
18. |
Xia, X.,
Hirschberg, B.,
Smolik, S.,
Forte, M.,
and Adelman, J.
(1998)
J. Neurosci.
18,
2360-2369 |
19. | Zhou, Y., Schopperle, W. M., Murrey, H., Jaramillo, A., Dagan, D., Griffith, L. C., and Levitan, I. B. (1999) Neuron 22, 809-818[CrossRef][Medline] [Order article via Infotrieve] |
20. | Butler, A., Tsunoda, S., McCobb, D. P., Wei, A., and Salkoff, L. (1993) Science 261, 221-224[Medline] [Order article via Infotrieve] |
21. | Pallanck, L., and Ganetzky, B. (1994) Hum. Mol. Genet. 3, 1239-1243[Abstract] |
22. |
Shipston, M. J.,
Duncan, R. R.,
Clark, A. G.,
Antoni, F. A.,
and Tian, L. J.
(1999)
Mol. Endocrinol.
13,
1728-1737 |
23. | Reinhart, P. H., and Levitan, I. B. (1995) J. Neurosci. 15, 4572-4579[Abstract] |
24. | Wang, J., Zhou, Y., Wen, H., and Levitan, I. (1999) J. Neurosci. 19, RC4[Medline] [Order article via Infotrieve] 1-7 |
25. |
Clark, A. G.,
Hall, S. K.,
and Shipston, M. J.
(1999)
J. Physiol. (Lond.)
516,
45-53 |
26. |
Saito, M.,
Nelson, C.,
Salkoff, L.,
and Lingle, C. J.
(1997)
J. Biol. Chem.
272,
11710-11717 |
27. |
Nara, M.,
Dhulipala, P. D.,
Wang, Y. X.,
and Kotlikoff, M. I.
(1998)
J. Biol. Chem.
273,
14920-14924 |