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This Article Abstract Full Text Full Text (PDF) All Versions of this Article: fj.06-6152fjev1 20/14/2588    most recent Alert me when this article is cited Alert me if a correction is posted Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Tian, L. Articles by Shipston, M. J. Search for Related Content PubMed PubMed Citation Articles by Tian, L. Articles by Shipston, M. J.

A noncanonical SH3 domain binding motif links BK channels to the actin cytoskeleton via the SH3 adapter cortactin

Lijun Tian^*,1, Lie Chen^*,1, Heather McClafferty^*, Claudia A. Sailer, Peter Ruth, Hans-Guenther Knaus and Michael J. Shipston^*,2

^* Centre for Integrative Physiology, School of Biomedical Science, University of Edinburgh, Edinburgh, UK;

Division for Molecular and Cellular Pharmacology, Medical University Innsbruck, Innsbruck, Austria; and

Pharmakologie und Toxikologie, Pharmazeutisches Institut der Universität Tübingen, Tübingen, Germany

²Correspondence: Centre for Integrative Physiology, School of Biomedical Science, University of Edinburgh, Edinburgh EH8 9XD, Scotland, UK. E-mail: mike.shipston{at}ed.ac.uk

SPECIFIC AIMS

Our aims were to test the hypothesis that putative noncanonical Src homology 3 (SH3) domain binding motifs in the intracellular C terminus of the pore-forming subunit of vertebrate large conductance calcium- and voltage-activated potassium (BK) channels allow SH3 domain proteins to interact with BK channels, and to characterize whether SH3-mediated interactions are relevant to BK channel activity.

PRINCIPAL FINDINGS

1. Identification of a novel, noncanonical, Src homology domain 3 (SH3) interaction motif in the BK channel intracellular C terminus

2. These noncanonical motifs allow targeting of multiple SH3 domain proteins to the channel in vitro and in vivo

3. The SH3 adapter protein cortactin acts as a molecular bridge between the noncanonical SH3 interaction motif of the BK channel and the cortical actin cytoskeleton

4. The first molecular explanation for the control of BK channel function by actin cytoskeletal dynamics reported in a number of systems and implicated in a number of disease states including epilepsy and stroke is provided

CONCLUSIONS AND SIGNIFICANCE

Large conductance calcium- and voltage-activated potassium BK channels are expressed in most cells of the body and play an important role in a diverse range of physiological processes ranging from control of blood flow, micturition and immunity, to the control of neuronal excitability and neurotransmitter release. Major human disorders, including hypertension, epilepsy, incontinence, and sexual dysfunction, may result from perturbations of BK channel function.

Increasing evidence supports the hypothesis that the large intracellular C-terminal domain of the BK channel, pore-forming -subunit acts as a signaling organizer by allowing the BK channel to interact with multiple signaling pathways via protein-protein interactions. However, in contrast to many other ion channels that bind to adapter proteins to assemble into macromolecular signaling complexes, the BK channel is largely devoid of canonical protein-protein interaction domains. Indeed, how the majority of proteins reported to assemble as a complex with the BK channel interact with channel is unknown.

Targeting of multiple SH3 domain proteins to BK channels
We demonstrate that the C terminus of the vertebrate BK channel, pore-forming subunit contains two adjacent noncanonical Src homology domain 3 (SH3) binding motifs (SBM1 and SBM2, Fig. 1 A) within a proline-rich domain. SH3 domains represent one of the most ubiquitous protein interaction modules, with >200 protein members in the human genome. These interaction motifs allow multiple SH3 domain proteins to interact with the C terminus of the BK channel (Fig. 1B ). Targeting of SH3 domain proteins to BK channels has important implications for BK channel physiology.

View larger version (25K): [in this window] [in a new window]   Figure 1. Noncanonical SH3 domain binding motifs in the intracellular C terminus of vertebrate BK channels. A) Schematic of a single murine BK channel, pore-forming -subunit. The noncanonical SH3 domain binding motifs (SBM1 and SBM2) in the proline-rich domain (PRD) between the two putative regulator of potassium conductance (RCK) domains of the C terminus are shown. An additional potential SH3 domain binding motif is also encoded by the murine alternatively spliced exon 22 (e22) at mammalian site of splicing C2. B) Representative overlay assay of a commercial glutathione S-transferase (GST)-SH3 domain fusion protein array (Panomics TranSignalTM SH3 domain array II) probed with a thioredoxin-fusion protein of the murine C terminus spanning amino acids V553 to H753, including (e22) or excluding (zero) the e22 alternatively spliced insert at site of splicing C2 (see Fig. 2A ). GST-SH3 domains are spotted in duplicate at the respective positions denoted by letter and number code. In this array, both e22 and zero fusion proteins interact with SH3 domains from mona/Gads (array position A2), CRKL (A5), and endophilin II (C8) but not with the GST control (D7). Note that under identical conditions, additional SH3 domains interact with the e22 but not zero fusion proteins. Thioredoxin (thio) showed no binding to any fusion protein. Membrane arrays were incubated with 30 µg ml–1 of the respective thioredoxin-fusion protein and interaction was detected by probing for the C-terminal –V5 epitope tag in the thioredoxin fusion protein using 1/1000 dilution of anti-V5 antibody (Ab). Immunoreactivity was detected by enhanced chemiluminescence (ECL).

First, it considerably expands the potential diversity of proteins that may interact directly with the channel.

Second, many proteins we identified are adapter proteins (e.g., CRKL, mona/Gads) that contain other common protein-protein interaction motifs, perhaps providing a mechanism for other protein families (e.g., SH2 domain proteins) to interact with the channel complex.

Third, the physiological features (including relatively low affinity and high promiscuity) and cellular distribution of SH3 domain proteins provide a mechanism to allow dynamic spatiotemporal assembly of BK channel signaling complexes, which may be important in cell-specific assembly of BK channel macromolecular signaling complexes. Supporting this, two robust interactions were with the SH3 domain adapter proteins CRKL and mona/Gads. CRKL allows integration of signaling pathways activated by growth and differentiation factors in a variety of cell types and is essential for neural crest development. Mona/Gads expression is restricted to hematopoietic cells, where it coordinates signaling cascades involved in macrophage development.

Fourth, identification of novel noncanonical SH3 domain binding motifs will have important implications for the analysis of SH3 interactions in other proteins. SH3 domain proteins represent one of the largest families in the human proteome and several SH3 domain proteins are implicated in major human diseases. Assembly of BK channels with distinct SH3 domain proteins may provide an important link between BK channel integration of voltage and calcium signaling events and regulation of diverse downstream signaling cascades.

SH3 domain adapter protein cortactin links BK channels to the actin cytoskeleton
A major SH3 domain adapter protein we identified as interacting with the C terminus is the SH3 adapter protein cortactin. Cortactin directly binds the actin cytoskeleton and is involved in coordinating the spatial organization of many transmembrane proteins. Activity and/or localization of several ion channels, including BK channels, can be dynamically regulated through alterations in the actin cytoskeleton.

We demonstrate that BK channels may exist in a complex with cortactin and actin in both native and heterologous expression systems. BK channel, pore-forming subunits expressed in HEK293 cells interact with endogenous cortactin and actin, and disruption of the cortical actin cytoskeleton results in activation of BK channels (Fig. 2 ). Activation is dependent on the interaction of cortactin with the BK channel C terminus mediated via the noncanonical SH3 domain binding motifs as mutation of the interaction motifs that abolish cortactin interaction prevent activation of BK channels by disassembly of the actin network (Fig. 2) .

View larger version (17K): [in this window] [in a new window]   Figure 2. Actin cytoskeletal regulation of BK channels is mediated via binding of cortactin to the noncanonical SH3 domain binding motifs. A) Representative traces from excised inside-out patches of HEK 293 cells expressing e22-hemagglutinin (HA), zero-HA, or e22P656:667A-HA channel variants before (control) and after (+ CD) application of 10 µM cytochalasin D to the intracellular face of the patch. Patches expressing e22-HA, zero-HA, and e22P656:667A-HA channels contained five, four, and two channels, respectively. Mean channel open probability (Po) determined from 60 s of recording at + 40 mV in the presence of 0.1 µM free calcium in equimolar potassium gradients is given under each trace. B) Summary bar chart of the effect of actin cytoskeletal manipulation on the activity of the respective BK channel variant expressed as percent change in mean open probability (Po) of channels in the patch with respect to pretreatment control. Patches were exposed for 10 min to 10 µM cytochalasin D under control conditions (CD) or in patches pretreated for 5 min with 5 µM of the actin-stabilizing drug Phalloidin (+ Phal). Data are means SEM (n=6 to 12 patches/group). **P < 0.01 ANOVA with Bonferroni post hoc test vs. respective CD-treated group under control conditions.

In conclusion, we demonstrate that noncanonical SH3 domain binding motifs in the intracellular C terminus of the BK channel, pore-forming -subunit target multiple signaling proteins to the BK channel complex (Fig. 3 ). A major challenge will be to characterize the BK channel macromolecular signaling complexes in different tissues and to determine how these complexes are spatiotemporally regulated according to the physiological requirement of the tissue. Elucidation of the functional role of SH3 domain protein interactions with the BK channel should provide significant insight into how BK channel complexes are organized and regulated in health and disease.

View larger version (21K): [in this window] [in a new window]   Figure 3. Schematic illustrating targeting of multiple SH3 domain proteins to BK channels, including the adapter protein cortactin that links BK channels to the actin cytoskeleton. Schematic of a single pore-forming subunit of the BK channel illustrating the intracellular proline-rich domain (PRD) that contains SH3 domain binding motifs SBM1 and SBM2. The adapter protein cortactin contains an SH3 domain that interacts with the BK channel and domains that interact directly with the cortical actin cytoskeleton. A diverse array of other SH3 domain proteins, including adapter proteins and proteins involved in structural organization and cell signaling, interact with the PRD. As BK channels are formed by assembly of four pore-forming subunits, multiple distinct SH3 domain proteins may be able to interact with the same BK channel.

FOOTNOTES

¹ These authors contributed equally to this work.

To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.06-6152fje

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This Article Abstract Full Text Full Text (PDF) All Versions of this Article: fj.06-6152fjev1 20/14/2588    most recent Alert me when this article is cited Alert me if a correction is posted Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Tian, L. Articles by Shipston, M. J. Search for Related Content PubMed PubMed Citation Articles by Tian, L. Articles by Shipston, M. J.