RIM ‐binding proteins recruit BK‐channels to presynaptic release sites adjacent to voltage‐gated Ca 2+ ‐channels
2018; Springer Nature; Volume: 37; Issue: 16 Linguagem: Inglês
10.15252/embj.201798637
ISSN1460-2075
AutoresAlessandra Sclip, Claudio Acuna, Fujun Luo, Thomas C. Südhof,
Tópico(s)Nicotinic Acetylcholine Receptors Study
ResumoArticle2 July 2018Open Access Source DataTransparent process RIM-binding proteins recruit BK-channels to presynaptic release sites adjacent to voltage-gated Ca2+-channels Alessandra Sclip Corresponding Author Alessandra Sclip [email protected] orcid.org/0000-0002-9313-4176 Department of Cellular and Molecular Physiology, Howard Hughes Medical Institute, Stanford University School of Medicine, Stanford, CA, USA Search for more papers by this author Claudio Acuna Claudio Acuna Department of Cellular and Molecular Physiology, Howard Hughes Medical Institute, Stanford University School of Medicine, Stanford, CA, USA CH Schaller Foundation and Institute of Anatomy and Cell Biology, Heidelberg University, Heidelberg, Germany Search for more papers by this author Fujun Luo Fujun Luo Department of Cellular and Molecular Physiology, Howard Hughes Medical Institute, Stanford University School of Medicine, Stanford, CA, USA School of Life Sciences, Guangzhou University, Guangzhou, China Search for more papers by this author Thomas C Südhof Corresponding Author Thomas C Südhof [email protected] Department of Cellular and Molecular Physiology, Howard Hughes Medical Institute, Stanford University School of Medicine, Stanford, CA, USA Search for more papers by this author Alessandra Sclip Corresponding Author Alessandra Sclip [email protected] orcid.org/0000-0002-9313-4176 Department of Cellular and Molecular Physiology, Howard Hughes Medical Institute, Stanford University School of Medicine, Stanford, CA, USA Search for more papers by this author Claudio Acuna Claudio Acuna Department of Cellular and Molecular Physiology, Howard Hughes Medical Institute, Stanford University School of Medicine, Stanford, CA, USA CH Schaller Foundation and Institute of Anatomy and Cell Biology, Heidelberg University, Heidelberg, Germany Search for more papers by this author Fujun Luo Fujun Luo Department of Cellular and Molecular Physiology, Howard Hughes Medical Institute, Stanford University School of Medicine, Stanford, CA, USA School of Life Sciences, Guangzhou University, Guangzhou, China Search for more papers by this author Thomas C Südhof Corresponding Author Thomas C Südhof [email protected] Department of Cellular and Molecular Physiology, Howard Hughes Medical Institute, Stanford University School of Medicine, Stanford, CA, USA Search for more papers by this author Author Information Alessandra Sclip *,1, Claudio Acuna1,2, Fujun Luo1,3 and Thomas C Südhof *,1 1Department of Cellular and Molecular Physiology, Howard Hughes Medical Institute, Stanford University School of Medicine, Stanford, CA, USA 2CH Schaller Foundation and Institute of Anatomy and Cell Biology, Heidelberg University, Heidelberg, Germany 3School of Life Sciences, Guangzhou University, Guangzhou, China *Corresponding author. Tel: +1 650 721 1418; E-mail: [email protected] *Corresponding author. Tel: +1 650 721 1418; Fax: +1 650 498 4585; E-mail: [email protected] The EMBO Journal (2018)37:e98637https://doi.org/10.15252/embj.201798637 PDFDownload PDF of article text and main figures. Peer ReviewDownload a summary of the editorial decision process including editorial decision letters, reviewer comments and author responses to feedback. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Abstract The active zone of presynaptic nerve terminals organizes the neurotransmitter release machinery, thereby enabling fast Ca2+-triggered synaptic vesicle exocytosis. BK-channels are Ca2+-activated large-conductance K+-channels that require close proximity to Ca2+-channels for activation and control Ca2+-triggered neurotransmitter release by accelerating membrane repolarization during action potential firing. How BK-channels are recruited to presynaptic Ca2+-channels, however, is unknown. Here, we show that RBPs (for RIM-binding proteins), which are evolutionarily conserved active zone proteins containing SH3- and FN3-domains, directly bind to BK-channels. We find that RBPs interact with RIMs and Ca2+-channels via their SH3-domains, but to BK-channels via their FN3-domains. Deletion of RBPs in calyx of Held synapses decreased and decelerated presynaptic BK-currents and depleted BK-channels from active zones. Our data suggest that RBPs recruit BK-channels into a RIM-based macromolecular active zone complex that includes Ca2+-channels, synaptic vesicles, and the membrane fusion machinery, thereby enabling tight spatio-temporal coupling of Ca2+-influx to Ca2+-triggered neurotransmitter release in a presynaptic terminal. Synopsis Direct interaction between large-conductance potassium BK-channels and scaffold RIM-binding proteins (RBPs) in the nerve presynaptic terminal is required for recruitment of BKα to active zone Ca2+-channels and efficient control of neurotransmitter release. Multi-domain protein RBP2 and BKα-channels directly interact in vitro as well as in mouse brain homogenates. RBP FN3-domains bind to both RCK-domains of BKα-channels. Expression of RBP2 shifts the voltage-dependence of BKα-channels in HEK293T cells and deletion of RBPs decreases presynaptic BK-currents. RBP1,2 double knockout mice show impaired presynaptic localization of BKα-channel protein. Introduction At the active zone of presynaptic terminals, action potentials (APs) cause voltage-gated Ca2+-channels to open. The resulting transient increase in cytoplasmic Ca2+ induces neurotransmitter release by triggering synaptic vesicle exocytosis (Walter et al, 2011; Sudhof, 2013). The number and location of voltage-gated Ca2+-channels at the active zone and the coupling of voltage-gated Ca2+-channels to synaptic vesicles are critical in determining the strength and plasticity of synapses (Meinrenken et al, 2002; Modchang et al, 2010; Eggermann et al, 2011). Several mechanisms control the size and duration of the Ca2+-signals at the active zone, thereby generating temporally restricted micro- and nano-domains of Ca2+ that determine the extent of release (e.g., see Fedchyshyn & Wang, 2005; Bucurenciu et al, 2008; Vyleta & Jonas, 2014). Among the most important of these mechanisms is the control of the AP duration by BK-channels (Fakler & Adelman, 2008; Contet et al, 2016; Griguoli et al, 2016). BK-channels are Ca2+-activated large-conductance K+-channels that limit the duration of an AP, thereby controlling the extent of Ca2+-channel opening and of neurotransmitter release per AP. BK-channels are enriched in presynaptic terminals and are present in a complex with voltage-gated Ca2+-channels, but it is unknown how BK-channels are recruited to presynaptic terminals and how they are molecularly connected with voltage-gated Ca2+-channels (Roberts, 1993; Berkefeld et al, 2006; Indriati et al, 2013). Voltage-gated Ca2+-channels are localized to active zones by binding to a multi-domain protein complex composed of RIMs and RIM-binding proteins (RBPs); this complex also directly interacts with synaptic vesicles and the exocytotic machinery and forms the core of the active zone (Deng et al, 2011; Han et al, 2011; Kaeser et al, 2011; Liu et al, 2011; Davydova et al, 2014; Acuna et al, 2015, 2016; Muller et al, 2015; Grauel et al, 2016; reviewed in Sudhof, 2012). In mammals, RBPs are encoded by three genes: RBP1 and RBP2 are differentially expressed throughout the brain, while RBP3 is primarily expressed outside the brain (Wang et al, 2000; Mittelstaedt & Schoch, 2007). RIM-binding proteins are composed of three SH3-domains and three fibronectin-type III domains (FN3-domains) that are flanked by variable interspersed sequences. The SH3-domains of RBPs directly bind to proline-rich sequences in RIMs and in L-, and P/Q- and N-type voltage-gated Ca2+-channels (Wang et al, 2000; Hibino et al, 2002; Kaeser et al, 2011). Via these interactions, RBPs contribute to the presynaptic recruitment of voltage-gated Ca2+-channels at Drosophila neuromuscular junctions (Liu et al, 2011), of N- and P/Q-type Ca2+-channels at standard chemical synapses (Acuna et al, 2015, 2016; Grauel et al, 2016), and of L-type Ca2+-channels at ribbon synapses (Krinner et al, 2017; Luo et al, 2017). RIMs interact with Ca2+-channels both indirectly via binding to RBPs and directly via their PDZ-domain that bind to the C-terminus of N- and P/Q-type Ca2+-channels (Kaeser et al, 2011). In addition, RIMs interact with Rab3-proteins on synaptic vesicles, with Bassoon in the cytomatrix, and with Munc13 in the vesicle fusion machinery (Wang et al, 1997, 2000; Betz et al, 2001; Schoch et al, 2002; Deng et al, 2011; Kaeser et al, 2011; Davydova et al, 2014). Thus, RIMs nucleate a macromolecular complex that includes Ca2+-channels, synaptic vesicles, and the release machinery in addition to RBPs (Sudhof, 2012). At standard chemical mammalian synapses in which N- and P/Q-type Ca2+-channels mediate presynaptic Ca2+-influx, RIMs are the primary drivers for synaptic recruitment of Ca2+-channels (Kaeser et al, 2011), whereas RBPs support RIMs in organizing nano-scale coupling of Ca2+-influx to release (Acuna et al, 2015; Grauel et al, 2016). As a result, at these synapses (as measured at the calyx of Held synapse), deletion of RIMs massively decreases presynaptic Ca2+-currents (Han et al, 2011; Kaeser et al, 2011), deletion of RBPs decreases the fidelity of neurotransmitter release without changing overall Ca2+-currents (Acuna et al, 2015), and deletion of both RIMs and RBPs nearly abolishes presynaptic Ca2+-currents (Acuna et al, 2016). At ribbon synapses that use L-type voltage-gated Ca2+-channels, however, RBPs are primarily responsible for recruiting voltage-gated Ca2+-channels because RBPs but not RIMs bind to L-type Ca2+-channels (Hibino et al, 2002; Kaeser et al, 2011). Here, deletion of RBPs has a much more pronounced effect on release at ribbon synapses than at standard chemical synapses (Luo et al, 2017). BK-channels are formed by tetramers of BKα-subunits that have seven transmembrane regions and a long cytoplasmic sequence containing two regulator-of-K+-conductance (RCK) domains (the RCK1- and RCK2-domains; Atkinson et al, 1991; Adelman et al, 1992; Butler et al, 1993; Lee & Cui, 2010). The RCK-domains regulate gating of BK-channels via Ca2+-binding, phosphorylation, and interactions with other proteins (Xia et al, 2002; Yusifov et al, 2008; Yuan et al, 2010). BKα proteins associate with cytoplasmic auxiliary BKβ- and BKγ-subunits that affect the gating properties of BK-channels and may contribute to their subcellular targeting (Meera et al, 1996; Tseng-Crank et al, 1996; Wallner et al, 1996; Orio et al, 2002). BK-channels are ubiquitously expressed in the brain (Sausbier et al, 2006) and enriched in presynaptic terminals (Robitaille et al, 1993a; Knaus et al, 1996; Zhou et al, 1999; Hu et al, 2001; Ishikawa et al, 2003; Misonou et al, 2006; Nakamura & Takahashi, 2007), where they form a molecular complex with Ca2+-channels, and are thus optimally located for coupling Ca2+-influx to the Ca2+-regulation of the duration of an AP (Roberts, 1993; Robitaille et al, 1993b; Berkefeld et al, 2006; Loane et al, 2007; Wang, 2008). High Ca2+-concentrations (~10 μM) are required for activation of BK-channels, implying that BK-channels are physiologically activated only in close proximity to voltage-gated Ca2+-channels and preferentially during AP trains (Berkefeld & Fakler, 2013). As a result, it has been suggested that nano-domain coupling of BK-channels to voltage-gated Ca2+-channels is necessary to trigger opening of BK-channels during APs (Berkefeld et al, 2006; Fakler & Adelman, 2008). Selective block of BK-channels with iberiotoxin (IbTX) or paxilline increased the probability of glutamate release in CA3-CA3 synapses (Raffaelli et al, 2004) and regulated neurotransmitter release in A17 amacrine cells in the retina (Grimes et al, 2009). Moreover, at the calyx of Held synapse (Ishikawa et al, 2003; Nakamura & Takahashi, 2007), in hippocampal mossy fibre synapses (Alle et al, 2011), and at CA3-CA1 Schaffer collateral synapses (Hu et al, 2001), a presynaptic function of BK-channels in regulating neurotransmitter release was uncovered when voltage-gated K+-channels were blocked with 4-aminopyridine. Overall, the current evidence suggests that BK-channels are an important mechanism of regulating neurotransmitter release especially during AP trains, but the molecular interactions that recruit BK-channels to presynaptic terminals are unclear. Here, we identified BK-channels in an unbiased yeast two-hybrid screen as novel interaction partner of RBPs. The binding of RBPs to BK-channels required the FN3-domains of RBPs and the RCK-domains of BK-channels. These findings led us to propose that RBPs function as scaffolds at the release site, and that by simultaneously binding to BK-channels (through the FN3-domains) and voltage-gated Ca2+-channels and RIMs (through their SH3-domains), RBPs contribute to the nano-domain coupling of Ca2+-influx to Ca2+-triggered synaptic vesicle exocytosis. Our results thus describe a function for the conserved FN3-domains of RBPs and suggest a mechanism by which BK-channels are recruited to voltage-gated Ca2+-channels at the release site in the active zone. Results RBPs bind to BKα-channels RBP1 and RBP2 are large multi-domain proteins that contain one N-terminal SH3-domain, three central FN3-domains, and two C-terminal SH3-domains (Fig 1A; Mittelstaedt & Schoch, 2007; Wang et al, 2000). To identify new binding partners for RBPs, we performed unbiased large-scale yeast two-hybrid screens using three fragments of rat RBP2 as baits: a fragment (residues 247–859) containing the FN3-domains and the linker region; a fragment (residues 1–859) containing the N-terminal SH3-domains, the FN3-domains, and the long central linker sequence; and a fragment (residues 247–1,068) containing the FN3-domains, linker region, and C-terminal SH3-domains (Fig 1A, Appendix Fig S1A). We isolated and re-confirmed a total of 71 prey clones (Appendix Fig S1B, Tables S1 and S2). Four of the prey clones that were isolated with the RBP2-247-859 bait encoded the cytoplasmic RCK2-domain of BKα-channels (Fig 1A, Appendix Table S1). Moreover, as previously described (Hibino et al, 2002; Kaeser et al, 2011), multiple clones representing the L-type Ca2+-channel α-subunit (Cacna1b) were recovered with baits containing the SH3-domains, validating the overall screen (Appendix Fig S1B and Table S1). In addition, several other preys of potential interest were recovered (Appendix Table S1), but were not pursued further because of the potential importance of a direct interaction of RBPs with BKα-channels. Figure 1. RBP2 interacts with the α-subunit of BK-channels A. Diagrams of RBP2 protein structure and bait construct used for the yeast two-hybrid screen which leads to the discovery of the interaction between RBP2 and BK channels (above), and location of the BKα prey sequences in the BKα domain structure (below; not depicted in scale). For details, see Appendix Fig S1, Table S1 and S2. B. Experimental strategy for validating the interaction of the RBP2 FN3-domains with BKα RCK-domains using co-immunoprecipitations (see C) or for imaging experiments (see D and E) on transfected HEK293T cells expressing various combinations of RBP2 and BKα proteins. C. Co-immunoprecipitation experiments to test the interaction of RBP2 with BKα. Cell lysates from HEK293T cells expressing YFP, YFP-tagged full-length RIM1α, or the YFP-tagged RCK2-domain of BKα (from prey clone 38) either alone or together with myc-tagged full-length RBP2 were subjected to immunoprecipitations with antibodies to GFP (which recognize YFP; left) or RBP2 (right). Input fractions (In; 1% of total) and immunoprecipitates (IP) were analysed by immunoblotting with antibodies to the myc-epitope (red; top) or GFP (green; bottom). Bands were visualized with fluorescently labelled secondary antibodies (B, negative control; arrows label specific YFP-positive bands). D. Imaging of transfected HEK293T cells expressing YFP alone or the YFP-tagged BKα RCK2-domain without or with myc-tagged full-length RBP2 (red, myc-epitope; blue, DAPI; green, YFP). Top, representative images (scale bar, 5 μm); bottom, fluorescence line scans across the cells to visualize the relative locations of YFP, DAPI, and myc signals (intensity was normalized to the maximal point and is expressed in arbitrary units). Note that under overexpression conditions, YFP and the YFP-tagged BKα RCK2-domain in the absence of RBP2 is partly nuclear, but YFP-tagged BKα is selectively recruited to the plasma membrane by RBP2, providing an imaging assay of the binding of BKα to RBP2. E. Quantitative analysis in transfected HEK293T cells of the co-localization of YFP (left) or the YFP-tagged BKα RCK2-domain (right) with the DAPI-stained nucleus as a function of the co-expression of RBP2. Graphs show the cumulative distribution (left) and the mean (right) of the Pearson correlation coefficient between the YFP and DAPI signals. Note that RBP2 dramatically reduces the correlation coefficient as a measure of the recruitment of the BKα RCK2-domain out of the nucleus to the cell membrane. Bar graphs show means ± SEM; n (cells/experiments): YFP only = 18/4, YFP + RBP2 = 17/4, YFP-BKα = 26/4, and YFP-BKα + RBP2 = 25/4. Statistical significance was calculated using Student's t-test (***P < 0.001). F, G. Co-immunoprecipitation of endogenous BKα and RBP2 from mouse brain homogenates (F, experimental strategy; G, representative immunoblots). Brain proteins solubilized with Triton X-100 (1%), NP-40 (1%), or SDS (0.3%) were immunoprecipitated with antibodies to RBP2. The input fraction (In; 1% of total protein) and immunoprecipitates (IP) as well as a negative control (B; preimmune serum) were analysed by immunoblotting using antibodies to RBP2 (4193) or BKα (APC-021). For more extensive studies, see Appendix Fig S2. Download figure Download PowerPoint To independently assess the interaction between RBP2 and BKα-channels, we performed co-immunoprecipitation experiments with proteins expressed in HEK293T cells (Fig 1B and C). We co-expressed myc-tagged full-length RBP2 with the YFP-tagged BKα RCK2-domain that we had isolated in the yeast two-hybrid screen. As negative and positive controls, respectively, we used YFP alone or YFP-tagged full-length RIM1α (Fig 1B). We then confirmed by co-immunoprecipitations with GFP antibodies (which recognize YFP) or with RBP2 antibodies that myc-tagged RBP2 can bind to both RIM and BKα-channels, whereas YFP alone could not bind to either molecule (Fig 1C). Next, we examined the interaction between RBP2 and BKα-channels by imaging their localizations in transfected HEK293T cells (Fig 1D and E). We expressed the YFP-tagged RCK2-domain of BKα alone or together with myc-tagged full-length RBP2 in HEK293T cells, immunostained the cells with myc-specific antibodies, and then quantified fluorescence signals relative to the nucleus, using DAPI as a marker of nuclei. The YFP-tagged RCK2-domain of BKα and YFP, when expressed alone, was localized to all cellular compartments (as is often observed for overexpressed proteins), whereas RBP2 expressed alone quantitatively localized to the plasma membrane (Fig 1D). Co-expression of RBP2 with the RCK2-domain, however, caused the translocation of the RCK2-domain to the membrane, confirming binding (Fig 1D). Co-expression of RBP2 with YFP, which is also localized in all cellular compartments, had no effect. Quantifications using correlation analyses confirmed that YFP-tagged RCK2-domain and YFP co-localized with the nuclear DAPI-stain in the absence of RBP2, and that YFP-tagged RCK2-domain but not YFP were recruited to the plasma membrane in the presence of RBP2, thus revealing their interaction in the context of the expressing cell (Fig 1E). Endogenous BKα and RBP2 form a complex in the brain The experiments up to now establish an in vitro interaction of RBP2 with the RCK2-domain of BKα. To test whether endogenous RBP2 and BKα-channels form a complex in vivo, we immunoprecipitated RBP2 or RIMs from brain homogenates, and immunoblotted the immunoprecipitated proteins for BKα, RBP2, and RIMs (Fig 1F and G, Appendix Fig S2). The challenge inherent to these experiments is that RIMs and RBPs are part of the active zone protein complex that is largely detergent-insoluble, prompting us to explore a variety of detergents for the immunoprecipitations, including low concentrations of SDS. We found that in these immunoprecipitations, BKα was co-enriched with both RBP2 and RIMs, whereas syntaxin-1 (used as a negative control) was not (Fig 1G, Appendix Fig S2B). These results are consistent with the presence of a complex composed of endogenous BKα, RBP2, and RIMs. Importantly, no signal for BKα was detected when immunoprecipitations were performed with samples from constitutive RBP1,2 double KO mice, confirming the specificity of the interaction (Appendix Fig S2C). RBP sequences containing FN3-domains bind to both RCK-domains of BKα-channels To map the domains of RBP2 that bind to BKα-channels, we co-expressed different fragments of myc-tagged RBP2 with YFP-tagged BKα-channels in HEK293T cells and tested binding by immunoprecipitation and imaging (Fig 2A). As assayed by co-immunoprecipitations, full-length RBP2 (aa 1–1,068) as expected bound robustly to BKα-protein (Fig 2B). A truncated version of RBP2 containing the FN3-domains and adjacent linker sequences (aa 247–859) bound reproducibly but weakly to BKα-channels, whereas a RBP2 construct containing a deletion of the FN3-domains and linker sequences (Δ247–859) failed to bind (Fig 2B). The same binding relationships were observed when measured by imaging the RBP2-dependent translocation of the YFP-tagged RCK2-domain of BKα out of the nucleus (Fig 2C and D). These results confirm the yeast two-hybrid experiment and suggest that the FN3-domains of RBPs are required for RBP binding to BKα-channels. Figure 2. Interaction of RBP2 with BKα requires RBP2 FN3-domains Domain structures of myc-tagged RBP2 constructs tested for interactions with BKα (from top: full-length RBP2 [residues 1–1,068], RBP2 sequence containing the FN3-domains [residues 247–859], and N- and C-terminal RBP2 sequences lacking the central FN3-domains [Δ247–859]). YFP-immunoprecipitations of proteins from HEK293T cells expressing YFP-tagged BKα alone, or the three myc-tagged RBP2 constructs alone or together with YFP-BKα. Input fractions (In; 1% of total) or immunoprecipitates (IP) were immunoblotted with antibodies to myc (red) or GFP (green, recognizes YFP). Note that RBP2 is only immunoprecipitated in the presence of YFP-BKα. Representative images (top; scale bar, 5 μm) and line scans (bottom) of HEK293T cells expressing myc-tagged RBP2 [247–859] or RBP2 [Δ247–859] without or with YFP-tagged BKα. Cells were stained for myc (red), DAPI (blue), or GFP (green). Line scans compare the localizations of myc, DAPI, and YFP. Cumulative plots and bar graphs of the correlation coefficients between the YFP-tagged BKα and the nuclear DAPI signals in HEK293T cells expressing myc-RBP2 (247–859) (top) or myc-RBP2 (Δ247–859) (bottom) proteins without or with YFP-BKα. Bar graphs show means ± SEM; n (cells/experiments): YFP-BKα = 26/4, YFP-BKα + RBP2 247–859 = 12/3, and YFP-BKα + RBP2 Δ247–859 = 12/3. Statistical significance was calculated using Student's t-test (*P < 0.05). Download figure Download PowerPoint We next examined the region of BKα-channels that mediates BKα-binding to RBPs. The intracellular sequences of BKα-channels are composed of tandem RCK-domains, the RCK1-domain (aa 391-692) and RCK2-domain (aa 761–1,178), that are homologous to each other (Appendix Fig S3A and B). We therefore tested if the interaction of RBP2 with the RCK2-domain, as shown above, is specific for this domain or if RBP2 can also bind to the RCK1-domain. Using co-immunoprecipitations (Fig 3A and B) and imaging experiments (Fig 3C and D), we found that full-length RBP2 binds to both RCK-domains of BKα-channels, consistent with the similar structure of these domains (Appendix Fig S3B). Similar results were found when the immunoprecipitations were performed with the truncated version of RBP2 containing only the FN3-domains and adjacent linker sequences (aa 247–859) (Appendix Fig S3C). Figure 3. The RCK1 or RCK2 domain of BKα both bind to RBP2 Domain structures of full-length BKα (top) and of the two BKα fragments containing the RCK1 [residues 391–692] or RCK2 domain [residues 761–1,178] that were tested for RBP2 interactions. Myc-immunoprecipitations of cell lysates from transfected HEK293T cells expressing either myc-tagged RBP2 alone or together with YFP or the indicated YFP-tagged BKα RCK-domains. Input fractions (In; 1% of total) or immunoprecipitates (IP) were analysed by immunoblotting with antibodies to myc (red) or GFP (green, recognizes YFP). For more experiments, see Appendix Fig S3. Representative images (top; scale bar, 5 μm) or line scans (bottom) of HEK293T cells expressing myc-tagged RBP2 alone, or the YFP-tagged BKα RCK1 or RCK2 domains alone or together with myc-tagged RBP2. Cells were stained for the myc-epitope (red) or DAPI (blue). Note that the BKα RCK-domains (green YFP fluorescence) are soluble when expressed alone, but are recruited to the cell membrane when co-expressed with RBP2. See also Fig 1D (experiments shown in Figs 1D and 3C were done in the same batch). Cumulative plots and bar graphs of the correlation coefficients between YFP-tagged BKα RCK1 (top) and RCK2 domains (bottom) and nuclear DAPI with or without co-expression of RBP2. Bar graphs show means ± SEM; n (cells/experiments) = YFP-BKα 391–692 = 26/4, YFP-BKα 391-692 + full-length RBP2 = 25/4, YFP-BKα 761–1,178 = 22/4, YFP-BKα 761–1,178 + full-length RBP2 = 20/4. Statistical significance was calculated with Student's t-test (**P < 0.01, ***P < 0.001). Download figure Download PowerPoint To confirm the presence of a direct interaction between RBPs and BKα, we produced recombinant GST-fused fragments of RBP1 (aa 771–1,086) and RBP2 (aa 298–604) containing only the FN3 domains (Appendix Fig S4A and B). We found that these fragments were sufficient to successfully pull-down YFP-BKα (aa761–1,178) containing the RCK2 domain from HEK293T cell lysates (Appendix Fig S4C and D). The presence of this interaction was further strengthened by co-immunoprecipitation experiments from transfected HEK293T cells co-expressing full-length RBP2 with Ca2+-channels (Cav2.2) and BKα-channels (Appendix Fig S5). Viewed together, these experiments indicate that the two RBP isoforms expressed in the brain (RBP1 and RBP2) can bind directly and simultaneously to Ca2+-channels via their SH3-domains and to BKα-channels via their FN3-domains. RBP2 alters the voltage dependence of BKα-channels expressed in HEK293T cells All experiments up to this point showing that RBPs bind to BKα were performed without regard to the channel function of BKα. To independently test whether RBP2 binds to functional BKα-channels in a membranous environment, we examined the effects of co-expressing full-length RBP2 or defined fragments of RBP2 on BKα-mediated K+-currents in transfected HEK293T cells (Fig 4A). In these experiments, we omitted the different auxiliary subunits of BK-channels because investigation of the relationship of the multiple auxiliary BK-subunits to RBP2 would have exceeded the scope of the current study. Figure 4. RBP2 shifts the voltage dependence of BKα-channels expressed in HEK293T cells Schematic of the experimental strategy for analysing the effects of various RBP2 proteins on BKα-mediated currents in HEK293T. Diagram of expressed proteins (top) and representative traces of BK-currents recorded in HEK293T in response to 10 mV step depolarizations from −80 mV to +110 mV (bottom, note that in the representative traces currents evoked by −20 mV depolarization are shown in yellow). BKα-currents were recorded in the absence or presence of full-length RBP2 [residues 1–1,068], or of RBP2 fragment containing [residues 247–859] or lacking [Δ247–859] the FN3-domains. Summary plots of the BKα-channel conductance as a function of voltage. Relative conductances (G/Gmax, where Gmax is the maximal conductance to control for differences in BKα expression levels) were used instead of current measurements to account for the contribution of the driving force and were calculated as G = I/(V−Vrev), with Vrev = RT/zF ln([K]out/[K]in) [here Vrev = −80.8203 mV]. Data were fitted with a Boltzmann equation. Bar graphs of the half-maximal activation voltage (Vhalf, left) and the slope of the conductance/voltage relation of BKα-channels (right) based on Boltzmann fits of the data in (C). Data information: Data in (C and D) are means ± SEM; n (cells): BKα only = 10, full-length RBP2 = 10, RBP2 247–859 = 14, and RBP2 Δ247–859 = 6. Statistical significance was assessed using two-way ANOVA for repetitive measurements (C) or one-way ANOVA (D), both followed by Bonferroni post hoc test (*P < 0.05, ***P < 0.001). Download figure Download PowerPoint HEK293T cells expressing BKα-channels exhibited large non-inactivating outward currents induced by voltage steps up to +110 mV in the presence of an intracellular Ca2+-concentration of 10 μM (Fig 4B, black trace). Co-expression of BKα-channel with both full-length RBP2 (Fig 4B, red trace) and a truncated version of RBP2 containing the FN3-domains and the linker region (Fig 4B, blue trace) caused a shift in the voltage dependence of BKα-channels, whereas co-expression of a deleted version of RBP2 in which the FN3-domains and the linker region were removed had no effect (Fig 4B, green trace). Quantification of the conductance–voltage (G-V) relationships for BKα-me
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