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Large conductance Ca2+- activated K+ channels (BKCa) encoded by the Slo1 gene play a role in the physiological regulation of many cell types. Here, we show that the β1 subunit of Na+/K+-ATPase (NKβ1) interacts with the cytoplasmic COOH-terminal region of Slo1 proteins. Reduced expression of endogenous NKβ1 markedly inhibits evoked BKCa currents with no apparent effect on their gating. In addition, NKβ1 down-regulated cells show decreased density of Slo1 subunits on the cell surface.
Large conductance Ca2+-activated K+ channels (BKCa channels) regulate physiology in a wide range of tissues . BKCa channels can be preferentially targeted to different portions of polarized cells. For example, in hippocampal neurons, BKCa channels extensively localize to axons and presynaptic terminals [2-3] whereas in cerebellar Purkinje cells, they are observed primarily in the somato-dendritic region . Similarly, in epithelial cells, BKCa channels can be preferentially expressed on either the apical or basolateral surface [4-5]. The pore-forming subunits of BKCa channels are encoded by the Slo1 gene, also known as KCNMA1. Alternative splicing and protein-protein interactions at the COOH-terminal tail region contribute significantly to regulation of BKCa channels [8-26].
The Na+/K+-ATPase is a ubiquitously expressed member of the P-type ATPase superfamily. The functional enzyme is a heterodimer comprised of a catalytic α-subunit as well as one of at least three highly conserved glycoprotein β-subunits [27-29]. Stoichiometrically unequal amounts of α- and β-Na+/K+-ATPase subunits are present in some tissues [30-32], and the two classes of subunits are subjected to different modes of degradation . This raises the possibility that some Na+/K+-ATPase β-subunits may be free to interact with other proteins. More recent studies suggest that these subunits contribute to cell adhesion and formation of tight junctions [34-35].
We now report that Slo1 proteins biochemically interact with NKβ1 and that this interaction plays a role in regulating the steady-state expression of BKCa channels on the cell surface.
An expression plasmid encoding NH2-terminal (ectofacial) Myc-tagged mouse Slo1 (VEDEC isoform) was provided by Dr. Min Li (Johns Hopkins University, Baltimore, MD). Other plasmids, including pGBKT7-Slo1G785-A985 and different pGEXKG-Slo1 constructs were created using PCR and confirmed by sequencing. Antibodies used in this work include: anti-Myc (9B11, Cell Signaling Technology, Inc); anti-Myc (06-549, Upstate Biotechnology, Inc); anti-NKβ1 (ab33144, Abcam Inc. Cambridge, MA), anti-α-Na+/K+-ATPase (anti-NKα1) (Developmental Studies Hybridoma Bank, University of Iowa, Iowa City, IA) and anti-Slo1 (APC-107, Alomone Laboratories, Jerusalem, Israel).
Yeast two-hybrid screens of an embryonic day 9 (E9) chick ciliary ganglion (CG) cDNA library were carried out using the Matchmaker™ system (BD Biosciences, San Jose, CA) according to the manufacturer's instructions, as described in detail previously [10,24]. The bait construct was comprised of amino acids G785-A985 of mouse Slo1. NKβ1 emerged repeatedly in this screen.
CG were excised from E9 chick embryos and lysed in PBST (phosphate-buffered saline with 1% Triton) containing protease inhibitors (P2714, Sigma-Aldrich, Inc.). The extracts were centrifuged briefly, and the supernatants were incubated with anti-Slo1 or anti-NKβ1, and precipitates were isolated using Protein A/G PLUS-Agarose beads (sc-2003, Santa Cruz Biotechnology, Inc.). Beads were washed in PBST, SDS-sample buffer was added and samples were boiled for 5 min and then loaded onto gels. SDS-PAGE separation, immunoblot analysis, cell-surface biotinulation assays, and GST pull-down assays were performed as described previously [10,19,24].
HEK293T cells were grown and transiently co-transfected with plasmids encoding Myc-Slo1 and si-NKβ1 (sc-36008, Santa Cruz Biotechnology, Inc.) using Lipofectamine 2000™ (Invitrogen) as described previously [10,19,24]. Control cells were co-transfected with Myc-Slo1 and non-specific siRNA (sc-37007, Santa Cruz Biotechnology, Inc.). Cells were used for physiology or biochemistry 48 hours after transfection. Neurons from E9 chick CG were dissociated and cultured as described previously [36-37].
E9 CG neurons were maintained in culture for 3 hours before fixation in 4% paraformaldehyde for 10 min. Preparations were rinsed and incubated with rabbit anti-Slo1 (1:500 dilution) and mouse anti-NKβ1 (1:500 dilution) overnight at 4°C. This was followed by incubation with secondary antibodies and images were collected as described previously .
Inside-out patch recordings were made at room temperature (22° C) from HEK293T cells transiently co-expressing si-NKβ1 or non-specific siRNA, along with Myc-Slo1 and green fluorescent protein (GFP), which was used to allow identification of transfected cells during recording. Protocols for recording and analysis were described previously [10,24]. Student's unpaired t-test was used to compare experimental groups to a single control group with α = 0.05.
We conducted a yeast two-hybrid screen of a chick CG cDNA library using as bait a conserved cytoplasmic domain (G785-A985) of mouse Slo1 (Fig. 1A). Based on the sequences of cDNAs that we isolated in these screens, the portion of NKβ1 that interacts with Slo1 channels includes residues between 228 to 299 in the extracellular COOH-terminal region of NKβ1 (Fig. 1B).
This interaction was also detected by co-immunoprecipitation from E9 chick CG extracts. NKβ1 could be detected in immunoprecipitates prepared using antibodies against Slo1 channels (Fig. 1C, left). Conversely, Slo1 could be detected when immunoprecipitation was carried out using anti-NKβ1 (Fig. 1C, right), whereas neither protein could be detected when immunoprecipitation was carried out with IgG. The Slo1- NKβ1 interaction was also seen using GST pull-down assays. Thus, GST-Slo1(G785-A985) could precipitate NKβ1 from CG extracts, whereas GST alone was ineffective (Fig. 2A). Additional GST fusion proteins, including GST-Slo1(L985-Q1108) were also able to precipitate NKβ1 from CG extracts (Fig. 2B). This construct comprises the most distal COOH-terminal portion of Slo1. It contains a caveolin-binding motif , and sorting motifs required for the apical expression of BKCa channels in certain epithelial cells . In addition, we made four smaller GST fusion constructs of the bait region used in the initial screens. We detected NKβ1 interactions with GST-Slo1(T884-N936) and GST-Slo1(I937-A985). However, GST-Slo1(G785-L843) and GST-Slo1(Q844-I883) were unable to pull NKβ1 out of CG lysates (Fig. 2C). We carried out a similar set of analyses in HEK293T cells and obtained the same interaction pattern (data not shown).
We also observed partial co-localization of NKβ1 and Slo1 by confocal microscopy in primary cultures of CG neurons (Fig. 3). Signals from anti-Slo1 and anti-NKβ1 show extensive overlap, especially in intracellular compartments of somatic areas. However, Slo1 signal in neurites does not appear to co-localize with NKβ1. Limits of resolution make it difficult to definitively observe co-localization at the cell surface.
In order to examine the functional significance of the Slo1-NKβ1 interaction, we used an siRNA targeting NKβ1 transcripts (si-NKβ1) to reduce the endogenous expression of NKβ1 protein in HEK293T cells (Fig. 4). Treatment with si-NKβ1 had no effect on the expression of Na+/K+-ATPase α-subunits (NKα) compared to controls (Fig. 4A). To determine the functional implications of Slo1- NKβ1 interactions, inside-out patch recordings were made from HEK293T cells transiently co-transfected with si-NKβ1 or non-specific siRNA together with Myc-tagged Slo1. Large BKCa currents were observed in patches excised from Slo1-expressing cells treated with non-specific siRNA. However, we observed significantly reduced currents at all membrane potentials in cells treated with si-NKβ1 (Fig. 5A, B). There was no difference in the voltage-dependence of BKCa activation (Fig. 5D) or activation or deactivation kinetics due to NKβ1 siRNA treatment (Fig. 5C). We also examined the effects of si-NKβ1 or non-specific siRNAs on the distribution of Myc-Slo1 protein using a cell-surface biotinylation assay. These analyses revealed a reduced surface expression of Slo1 in si-NKβ1-transfected cells in comparison to control cells (Fig. 6A, top). There was no change in the protein expression levels of Slo1 as revealed by immunoblot analyses of the whole cell lysates (Fig. 6A, bottom). Quantitative analysis of the cell surface assays showed a significant reduction in the signal derived from the cell surface in NKβ1 down-regulated cells (Fig. 6B). Probing the same blots with anti-NKβ1 antibody showed a corresponding reduction in the cell surface expression of NKβ1.
The functional expression of BKCa channels on the cell membrane is a highly regulated process in many tissues. Here we show that NKβ1 is an interaction partner of Slo1. The region of NKβ1 that interacts with Slo1 is highly conserved in all known Na+/K+-ATPase and H+/K+-ATPase β-subunits . Down-regulation of NKβ1 expression caused a marked reduction in the surface expression of Slo1 in HEK293T cells. The effects of NKβ1 on the cell surface expression of BKCa channels may be related to other physiological effects recently ascribed to NKβ1. For example, NKβ1 plays a major role in the localization of tight junction-associated proteins in certain cell types and in the development of cell polarity [43-45]. In morphologically polarized cells such as neurons and epithelial cells, the interaction between Slo1 and NKβ1 may be responsible for targeting of BKCa channels to specific regions on the plasma membrane. There is evidence that formation of tight junctions in epithelia is sensitive to ionic conditions, including localized increases in intracellular Na+ concentration that lead to alterations in the subjacent cytoskeleton . It is possible that co-localization of Slo1 channels with Na+/K+-ATPases contributes to local ionic homeostasis and maintenance of tight-junction integrity. Moreover, in cardiac myocytes and other tissues, NKβ1 regulates the concentration of Na+/K+-ATPases in caveolae  where these enzymes are pre-assembled with their partners . Since NKβ1 is a ubiquitous protein, its ability to target proteins to caveolae may be a generalized mechanism that affects additional proteins, including BKCa channels [15-16].
Is NKβ1 an independent mediator of physiological regulation inside cells? Unequal amounts of α- and β-ATPase proteins are observed in many tissues . In addition, mature β-subunits can be recovered independent of α-subunits, at least in the case of H+/K+-ATPases . We have observed that Slo1 and NKα subunits interact with different regions on NKβ1 (see  for NKα binding sites), and it is possible that NKβ1 can interact with Slo1 and NKα simultaneously. We cannot exclude that the interaction between NKβ1 and BKCa channels may be facilitated by the presence of NKα subunits, or that the Slo1-NKβ1 interaction occurs within a larger complex formed between Na+/K+-ATPase, BKCa channels, and other proteins.
The possible existence of Slo1-NKβ1 interaction within a larger complex is intriguing in light of growing evidence that Na+/K+-ATPases can function as transducers for signaling pathways [49-50]. A number of studies point to the functional coupling between Na+/K+-ATPases and other ion channels [51-52] possibly including L-type Ca2+ channels . Therefore, Na+/K+-ATPases may co-exist with a Cav-BKCa complex at the plasma membrane that plays a role in regulation intracellular Ca2+ levels. NKβ1 subunits may be important for formation or stabilization of such complexes.
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