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First published online 20 February 2007
doi: 10.1242/jcs.03399

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Co-assembly of N-type Ca²⁺ and BK channels underlies functional coupling in rat brain

David J. Loane^*, Pedro A. Lima and Neil V. Marrion

Department of Pharmacology and MRC Centre for Synaptic Plasticity, University of Bristol, Bristol, BS8 1TD, UK

Author for correspondence (e-mail: N.V.Marrion{at}bris.ac.uk)

Accepted 9 January 2007

	Summary

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Activation of large conductance Ca²⁺-activated potassium (BK) channels hastens action potential repolarisation and generates the fast afterhyperpolarisation in hippocampal pyramidal neurons. A rapid coupling of Ca²⁺ entry with BK channel activation is necessary for this to occur, which might result from an identified coupling of Ca²⁺ entry through N-type Ca²⁺ channels to BK channel activation. This selective coupling was extremely rapid and resistant to intracellular BAPTA, suggesting that the two channel types are close. Using reciprocal co-immunoprecipitation, we found that N-type channels were more abundantly associated with BK channels than L-type channels (Ca_V1.2) in rat brain. Expression of only the pore-forming -subunits of the N-type (Ca_V2.2) and BK (Slo₂₇) channels in a non-neuronal cell-line gave robust macroscopic currents and reproduced the interaction. Co-expression of Ca_V2.2/Ca_V3 subunits with Slo₂₇ channels revealed rapid functional coupling. By contrast, extremely rare examples of rapid functional coupling were observed with co-expression of Ca_V1.2/Ca_V3 and Slo₂₇ channels. Action potential repolarisation in hippocampal pyramidal neurons was slowed by the N-type channel blocker -conotoxin GVIA, but not by the L-type channel blocker isradipine. These data showed that selective functional coupling between N-type Ca²⁺ and BK channels provided rapid activation of BK channels in central neurons.

Key words: Co-immunoprecipitation, Channel coupling, Hippocampus, Action potential

	Introduction

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Membrane proteins can be organised into complexes with other proteins. There has been particular focus on the association of effector molecules with their targets. For example, the large conductance Ca²⁺-activated potassium (BK) channel has been reported to be present in a large signalling complex in rat brain that includes the ₂ adrenergic receptor, the cytosolic A-kinase-anchoring protein (AKAP79), protein kinase A (PKA) and the L-type Ca²⁺ channel (Liu et al., 2004). Results such as this led to the idea that ion channels can reside and be modulated within a specialised membrane micro-domain (Davare et al., 1999; Liu et al., 2004).

Considerable evidence exists showing that ion channel subtypes may co-exist within membrane micro-domains. There have been a number of examples where Ca²⁺ ion entry through different channel types leads to activation of Ca²⁺-activated channels. For example, activation of small conductance Ca²⁺-activated (SK) channels within the CNS can result from Ca²⁺ entry through voltage-gated L-type (Marrion and Tavalin, 1998), T-type (Wolfart and Roeper, 2002), P-type (Edgerton and Reinhart, 2003) and N-type (Hallworth et al., 2003) Ca²⁺ channels. In addition, SK channel activation can arise from Ca²⁺ entry through NMDA receptors (Faber et al., 2005). Activation of BK channels can occur by Ca²⁺ entry through particular ion channel types. For example, it has been reported that Ca²⁺ entry through NMDA receptors (Isaacson and Murphy, 2001), voltage-dependent N-type (Marrion and Tavalin, 1998) or L- and N-type Ca²⁺ channels (Sun et al., 2003) can activate BK channels in different central neurons. It is likely that the identity of channel subtypes involved in functional coupling may vary according to cell type. In most cases, functional coupling has been inferred from the effects of block of a Ca²⁺ channel subtype on the activation of Ca²⁺-activated current (Edgerton and Reinhart, 2003; Faber et al., 2005; Hallworth et al., 2003; Isaacson and Murphy, 2001; Sun et al., 2003; Wolfart and Roeper, 2002). These data fail to distinguish between functional coupling resulting from a specific interaction between channel subtypes and that arising because channels share the same subcellular location. A common subcellular location may be the case of functional coupling to the activation of SK channels, because these channels are more Ca²⁺ sensitive (Hirschberg et al., 1998; Xia et al., 1998) and require some distance between Ca²⁺ source and channel to experience the correct Ca²⁺ concentration for activation (Marrion and Tavalin, 1998). This is expected to be different for BK channels, as they are required to be closer to the source of Ca²⁺ entry because they are less sensitive to Ca²⁺ concentration than SK channels (Vergara et al., 1998). The BK channel must be extremely close to the source of Ca²⁺ because its activation has to occur <1 msecond to hasten the repolarisation of the action potential in hippocampal neurons (Lancaster and Nicoll, 1987; Storm, 1987). Three studies have identified a selective colocalisation of BK and Ca²⁺ channels: one using single-channel analysis that directly showed functional coupling of N-type and BK channels in hippocampal CA1 pyramidal neurons (Marrion and Tavalin, 1998) and two using biochemical techniques to demonstrate the association of L-type and BK channels in rat whole brain (Grunnet and Kaufmann, 2004; Berkefeld et al., 2006). However, no study has yet identified the rapid functional coupling between BK and Ca²⁺ channels and resolved how this might be achieved.

We show that the functional coupling of N-type Ca²⁺ and BK channels in whole rat brain and hippocampus results from the co-assembly of the two proteins. This association was markedly more abundant than the association of L-type (Ca_V1.2) and BK channels in brain. Association of N-type Ca²⁺(Ca_V2.2) and BK (rSlo₂₇) channels was reconstituted by expression of only the pore-forming -subunits. Functional coupling between Ca_V2.2 and rSlo₂₇ channels expressed in tsA-201 cells was frequent and was observed to be identical to that seen in hippocampal neurons: a coupling that was resistant to the buffering of intracellular Ca²⁺ by BAPTA (Marrion and Tavalin, 1998). Coupled events were very rarely observed in tsA-201 cells expressing Ca_V1.2 and rSlo₂₇ subunits. In contrast to the block of L-type Ca²⁺ channels with isradipine, application of the N-type channel blocker -conotoxin GVIA to hippocampal slices slowed CA1 pyramidal cell action potential repolarisation, showing that the specific intimate association of channel pore-forming subunits permits the rapid activation of BK channels to hasten action potential repolarisation (Lancaster and Nicoll, 1987; Shao et al., 1999).

	Results

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N-type Ca²⁺ channels and BK channels co-assembled in rat brain
The functional coupling between N-type Ca²⁺ and BK channels identified in hippocampal CA1 neurons suggested a close association between channel subunits (Marrion and Tavalin, 1998). Co-immunoprecipitation (co-IP) from native rat brain using antibodies directed against the pore-forming -subunits was performed to reveal whether interactions between channel subunits were responsible for functional coupling. Immunoblotting the anti-BK_1118-1135 co-IP (BK-IP) sample with an antibody directed against the N-type Ca²⁺ channel (anti-Ca_V2.2) revealed a strong immunoreactive band of the predicted molecular mass of the native Ca_V2.2 subunit (210 kDa) (Fig. 1Ai). Enrichment of BK channel -subunit immunoreactivity in the BK-IP confirmed the specificity of the immunoprecipitation (Fig. 1Aii). This specificity of the interaction was further confirmed by the absence of the band corresponding to the Ca_V2.2 subunit in the control rabbit IgG co-IP (IgG-IP) (Fig. 1Ai).

View larger version (25K): [in this window] [in a new window]   Fig. 1. Reciprocal co-immunoprecipitation from rat brain and hippocampus demonstrated selective co-assembly of BK and N-type Ca2+ channels. Western immunoblots of proteins isolated from soluble whole brain (A,C) or hippocampal (B) extracts by immunoprecipitation using antibodies for the -subunits of the BK channel (anti-BK1118-1135; BK-IP) or the CaV2.2 channel (anti-CaV2.2; CaV2.2-IP). (Ai) Probing BK-IP and rabbit IgG IP (IgG-IP) samples with anti-CaV2.2 revealed a band of 210 kDa in the BK-IP sample lane but not in the IgG control co-IP lane, indicative of the lower molecular mass form of the CaV2.2-subunit (n=4). (Aii) Enrichment of the immunoreactive band for the BK channel -subunit (120 kDa) in the BK-IP sample demonstrated the specificity of the immunoprecipitation. (B) The CaV2.2-subunit was co-immunoprecipitated with the BK channel -subunit from solubilised rat hippocampal tissue. This was seen as a band of 210 kDa in the BK-IP sample, which was absent in the control IgG-IP lane. (Ci) Probing CaV2.2-IP and rabbit IgG co-IP (IgG-IP) samples with anti-BK produced an immunoreactive band of the predicted molecular mass of BK channel -subunit (not observed in the IgG control co-IP lane), showing that the BK channel -subunit reciprocally co-immunoprecipitated with the CaV2.2-subunit (n=3). (Cii) Enrichment of the immunoreactive band for the CaV2.2-subunit (210 kDa) in the CaV2.2-IP sample demonstrated the specificity of the immunoprecipitation. In each of the above, a solubilised whole brain extract (input) was run alongside the co-immunoprecipitation samples. Input was 5% of total protein extract used in the assay. The positions of channel proteins and the heavy chain IgG (HC IgG) of the immunoprecipitating antibodies are indicated by arrows, and molecular mass standards are shown in each immunoblot.

Co-assembly of N-type Ca²⁺ and BK channels with expression of pore-forming -subunits
Co-immunoprecipitation of native brain channel proteins demonstrated that N-type Ca²⁺ and BK channels co-assemble to form a protein complex in rat brain. However, this approach cannot determine whether the interaction is direct, or requires other proteins present in brain. To circumvent this problem, we chose to express only the pore-forming -subunits of each channel subtype in a non-neuronal cell-line (tsA-201) to determine whether association of channel subunits occurred with these `minimal' channels.

It was imperative that functional channels were expressed under the conditions used to identify interaction between -subunits. Transient transfection of tsA-201 cells with N-terminal GFP-Ca_V2.2 (GFP-Ca_V2.2) (Raghib et al., 2001) alone gave a voltage-dependent inward current (Fig. 2Ai) that was half activated at +21 mV (n=12) (Fig. 2Aii). Whole-cell currents exhibited voltage-dependent inactivation during the sustained depolarisation (Fig. 2Ai). Steady-state inactivation determined by a pre-pulse protocol (see Fig. 2, legend) was fit with a Boltzmann distribution, with a V of –21 mV (n=10) (Fig. 2Aii). In addition, 90% of current recorded in 60 mM Ba²⁺ was blocked by the N-type Ca²⁺ channel antagonist -conotoxin GVIA (300 nM) (data not shown). Inward currents were not observed in cells expressing EGFP alone. These properties were similar to currents recorded from HEK293 cells expressing Ca_V2.2 subunits alone (Yasuda et al., 2004; Butcher et al., 2006). Expression of rSlo₂₇ (Ha et al., 2000) gave Ca²⁺-dependent and voltage-dependent whole cell and single channel currents. Whole-cell currents were observed only when cells were dialysed with 1 µM Ca²⁺ (Fig. 2Bi), with currents showing clear voltage dependence (Fig. 2Bi,ii). Excised inside-out patches exhibited Ca²⁺-dependent channel activity of 217±17 pS (n=4) conductance (data not shown). Ca²⁺-activated outward currents were not observed in cells expressing EGFP alone. rSlo₂₇ current activated at more negative voltages than seen when expressed in Xenopus oocytes (Ha et al., 2000): a property also observed when human and mouse Slo subunits were expressed in mammalian cell lines (Alioua et al., 2002; Ling et al., 2000). These data showed that expression of only the pore-forming -subunits of each channel subtype gave functional current. GFP-Ca_V2.2 and rSlo₂₇ subunit expression was visualised by western immunoblotting with anti-BK or an antibody directed against the N-terminal GFP tag of the Ca_V2.2 subunit (anti-GFP). Co-IP with anti-GFP from tsA-201 cells co-transfected with GFP-Ca_V2.2 and rSlo₂₇ (GFP-IP) was probed with anti-BK to reveal a band of the predicted molecular mass of the rSlo₂₇ channel -subunit protein (Fig. 2Ci). Probing the GFP-IP with either anti-GFP or anti-Ca_V2.2 showed two bands that were of the predicted molecular mass of GFP-Ca_V2.2 (Fig. 2Cii). This demonstrated that anti-GFP specifically immunoprecipitated GFP-Ca_V2.2 channel protein complexes from co-transfected tsA-201 cells. These data indicated that the -subunits of each channel interacted in a non-neuronal cell-line and suggested that an interaction between -subunits underlies association in brain (see below).

View larger version (24K): [in this window] [in a new window]   Fig. 2. Expression of only pore-forming -subunits produced functional current. (Ai) Family of whole-cell currents from a cell transfected with GFP-CaV2.2 evoked by step depolarisations (–60 to +60 mV) from a holding potential of –100 mV. (Aii) Normalised activation curve () was fit by a Boltzmann distribution with a V of +21 mV and a slope of e-fold in 7.8 mV (n=12). By contrast, steady-state inactivation was determined by (prepulse) voltage steps (1-second duration) preceding a test pulse (+30 mV) (n=10). The relationship () was fit by a Boltzmann distribution of V –21 mV. (Bi) Representative macroscopic current sweeps from two cells expressing rSlo27, one dialysed with an electrode solution containing 1 µM free Ca2+ (upper) and another dialysed with a solution containing 60 nM free Ca2+ (lower). (Bii) Mean normalised current-voltage relationship for cells dialysed with either 1 µM (, n=5) or 60 nM (, n=5) free Ca2+. (Ci) Expression of rSlo27 channels was confirmed by western immunoblotting with anti-BK (tsA). Probing the GFP-IP sample from cells co-transfected with rSlo27 and GFP-CaV2.2 subunits with anti-BK produced a band of 120 kDa, the predicted molecular mass of the rSlo27 channel -subunit. (Cii) Expression of the GFP-CaV2.2 subunit in tsA-201 cell lysates (tsA) was confirmed by western immunoblotting using anti-GFP, with anti-GFP immunoreactivity being absent in the rat whole brain (wb) tissue lane. Immunoreactive bands of 240 and 270 kDa, the predicted molecular masses of the GFP-CaV2.2 channel protein, were detected in the GFP-IP by both anti-CaV2.2 (lane 1) and anti-GFP (lane 2).

View larger version (42K): [in this window] [in a new window]   Fig. 3. Functional coupling of N-type Ca2+ channels with BK channels reconstituted in tsA-201 cells. (A) Representative macroscopic currents from a cell transfected with GFP-CaV2.2/CaV3 subunits, evoked from a holding potential of –100 mV by step depolarisations (shown are 20 mV increments) from –60 mV. Below is the normalised current-voltage relationship (n=5). (B) Single channel activity conducted by 160 mM Ca2+ evoked by step depolarisations to +20 mV from a holding potential of –120 mV. (Ci) Cell-attached patch records from a cell co-transfected with rSlo27 and GFP-CaV2.2/CaV3 subunits showing near coincident opening of rSlo27 channels with inward opening of GFP-CaV2.2/CaV3 Ca2+ channels. The close temporal association of these two expressed channels is seen in the expanded trace (Cii), where the full amplitude of the rSlo27 openings has been truncated to resolve coincident openings. (Di) Intracellular BAPTA did not disrupt the coupling between expressed GFP-CaV2.2/CaV3 and rSlo27 channels. Cell-attached patch current sweeps from a BAPTA-AM (10 µM)-treated cell displayed near coincident activation of rSlo27 channels following the opening of GFP-CaV2.2/CaV3 channels. The close temporal coupling of expressed channels is seen in the expanded trace (Dii).

Association of L-type Ca²⁺ channels and BK channels
Ca²⁺ entry through both L- and N-type Ca²⁺ channels activates BK channels in neocortical neurons (Sun et al., 2003) and association between L-type Ca²⁺ and BK channels has been observed in rat whole brain (Grunnet and Kaufmann, 2004; Berkefeld et al., 2006). Immunoblotting an anti-BK_1118-1135 co-IP (BK-IP) sample from rat brain with an antibody directed against the L-type Ca²⁺ channel (anti-Ca_V1.2) revealed an immunoreactive band of the predicted molecular mass of the native Ca_V1.2 subunit (210 kDa). Enrichment of Ca_V1.2 immunoreactivity in the BK-IP confirmed the specificity of the immunoprecipitation. The specificity of interaction was further confirmed by the presence of only a very faint band corresponding to the Ca_V1.2 subunit in the control rabbit IgG co-IP (IgG-IP) (Fig. 4A). Densitometric comparison of this data with the co-IP of N-type Ca²⁺ channels by the same antibody (Fig. 1Ai) showed that Ca_V1.2 immunoreactivity was 45% weaker than Ca_V2.2 immunoreactivity. This weak interaction between L-type Ca²⁺ and BK channels was reminiscent of the co-IP data reported by Grunnet and Kaufmann (Grunnet and Kaufmann, 2004). Thus, the data clearly indicated that the two channel subunits could associate in rat brain, but the association was more prevalent between N-type Ca²⁺ and BK channels.

View larger version (18K): [in this window] [in a new window]   Fig. 4. Association of L-type Ca2+ and BK channels. (A) Western immunoblots of proteins isolated from soluble whole brain extracts by immunoprecipitation using anti-BK1118-1135 (BK-IP). Probing BK-IP and rabbit IgG co-IPs (IgG-IP) samples with anti-CaV1.2 revealed a weak band of 210 kDa in the BK-IP sample lane, with a weaker reactivity observed in the IgG control co-IP lane. Enrichment of the immunoreactive band for CaV1.2 in the BK-IP sample demonstrated the specificity of the immunoprecipitation (n=3). (B,C) Cell-attached patch records from two separate cells co-transfected with rSlo27 and CaV1.2/CaV3 subunits. The vast majority of outward channel openings (derived from rSlo27) were not associated with any inward channel opening (derived from CaV1.2/CaV3). In very rare examples, a near coincident opening of rSlo27 channels with inward opening of CaV1.2/CaV3Ca2+ channels was observed. The close temporal association of these two expressed channels is seen in the expanded trace. The full amplitude of the rSlo27 openings has been truncated to resolve the small amplitude inward channel openings.

Functional interplay between N-type Ca²⁺ channels and BK channels during action potential repolarisation in hippocampal CA1 neurons
The effects of the N-type Ca²⁺ channel blocker -conotoxin GVIA on action potential (spike) duration was assessed in hippocampal slices to determine whether functional coupling underlies the physiological activation of BK channels. Evoked spikes were of short duration and exhibited an obvious fast afterhyperpolarisation (fAHP) (Fig. 5Ai, asterisk in inset). Under control conditions, an increase in action potential duration was observed between the first and subsequent spikes within the train, with the broadening of the second spike relative to the first spike being 23.5±1.3% (n=5 cells; 10 spikes per cell, P<0.05). This degree of spike broadening was sustained throughout the train (Fig. 5Aii,iv, open bars). BK channel inactivation (Hicks and Marrion, 1998) has been proposed to contribute to this spike broadening during repetitive firing (Faber and Sah, 2003; Shao et al., 1999). Block of BK channels by iberiotoxin (IbTx, 100 nM) (Fig. 5Ai) or charybdotoxin (ChTx, 10 nM) (Fig. 5Aiii) caused a marked broadening of the lower half of the spike and a concomitant reduction in the fAHP, confirming that BK channel activation hastened action potential repolarisation. The spike broadening caused by either BK channel blocker was sustained throughout the spike train but was contributed less in later spikes by a toxin-sensitive component. Ibtx (100nM) produced significant broadening of the first (24.1±1.7%, P<0.05), second (14.7±1.5%, P<0.05), third (15.6±0.6%, P<0.05) and fourth spikes (17.4±0.9%, P<0.05; n=6, 10 spikes per cell). ChTx (10 nM) also slowed significantly the first (14.2±1.2%, P<0.05), second (7.2±0.6%, P<0.05), third (7.2±0.9%, P<0.05) and fourth spikes (8.1±0.8%, P<0.05; n=3, 10 spikes per cell). This also showed that spike broadening resulting from BK channel inactivation was seen primarily in the second spike, after which inactivated BK channels did not further contribute to spike repolarisation.

View larger version (42K): [in this window] [in a new window]   Fig. 5. Selective block of N-type Ca2+ channels prolonged action potential duration. (Ai) Example action potentials showing action potential broadening and block of the fast afterhyperpolarisation (fAHP) by application of the BK channel blocker iberiotoxin (IbTx, 100 nM). All spike traces refer to the first spike of the train. Inset shows an action potential train of five spikes in a CA1 pyramidal neuron from a hippocampal slice evoked by a 200 msecond depolarising current injection. * indicates the fAHP. (Aii) Normalised pooled data showing the slowing of action potential duration during a train of action potentials resulting from BK channel inactivation (open bars, see text). Shaded bars show that the slowing of action potential duration by IbTx (100 nM) is observed throughout the train, with the effect being sustained after the second action potential (n=6 cells, 10 spikes per cell, this and subsequent pooled data plots; *P<0.05). (Aiii) Example action potentials illustrating the slowing of action potential repolarisation and block of the fAHP after addition of the BK channel blocker charybdotoxin (ChTx, 10 nM). (Aiv) Normalised pooled data showing that the prolongation of action potential duration by ChTx was sustained throughout the train (n=3 cells, 10 spikes per cell). (Bi) Block of N-type Ca2+ channels by -conotoxin GVIA (-Ctx GVIA, 300 nM) slowed the lower half of action potential repolarisation and abolished the fAHP. (Bii) The normalised pooled data showed that -Conotoxin GVIA (300 nM) significantly slowed spike repolarisation of the first and subsequent spikes in a train (n=7 cells, 10 spikes per cell). Inset shows P/4 leak-subtracted whole-cell currents evoked by a 300 msecond depolarising voltage step to 0 mV from a holding potential of –70 mV. Evoked current was greatly reduced by application of isradipine (5 µM), indicating that L-type Ca2+ channels carried the majority of inward current. (Ci) Example action potentials showing that selective block of L-type Ca2+ channels by the dihydropyridine antagonist isradipine (10 µM) had no effect on action potential duration. (Cii) The lack of an effect for all action potentials within the train is shown in the normalised pooled data (n=9 cells, 10 spikes per cell).

Block of N-type Ca²⁺ channels by -conotoxin GVIA (300 nM) caused a broadening of the lower half of the action potential and a block of the fAHP (Fig. 5Bi), similar to that seen with either BK channel blocker (Fig. 5A). The -conotoxin GVIA-induced broadening was sustained throughout the spike train but was contributed less in later spikes by the toxin-sensitive component (first, 18.2±0.9%, P<0.05; second, 10.7±1.1%, P<0.05; third, 11.2±0.9%, P<0.05; fourth spike, 10.9±0.8%, P<0.05; n=7; 10 spikes per cell; Fig. 5Bii). The effect of -conotoxin GVIA was concentration dependent, with 1 µM of the toxin producing a significant broadening of first (45.0±2.3%, P<0.05), second (37.8±1.9%, P<0.05), third (38.2±1.5%, P<0.05) and fourth spikes (37.4±2.0%, P<0.05; n=5; 10 spikes per cell; data not shown). These data show that the effect of -conotoxin GVIA was most prominent in the first one or two spikes of the train, when spike broadening owing to BK channel inactivation was less significant.

The role of L-type Ca²⁺ channels coupling to BK channel activation was assessed, because it has been previously reported that L-type Ca²⁺ and BK channels were biochemically associated in rat brain (Fig. 4) (Grunnet and Kaufmann, 2004; Berkefeld et al., 2006). Application of the dihydropyridine (DHP) antagonist isradipine (10 µM) had no effect on spike duration throughout the train (Fig. 5Ci,ii, Fig. 6A,B; n=9; P>0.1). The lack of effect was not the result of access problems, because lower concentrations of isradipine significantly reduced the slow AHP (data not shown) and the Ca²⁺ current recorded in these neurons (Fig. 5B,C inset). In addition, a prolongation of the spike duration was observed when IbTx was applied subsequently in the presence of isradipine (Fig. 6Aii). Specificity of coupling was demonstrated when isradipine (10 µM) failed to affect spike duration, but subsequent application of -Ctx GVIA (300 nM) in the continued presence of the DHP antagonist broadened spike duration as seen before (Fig. 6Bi,ii). Finally, application of -Ctx GVIA (300 nM) had no effect when it was applied after BK channels had been blocked by prior application of IbTx (Fig. 6Ci,ii). These data confirmed that the effect of N-type Ca²⁺-channel block was not additive to direct the block of BK channels by IbTx. This demonstrated that block of N-type Ca²⁺ channels by -conotoxin GVIA caused spike broadening by preventing BK channel activation during the falling phase of the action potential. In addition, these data show that action potential repolarisation is primarily hastened by activation of coupled BK channels. The results demonstrated that specific functional coupling underlies rapid activation of BK channels to hasten spike repolarisation in hippocampal neurons.

View larger version (36K): [in this window] [in a new window]   Fig. 6. Cumulative addition of channel blockers demonstrates selective coupling of N-type Ca2+ and BK channels. (Ai) Diary plot of the duration of the first action potential in the evoked train from a single hippocampal neuron recorded in a slice preparation. Application of the L-type Ca2+ channel dihydropyridine antagonist isradipine (10 µM) had no effect on action potential duration. By contrast, subsequent application of the BK channel blocker iberiotoxin (IbTx, 100 nM) in the presence of the DHP antagonist slowed action potential repolarisation. (Aii) Examples of action potentials taken from the cell used in Ai, recorded before (control) and after addition of isradipine (10 µM) and isradipine (10 µM) + IbTx (100 nM). A clear prolongation of action potential duration is seen after IbTx addition (superimposed traces in black, control; light grey, isradipine; dark grey, isradipine+IbTx). In addition, a bar chart is shown of normalised action potential duration showing the broadening of action potentials during a train in the absence and presence of channel blockers. No effect of isradipine (10 µM) is seen, while further concomitant addition of IbTx (100 nM) slowed action potential repolarisation throughout the train (n=3, 10 spikes per cell). (Bi) Diary plot of duration of the first action potential in the evoked train showing that isradipine (10 µM) had no effect. In contrast, addition of the N-type Ca2+ channel blocker -conotoxin GVIA (-Ctx GVIA, 300 nM) slowed action potential repolarisation. (Bii) Example action potentials from the experiment shown in Bi, showing the action potential broadening only after application of -Ctx GVIA (300 nM). Normalised pooled data showed that the effect of -Ctx GVIA was observed throughout the train of action potentials (n=5 cells, 10 spikes per cell). (Ci) Diary plot of action potential duration showing that -Ctx GVIA (300 nM) had no effect if BK channels were pre-blocked by IbTx (100 nM). (Cii) Example action potentials from the experiment illustrated in Ci, showing action potential duration was prolonged by IbTx (100 nM) with no further effect of -Ctx GVIA (300 nM). The normalised pooled data showed that the effect of IbTx was observed throughout the train of action potentials, with no effect of subsequent addition of -Ctx GVIA (n=4, 10 spikes per cell).

	Discussion

Top Summary Introduction Results Discussion Materials and Methods References

 
Both N-type Ca²⁺ and BK channels have been shown to be associated with different proteins. Apart from the auxillary - and ₂-subunits, the pore-forming subunit for N-type channels (Ca_V2.2) is known to associate with syntaxin (Sheng et al., 1994), Ras (Richman et al., 2004), G protein subunits (Zamponi et al., 1997) and calmodulin (Liang et al., 2003). By contrast, mammalian BK channels associate with syntaxin (Cibulsky et al., 2005; Ling et al., 2003) and the ₂-adrenergic receptor (Liu et al., 2004). The reported interaction of mSlo BK channels with syntaxin excluded the interaction between syntaxin and Ca_V2.2 (Cibulsky et al., 2005). Lack of endogenous syntaxin in tsA-201 cells (data not shown) and previous work (Leveque et al., 1994) suggests that a common association with syntaxin did not mediate the association of rSlo₂₇ and Ca_V2.2 subunits.

The use of pharmacological inhibition to identify coupling between Ca²⁺ and Ca²⁺-activated channels cannot distinguish between discrete channel co-localisation and a looser association where channels share a common subcellular localisation. Our data demonstrated an association between N-type and BK channel -subunits in rat brain, which was reconstituted when only the -subunits were expressed in a non-neuronal cell line. Expression of both -subunits (plus Ca_V3) in tsA-201 cells provided functional coupling in cell-attached patches that was identical to that observed in hippocampal CA1 pyramidal neurons (Marrion and Tavalin, 1998). This was in contrast to data obtained from cells expressing L-type (Ca_V1.2 plus Ca_V3) and rSlo₂₇ subunits, where only extremely rare examples of coupled openings were observed. This paucity of functional coupling was consistent with the weaker association of the two channel subtypes in brain and the lack of coupling between native L-type and BK channels in hippocampal neurons (Marrion and Tavalin, 1998). The use of intracellular Ca²⁺ buffers, such as EGTA and BAPTA can provide invaluable information regarding the relative location of Ca²⁺ and Ca²⁺-activated channels. For example, activation of BK channels in chromaffin cells required Ca²⁺ entry through both L-type and Q-type channels (Prakriya and Lingle, 1999). Most BK channels were found to be close enough to Ca²⁺ channels to be resistant to high concentrations of EGTA, but activation was blocked by BAPTA (Prakriya and Lingle, 2000; Berkefeld et al., 2006). These data suggested that some distance [estimated to be between 50 and 160 nm by Prakriya and Lingle (Prakriya and Lingle, 2000)] between Ca²⁺ and BK channels exists in this cell type. This distance was proposed to separate L-type Ca²⁺ and SK channels in hippocampal neurons, partly because this coupling was sensitive to intracellular BAPTA (Marrion and Tavalin, 1998). By contrast, the rapid coupling observed between N-type and BK channels in this study and in the hippocampus was insensitive to BAPTA. Our data have demonstrated that the channel -subunits are very close and it is possible that the interaction is direct. The interaction occurred when only the -subunits were expressed in a non-neuronal cell-line. This was complicated by the inability to resolve single Ca_V2.2 channel openings when expressed alone, requiring co-expression of Ca_V3 subunits to enable recording of single channel activity. The observed functional coupling could have resulted from association between Ca_V3 and Slo₂₇ subunits. This is unlikely to be the case, because co-expression of Ca_V1.2/Ca_V3 and rSlo₂₇ channels gave only very rare examples of rapid functional coupling. These data suggested that it is probable that the association between Ca_V2.2 and rSlo₂₇ channels was direct between the pore-forming subunits, but if the interaction was mediated by an intermediate protein it would have to be common to both tsA-201 cells and hippocampal neurons.

This is the first study to show that association between -subunits underlies rapid functional coupling between Ca²⁺ and Ca²⁺-activated channels. This type of association is best suited to the activation of BK channels, because of their requirement for high micromolar Ca²⁺ concentrations (Vergara et al., 1998). A direct association of the N-type Ca²⁺ and BK channel is particularly well suited to provide the rapid activation of BK channels that is required to hasten action potential repolarisation. Recorded action potentials had a duration of approximately 1.2 mseconds (Figs 5 and 6). N-type Ca²⁺ channels only open during the second half of the falling phase of the spike (Helton et al., 2005). For a selective functional coupling between N-type Ca²⁺ and BK channels to achieve spike brevity, it is necessary that N-type Ca²⁺ channels open, Ca²⁺ ions enter the cell and BK channels are activated in less than a few hundred microseconds. We have observed that there was an almost instantaneous activation of rSlo₂₇ channels upon the opening of a functionally coupled Ca_V2.2 channel. This behaviour was identical to that seen with native N-type Ca²⁺ and BK channels in hippocampal neurons (Marrion and Tavalin, 1998). These data were consistent with the biochemical data showing association of pore-forming -subunits. All these data clearly indicate that selective co-assembly is required for BK channels to play their role in hastening action potential repolarisation.

This study has confirmed the physiological relevance of association between -subunits of N-type Ca²⁺ and BK channels. The hastening of action potential repolarisation in hippocampal neurons results from activation of BK channels by Ca²⁺ entry through colocalised N-type Ca²⁺ channels, showing that functional coupling between specific channel subtypes is used to maintain spike brevity. The lack of an effect on action potential duration of the L-type Ca²⁺ channel blocker isradipine appeared to contrast with the biochemical data reporting an association between L-type Ca²⁺ and BK channels in brain (Grunnet and Kaufmann, 2004; Berkefeld et al., 2006). However, we showed that association of L-type and BK channels in brain was much weaker than that seen with N-type and BK channels: an association that was mirrored in co-IP data reported by Grunnet and Kaufmann (Grunnet and Kaufmann, 2004). It is likely that association between L-type Ca²⁺ and BK channels may underlie the activation of BK channels in other CNS neurons (Sun et al., 2003). The finding that mainly N-type channels provide the Ca²⁺ entry for activation of BK channels is in accord with both channel subtypes displaying a common subcellular location (Sausbier et al., 2006; Westenbroek et al., 1992), while L-type and BK channels are not necessarily in the same subcellular compartment in CNS neurons (Hell et al., 1993; Bowden et al., 2001; Sausbier et al., 2006). Application of -conotoxin GVIA to hippocampal neurons had no significant effect on spike firing patterns and adaptation during a burst, which are regulated by other Ca²⁺-activated potassium conductances [medium and slow AHPs (data not shown) (Lancaster and Nicoll, 1987; Vergara et al., 1998)]. This supports cell-attached patch data from hippocampal neurons (Marrion and Tavalin, 1998) showing that the functional coupling between N-type Ca²⁺ and BK channels is selective.

	Materials and Methods

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Solubilisation of rat brain membranes
Brains or dissected hippocampi from 9-12 day old Wistar rats were homogenised in ice-cold buffer (10 mM HEPES, 350 mM sucrose, 5 mM EDTA, pH 7.4) and centrifuged (2000 g) at 4°C for 5 minutes. All buffers contained the protease inhibitors: pepstatin A (1 µg/ml), leupeptin (1 µg/ml), aprotinin (1 µg/ml), Pefabloc SC (0.2 mM), benzamidine (0.1 mg/ml) and calpain inhibitors I and II (8 µg/ml each). The supernatant was centrifuged (100,000 g) at 4°C for 1 hour and the membrane pellet resuspended in ice-cold buffer (2 mg/ml). Membrane proteins were solubilised (10 mM HEPES, 5 mM EDTA, pH 7.4 containing 1.2% digitonin) at 4°C for 1 hour and insoluble material removed by centrifugation (100,000 g) at 4°C for 1 hour.

Co-immunoprecipitation (co-IP) from rat brain
Solubilised rat brain lysates (1.0-2.0 mg/ml) were incubated with protein A-Sepharose (BK channel co-IPs) or protein A/G agarose (N-type Ca²⁺ channel/Ca_V2.2 co-IPs) for 3 hours at 4°C to remove non-specific interactions. Precleared solubilised proteins were incubated with either rabbit polyclonal anti-BK_1118-1135 (Wanner et al., 1999) or rabbit polyclonal anti-Ca_V2.2 (Chemicon International, CA) for 12-15 hours at 4°C. Protein A-Sepharose or protein A/G agarose was added and the mixture incubated at 4°C for 3 hours to capture immunoprecipitated proteins. The beads were washed with buffer (10 mM HEPES, 150 mM NaCl, 5 mM EDTA, pH 7.4 containing 0.2% digitonin) and bound proteins were eluted with 1x SDS sample buffer, resolved by 6-10% SDS-PAGE and transferred to PVDF membrane. Membranes were blocked with 5% non-fat milk (dissolved in PBS-T: 137 mM NaCl, 2.7 mM KCl, 8.1 mM Na₂HPO₄, 1.5 mM KH₂PO₄, 0.1% Tween-20) for 12-15 hours at 4°C and either incubated with rabbit polyclonal anti-BK (Chemicon International, CA; 1:250), rabbit polyclonal anti-Ca_V2.2 (Chemicon International; 1:150) or rabbit polyclonal anti-Ca_V1.2 (Alomone Laboratories, Jerusalem, Israel; 1:250). Immunolabelled bands were resolved using a rabbit IgG HRP-conjugated secondary antibody and visualised using ECL (Amersham Pharmacia, UK). Enrichment of protein in the immunoprecipitation was used to confirm the specificity of the immunoprecipitating antibody, with enrichment being observed when a solubilised whole brain extract (input) was run alongside the co-immunoprecipitation samples. Input was 5% of total protein extract used in the assay.

Co-immunoprecipitation from tsA-201 cells
Cells were transfected using Superfect^TM (Qiagen, UK) with plasmids encoding the -subunits GFP-Ca_V2.2 (Raghib et al., 2001) and rSlo₂₇, a variant of the BK channel -subunit that is highly expressed in the hippocampus (Ha et al., 2000), at a 1:1 plasmid DNA ratio. Cells were harvested after 24-36 hours in lysis buffer (25 mM Tris-HCl, 250 mM NaCl, 5 mM EDTA, pH 7.5 containing 1% digitonin), incubated at 4°C for 1 hour, and insoluble material was removed by centrifugation (12,000 g) at 4°C for 10 minutes. Solubilised cell lysates (1.0 mg/ml) were incubated with protein A-Sepharose and precleared solubilised proteins were incubated with rabbit polyclonal anti-GFP (Santa Cruz Biotechnology, CA) for 12-15 hours at 4°C. Protein A-Sepharose was added and the mixture incubated at 4°C for 3 hours to capture immunoprecipitated proteins. The beads were washed with lysis buffer and bound proteins were processed as above. Membranes were western immunoblotted with anti-BK to resolve rSlo₂₇ expression, and either anti-GFP or anti-Ca_V2.2 to resolve GFP-Ca_V2.2 expression.

Recording from tsA-201 cells
tsA-201 cells were co-transfected with plasmids encoding GFP-Ca_V2.2, Ca_V1.2, CaV₃, rSlo₂₇ and EGFP using 1:1:1:0.1 ratio of plasmids and used after 24-36 hours. For GFP-Ca_V2.2, cells were superfused with an external solution of composition: 100 mM TEACl, 2.5 mM KCl, 1 mM MgCl₂, 10 mM HEPES, 60 mM BaCl₂, pH 7.4. Electrodes were fabricated from KG-33 glass and filled with: 120 mM CsMeSO₄, 20 mM TEA, 1.5 mM MgCl₂, 15 mM Na₂ATP, 10 mM EGTA, 80 mM free CaCl₂, pH 7.4. Whole-cell recordings for GFP-Ca_V2.2/Ca_V3 channels were as above, except extracellular CaCl₂ was 5 mM and intracellular EGTA was 0.5 mM. Currents were recorded using an Axopatch 200A with electrode resistances of 2-4 M. Capacitance and series-resistance compensation was used throughout. Currents were evoked by 200-msecond voltage steps (–50 to +30 mV) from a holding potential of –100 mV, low pass filtered at 1 kHz through an eight-pole Bessel filter and acquired at 100 µsecond intervals using PULSE (Heka, Germany). Currents were leak-subtracted using a P/4 protocol. For rSlo₂₇, cells were superfused with a solution (^*) containing: 144 mM NaCl, 2.5 mM KCl, 1.2 mM MgCl₂, 2.5 mM CaCl₂, 10 mM HEPES, 5.6 mM D-glucose, pH 7.4. Whole-cell electrodes were filled with (+): 130 mM KMeSO₄, 20 mM KCl, 1.5 mM K₂ATP, 10 mM HEPES, 10 mM EGTA, 3 mM MgCl₂, 9.62 mM CaCl₂ (1 µM free) or 3.62 mM MgCl₂, 6.02 mM CaCl₂ (60 nM free), pH 7.4.

Inward GFP-Ca_V2.2/Ca_V3 channel currents were recorded using quartz electrodes (7-10 M) filled with 160 mM CaCl₂ (Marrion and Tavalin, 1998). For rSlo₂₇, excised inside-out patch recordings were made using quartz electrodes filled with the solution ^* described above and bathed in solution +, with rSlo₂₇ channel activity evoked by raising the bath Ca²⁺ concentration to 1 µM. All single channel currents were filtered at 1 kHz (eight-pole Bessel) and acquired at 100-µsecond intervals. Single channels were analysed with TAC (Bruxton) using the 50% threshold technique. Cell-attached patches from cells co-transfected with Ca_V2.2/Ca_V3 or Ca_V1.2/Ca_V3 and rSlo₂₇ subunits were obtained with the solutions used for resolving Ca_V2.2/Ca_V3 channel activity described above, with functional coupling resolved by a step depolarisation from a holding potential of –100 mV (Ca_V2.2/Ca_V3) or –90 mV (Ca_V1.2/Ca_V3) to 0 mV for 500 mseconds (Marrion and Tavalin, 1998).

Hippocampal slice preparation and recording
Hippocampal slices (350 µm thick) were cut from Wistar rats (20-24 days old) in ice-cold artificial CSF (ACSF), which contained: 125 mM NaCl, 2.5 mM KCl, 25 mM NaHCO₃, 16 mM D-glucose, 1.25 mM KH₂PO₄, 1 mM MgCl₂ and 1 mM CaCl₂, pH 7.4 (bubbled with 95% O₂/5% CO₂). Slices were stored in ACSF at room temperature in a humidified interface chamber for >1 hour and then transferred to the recording chamber mounted on an Axioskop 2 FS (Zeiss, Germany) and continuously superfused at 30°C with ACSF (with CaCl₂ raised to 2.5 mM). CA1 pyramidal neurons were visualised using infrared DIC. Whole-cell electrodes (5-9 M) filled with a pipette solution containing: 135 mM potassium-D-gluconate, 10 mM KCl, 10 mM HEPES, 1 mM MgCl₂, 2 mM Na₂ATP and 0.4 mM Na₃GTP (280-300 mOsm), pH 7.2-7.3 with KOH. Membrane voltage was recorded using an AxoClamp 2B amplifier (Axon Instruments, CA) in bridge current-clamp mode, low-pass filtered at 3 kHz (eight-pole Bessel, Frequency Devices, CT) and acquired at 20 kHz using PULSE (HEKA, Germany). Only cells with a membrane potential more negative than –60 mV were used, with the membrane potential maintained at –60 mV by injection of depolarising current. Action potentials (5 spikes/pulse) were evoked by depolarising current injection (200 mseconds duration every 30 seconds). Action potential duration was measured at one-third of the total spike amplitude (measured from the threshold level). Analysis of the fifth spike was not included because it occasionally coincided with the end of the current injection. Drug effects on spike duration were assessed by comparing mean action potential duration (obtained from measurement of ten action potentials) before and during application of channel blocker (determined after 10 minutes if no effect was apparent or after the effect had stabilised – see time-courses in Fig. 6). Effect on spike duration was presented, with duration normalised to the longest duration measured in the same experiment. Significance was determined using the student's t-test, with values expressed as mean ± s.e.m. Ca²⁺ current measurements were carried out by whole-cell recording using an electrode solution containing: 120 mM CsMeSO₄, 30 mM TEA, 10 mM BAPTA, 5 mM MgCl₂, 5 mM Na₂ATP, 10 mM HEPES (pH 7.2), with slices bathed in the ACSF detailed above (with extracellular CaCl₂ raised to 10 mM). Membrane currents were evoked from a holding potential of –70 mV, filtered at 1 kHz and acquired at 10 kHz.

	Acknowledgments

 
We thank Hans-Günther Knaus (University of Innsbruck, Austria) for anti-BK_1118-1135, Chul-Seung Park (Gwangju Institute of Science and Technology, Korea) for rSlo₂₇, Annette Dolphin (UCL, UK) for providing GFP-Ca_V2.2, Yoichi Yamada (Sapporo Medical University, Japan) for Ca_V1.2 and Kevin Campbell (University of Iowa, USA) for donating Ca_V3. tsA-201 cells were kindly provided by David Brown. We thank Dewi Roberts and Jenna Montgomery for technical assistance. We wish to thank Michael Shipston and Dawn Shepherd for critical reading of the manuscript. This work was supported by the MRC (UK).

	Footnotes

 
^* Present address: Laboratory for the Study of CNS Injury, Department of Neuroscience, Georgetown University Medical Center, Washington, DC 20057, USA

Present address: Dep. Fisiologia, Fac. Ciências Médicas, UNL, 1169-056 Lisboa, Portugal

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