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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

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.

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).

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.

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).

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).