Featured ArticleArticles, Cellular/Molecular

Repolarizing Responses of BKCa–Cav Complexes Are Distinctly Shaped by Their Cav Subunits

Henrike Berkefeld and Bernd Fakler
Journal of Neuroscience 13 August 2008, 28 (33) 8238-8245; https://doi.org/10.1523/JNEUROSCI.2274-08.2008

Abstract

Large-conductance Ca2+- and voltage-activated potassium (BKCa) channels shape the firing pattern in many types of excitable cell through their repolarizing K+ conductance. The onset and duration of the BKCa-mediated currents typically initiated by action potentials (APs) appear to be cell-type specific and were shown to vary between 1 ms and up to a few tens of milliseconds. In recent work, we showed that reliable activation of BKCa channels under cellular conditions is enabled by their integration into complexes with voltage-activated Ca2+ (Cav) channels that provide Ca2+ ions at concentrations sufficiently high (≥10 μm) for activation of BKCa in the physiological voltage range. Formation of BKCa–Cav complexes is restricted to a subset of Cav channels, Cav1.2 (L-type) and Cav2.1/2.2 (P/Q- and N-type), which differ greatly in their expression pattern and gating properties. Here, we reconstituted distinct BKCa–Cav complexes in Xenopus oocytes and culture cells and used patch-clamp recordings to compare the functional properties of BKCa–Cav1.2 and BKCa–Cav2.1 complexes. Under steady-state conditions, K+ currents mediated by BKCa–Cav2.1 complexes exhibit a considerably faster rise time and reach maximum at potentials markedly more negative than complexes formed by BKCa and Cav1.2, in line with the distinct steady-state activation and gating kinetics of the two Cav subtypes. When AP waveforms were used as a voltage command, K+ currents mediated by BKCa–Cav2.1 occurred at shorter APs and lasted longer than that of BKCa–Cav1.2. These results demonstrate that the repolarizing K+ currents through BKCa–Cav complexes are shaped by the respective Cav subunit and that the distinct Cav channels may adapt BKCa currents to the particular requirements of distinct types of cell.

Introduction

Large-conductance calcium- and voltage-activated K+ channels (also termed BKCa, KCa1.1, or Slo1) act as key modulators of electrical signaling in many types of excitable cell (Sah and Faber, 2002; Latorre and Brauchi, 2006). Activated by the concerted action of membrane depolarization and elevation in intracellular Ca2+ concentration ([Ca2+]i) BKCa channels provide the robust K+ currents that contribute to action potential (AP) repolarization (Storm, 1987b; Edgerton and Reinhart, 2003), mediate the fast phase of afterhyperpolarization (fAHP) (Lancaster and Nicoll, 1987; Storm, 1987a; Yazejian et al., 2000), shape dendritic Ca2+ spikes (Golding et al., 1999; Rancz and Häusser, 2006), and influence transmitter release (Robitaille et al., 1993). Both onset and duration of the BKCa-mediated K+ currents vary widely among different cell types or among different subcellular compartments (Hu et al., 2001; Pattillo et al., 2001). Thus, in cerebellar Purkinje cells and hippocampal granule and pyramidal cells (Shao et al., 1999; Edgerton and Reinhart, 2003; Loane et al., 2007; Müller et al., 2007), BKCa channels are activated by short APs and remain open for a few milliseconds, whereas in chromaffin cells, vomeronasal sensory neurons, suprachiasmatic nucleus neurons, and smooth muscle cells the periods of channel activity extend to a few tens of milliseconds (Heppner et al., 1997; Lovell and McCobb, 2001; Jackson et al., 2004; Ukhanov et al., 2007).

Mechanistically, membrane depolarization and elevation in [Ca2+]i converge allosterically on the gating machinery of BKCa channels, with increasing Ca2+ concentrations shifting the steady-state activation curve into the physiological voltage range (Marty, 1981; Cui et al., 1997). Robust activation of BKCa channels at potentials ≤20 mV requires values for [Ca2+]i of ≥10 μm, which in the cellular context may be achieved either by a global or local/focal rise in [Ca2+]i. In CNS neurons and most other cell types, high micromolar levels of [Ca2+]i are restricted to so-called “Ca2+-nano/microdomains” that form around active Ca2+ sources, particularly voltage-gated Ca2+ (Cav) channels (Neher, 1998; Augustine et al., 2003). In recent work using functional proteomics, we showed that BKCa channels in the rat brain, tetramers of α subunits (BKα) either alone or together with the auxiliary subunits BKβ2/4 (Berkefeld et al., 2006), are able to coassemble with a subset of Cav channels into macromolecular BKCa–Cav complexes placing both types of channel within nanometers of each other. Within these complexes, BKCa channels are supplied with [Ca2+]i sufficiently high to ensure robust channel activation at physiological membrane potentials with onset of BKCa currents after Cav channel activation by less than a millisecond (Berkefeld et al., 2006). Moreover, this coupling exhibits the distinct sensitivity to Ca2+ buffers defining for Ca2+ nanodomains: it can be interfered with the fast chelator BAPTA, whereas EGTA is entirely ineffective.

The Cav channels that physically interact with BKCa through their α-subunits are Cav1.2 (L-type), Cav2.1 (P/Q-type), and Cav2.2 (N-type) (Berkefeld et al., 2006). These Cav subtypes are greatly distinct with respect to cellular distribution, subcellular localization, and functional properties. Thus, Cav2.1 and Cav2.2 are predominantly found in the synaptic compartment (Kulik et al., 2004), where they are crucial for neurotransmitter release (Castillo et al., 1994; Wu et al., 1999; Pelkey et al., 2006). In contrast, Cav1.2 channels are mostly localized in cell somata and dendrites (Reid et al., 2003; Obermair et al., 2004), in which they are involved in the control of bioelectrical regenerative properties (Dobremez et al., 2005).

The present work investigated the impact of distinct Cav subtypes on the characteristics of the repolarizing K+ current output of BKCa–Cav complexes in response to voltage steps and AP commands. For this purpose, defined channel–channel complexes composed of BKCa and either Cav2.1 or Cav1.2 channels were heterologously reconstituted and analyzed by giant and conventional patch-clamp recordings.

Materials and Methods

Molecular biology and reconstitution of protein complexes.

BKCa–Cav complexes were heterologously reconstituted in Xenopus oocytes and Chinese hamster ovary (CHO) cells by injection of cRNAs or transfection of cDNAs coding for the subunits of BKCa (BKα, BKβ4) and Cav channels (Cav2.1 or 1.2, Cavβ3 or Cavβ1b, α2δ). Preparation and injection of cRNA into Xenopus oocytes were done as described previously (Fakler et al., 1995). Briefly, Xenopus oocytes were surgically removed from adult females and manually dissected. Dumont stage VI oocytes were injected with aquatic solution containing the aforementioned cRNAs, treated with collagenase type II 2–3 d after injection, and incubated at 18°C for another 1–3 d before use. CHO cells were transfected with the JetPEI transfection reagent (Biomol), incubated at 37°C and 5% CO2, and measured 2–3 d after transfection. GenBank accessions of the clones used are A48206 (BKα, gift from Dr. L. Salkoff, School of Medicine, Washington University, St. Louis, MO), NM_021452.1 (BKβ4), M67515.1 (Cav1.2), NM_017346.1 (Cavβ1b), X57477.1 [Cav2.1(I1520H), gift from Dr. J. Yang, Columbia University, New York, NY], NM_012828.1 (Cavβ3), and AF286488.1 (α2δ). EGFP was simultaneously added as positive transfection control. All cDNAs were verified by sequencing.

Electrophysiology.

Standard whole-cell patch-clamp recordings on CHO cells were done at room temperature (22–24°C) as described previously (Bildl et al., 2004). Briefly, currents were recorded with an Axopatch 200B amplifier, filtered at 10 kHz and sampled at 25 kHz. All voltage stimuli were performed as P/4 protocols. Recording pipettes were made from quartz glass capillaries and had resistances of 1–5 MΩ when filled with intracellular solution containing (in mm) 135 KCl, 3.5 MgCl2, 2.5 NaATP, 5 K2EGTA, and 5 HEPES, pH adjusted to 7.3 with HCl. The extracellular solution was composed of (in mm) 144 NaCl, 5.8 KCl, 0.9 MgCl2, 1.3 CaCl2, 0.7 NaH2PO4, 5.6 d-glucose, and 10 HEPES, pH adjusted to 7.4 with NaOH. Recordings from giant patches excised from Xenopus oocytes were performed at room temperature as described previously (Fakler et al., 1995). Briefly, currents were recorded with an EPC9 amplifier (HEKA Elektronik), low-pass filtered at 3 kHz, and sampled at 25 kHz. Pipettes were made from thick-walled borosilicate glass and had resistances of 0.3–0.8 MΩ when filled with (in mm) 115 NaMES, 5 KMES, 10 HEPES, and 1.3 CaCl2, pH adjusted to 7.2 with HMES. Intracellular solution applied via a gravity-driven multibarrel pipette was composed of (in mm) 120 KMES, 5 K2EGTA, and 5 HEPES, pH adjusted to 7.2 with HMES. The bath solution contained (in mm) 120 KMES, 5 HEPES, 1 K2EGTA, and 1 MgCl2, pH adjusted to 7.2 with HMES.

Data analysis.

Data analyses were done with Igor Pro 4.05A on a Macintosh G4. K+ currents mediated by BKCa–Cav complexes were isolated and characterized via tail-current protocols (see Figs. 1 A, 2 A, 5 A), where the current measured instantaneously after the tail step was taken as the relevant “tail current” carried by K+ ions. Current–voltage (I–V) relationships of Cav channels (see Fig. 3 B) were fitted using the following formula: Embedded Image where P is an amplitude factor, C and D determine current rectification and reversal potential, V 1/2 is voltage required for half-maximal activation, and k is the corresponding slope factor.

Time constants of channel activation (Fig. 3 C) were derived from monoexponential fits to the rising phase of the recorded Ca2+ currents. All data are given as mean ± SEM.

Results

Steady-state activation of BKCa channels is determined by the associated Cav subtype

For characterization of their response properties, distinct BKCa–Cav complexes were heterologously reconstituted; BKCa channels (composed of BKα and BKβ4, the BKβ most abundantly coassembled with BKα in rat brain) (Berkefeld et al., 2006) were coexpressed with either Cav2.1 (Cav2.1, Cavβ3, α2δ) or Cav1.2 channels (Cav1.2, Cavβ1b, α2δ) in Xenopus oocytes and CHO cells, respectively. Figure 1 A illustrates typical current responses of BKCa–Cav2.1 complexes to step depolarizations recorded in giant inside-out patches from Xenopus oocytes under physiological ion conditions and with a high concentration of EGTA (5 mm) present on the cytoplasmic side (to prevent activation of noncomplexed BKCa channels) (Berkefeld et al., 2006). For voltage steps exceeding the activation threshold of Cav2.1 channels (approximately −30 mV), current responses were biphasic (Fig. 1 A, inset): an initial inward current carried by Ca2+ that was followed by an outwardly directed K+ current as anticipated for activation of BKCa channels by Ca2+ influx through Cav2.1 channels. Accordingly, Ca2+ coupling between both channels was mandatory for activation of BKCa channels in the voltage range from −50 to 70 mV, as seen in control experiments with BKCa channels devoid of their Cav partners (Fig. 1 B).

Figure 1.
Figure 1.

Steady-state activation of BKCa channels is distinct in macromolecular complexes with the Cav subtypes Cav2.1 (P/Q-type) and Cav1.2 (L-type). A , Current response of BKCa–Cav2.1 complexes to the indicated voltage steps (−50 to 10 mV, 10 mV increment, holding potential −80 mV) recorded under physiological ion conditions (1.3 mm extracellular Ca2+) in giant inside-out (i–o) patches from Xenopus oocytes expressing Cav2.1 [Cav2.1(I1520H), a2δ, Cavβ3] and BKCa (BKα and BKβ4). Cytoplasmic solution was buffered with 5 mm EGTA (as throughout all experiments). Time scaling is as indicated, and the current scale is 10 nA. Inset, Current traces at expanded time scale (red, current trace at 0 mV). B , Normalized currents through BKCa–Cav2.1 complexes (triangles) or through BKCa channels alone (circles) as a function of membrane potential recorded from giant i–o patches as in A . Data points are the mean ± SEM of 28 experiments. Filled triangles, Steady-state currents (I step) measured 90 ms after the voltage step. Open triangles, Tail currents (I tail) measured immediately after voltage step to the tail potential; I tail represents the activation curve of BKCa channels in BKCa–Cav2.1 complexes. C , Activation curves of BKCa channels in complexes with either Cav2.1 (triangles, from B ) or Cav1.2 channels (squares). Data points for BKCa–Cav1.2 complexes are the mean ± SEM of six experiments. rel., Relative (for all figures).

The amplitude of the combined Ca2+ and K+ currents through BKCa–Cav2.1 complexes determined under steady-state conditions (I step, recorded 90 ms after step depolarization) was strongly voltage dependent and exhibited a bell-shaped I–V relationship peaking at potentials between 0 and 10 mV (Fig. 1 B). A similar bell-shaped I–V relationship with a slightly left-shifted peak amplitude was obtained when the sole K+ current component was plotted over the membrane potential (Fig. 1 B). Experimentally, the K+ current component was isolated by a tail-current protocol stepping the membrane potential to 60 mV at the end of each depolarizing voltage step (Fig. 1 A, bottom). At this potential, the Ca2+ current was extinguished instantaneously because of the curtailed driving force for Ca2+ ions (Fig. 3), whereas the outward-going K+ current was transiently enhanced (before it declined to zero because of the ceased Ca2+ influx) (Fig. 1 A). Because the current amplitude recorded right after the tail step directly reflects channel activity at end of the preceding 100 ms depolarization, the K+ current I–V relationship may be regarded as steady-state activation curve of BKCa channels in BKCa–Cav2.1 complexes. Accordingly, the ascending phase of the bell-shaped I–V relationship reflects onset and increase in BKCa channel activity, whereas the descending phase of the BKCa channel I–V corresponds to cessation in channel activity.

These bell-shaped characteristics were repeated for K+ currents through BKCa channels integrated into complexes with the L-type channel encoding Cav1.2 (supplemental Fig. 1, available at www.jneurosci.org as supplemental material). As illustrated in Figure 1 C, the respective activation curve exhibited ascending and descending phases with a peak value at ∼30 mV and an onset of the ascending phase at potentials of ∼0 mV, both values markedly shifted toward positive voltages compared with BKCa–Cav2.1 complexes.

Next, we investigated the time course of the K+ currents mediated by BKCa channels integrated into complexes with either of the two Cav subtypes in response to step depolarizations. A modified tail-current protocol was used to both isolate and scan the time course of the K+ current component (Fig. 2 A, top). Figure 2 A (middle) illustrates the result of such a “scanning tail-current protocol” obtained with BKCa–Cav2.1 complexes after a step depolarization to 0 mV: after an initial lag phase, BKCa-mediated currents rose continuously, reaching steady-state amplitude after a few tens of milliseconds (Fig. 2 A, bottom). The time course of the BKCa current was voltage-dependent (Fig. 2 B), as was the initial lag phase defined by the interval between voltage step and K+ current exceeding the 2% relative current threshold (Figs. 2 C,D, gray line). Thus, in the voltage range between −20 and 0 mV where BKCa channels exhibited peak steady-state activity (Figs. 1 B,C), K+ currents followed the voltage step with a delay of ≤1.5 ms and reached ∼25% of their maximal amplitude within 4 ms after the depolarization step (Figs. 2 C,E). In contrast, at more positive potentials the lag phase increased to values ≥3 ms (Figs. 2 C,E). When repeated with BKCa channels in complex with Cav1.2, K+ currents obtained with the scanning tail-current protocol also displayed voltage-dependent increase in current after an initial delay, albeit with slower kinetics (Figs. 2 B–E). Thus, at potentials with peak activity (∼30 mV), the K+ currents reached <10% of their maximum after the first 4 ms period, and the lag phase was ≥3 ms (Figs. 2 B,D,E).

Figure 2.
Figure 2.

Time course of K+ currents differs between BKCa–Cav2.1 and BKCa–Cav1.2 complexes. A , Top, Middle, Currents through BKCa–Cav2.1 complexes recorded in inside-out (i–o) patches from oocytes in response to the indicated scanning tail-current protocol; steps to the tail potential (80 mV) were applied on top of a step depolarization to 0 mV at intervals of 0.5, 1, 1.5, 2, 3, 4, 6, 8, 16, 32, and 50 ms after the depolarization step. Current traces with tail steps at 1, 3, 8, and 32 ms are in black; the current scale is 2 nA. Bottom, Lot of I tail recorded in the experiment above as function of time. B , Plot of normalized I tail recorded in experiments as in A in response to step depolarizations to the indicated voltages with BKCa–Cav2.1 (left) and BKCa–Cav1.2 complexes (right); individual experiments comprised the complete set of step depolarizations. I tail was normalized to the peak current recorded in each experiment. Data points are mean ± SEM of 13 and 7 experiments for BKCa–Cav2.1 and BKCa–Cav1.2 complexes, respectively. C , D , First 4 ms-period of the I tail-plots from B (gray line in B ) at an enlarged scale. The horizontal bar denotes the 2% threshold. BKCa currents under Cav2.1 ( C ) and Cav1.2 ( D ) from B . For Cav2.1, voltage steps to −20, 0, and 20 mV evoked currents larger than the 2% threshold. For Cav1.2, voltage steps to 20, 40, and 60 mV evoked currents larger than the 2% threshold. Currents crossing the 2% threshold occurred within the first 2 ms after pulse start for BKCa under Cav2.1, whereas BKCa under Cav1.2 took longer than 2 ms to exceed the 2% at any voltage. E , Summary plot of the lag phase [Δt (I BK>2%)] defined by the interval between voltage step and K+ current exceeding the 2% threshold for the two distinct BKCa–Cav complexes indicated. Note that BKCa channels associated with Cav2.1 channels activated faster and at more negative potentials than those in complex with Cav1.2 channels.

Together, these data indicated that BKCa channels exhibit quite distinct activation properties when associated in bimolecular complexes with either Cav2.1 or Cav1.2 channels.

Distinct response properties of Cav2.1 and Cav1.2 channels

We, therefore, analyzed the properties of these two Cav channel subtypes (molecular composition as indicated above) under the same physiological ion conditions as before, in particular using an extracellular Ca2+ concentration of 1.3 mm together with 5 mm of the Ca2+ chelator EGTA on the cytoplasmic side.

As shown in Figures 3, A and B, both types of Cav channel provided robust currents in response to step depolarizations and their maximal current amplitudes defined bell-shaped I–V relationships with distinct characteristics. Thus, for Cav2.1 channels, the threshold for activation and the peak current amplitude were observed at more negative potentials than for Cav1.2 channels, with values for the voltage generating peak current amplitudes of −10.2 mV (fit to the mean) and 19.3 mV for Cav2.1 and Cav1.2, respectively (Fig. 3 B). The midpoint potentials (and slope factor) for steady-state activation obtained from fitting Equation 1 to the respective I–V relationships were −18.7 ± 1.3 mV (and 5.9 ± 0.4 mV) for Cav2.1 and 7.0 ± 2.5 mV (and 8.1 ± 0.4 mV) for Cav1.2. In addition to steady-state activation, both Cav channels exhibited distinct activation kinetics as indicated by the time constants obtained from monoexponential fits to the current onset (Fig. 3 C). Thus, for membrane potentials ≥0 mV, activation of Cav2.1 channels was significantly faster (p ≤ 0.01) than that of Cav1.2 channels, with values for τactivation ranging from 1.07 ± 0.18 to 0.37 ± 0.08 ms for Cav2.1 and from 1.83 ± 0.24 ms to 0.78 ± 0.07 ms for Cav1.2 channels.

Figure 3.
Figure 3.

Distinct gating properties of Cav2.1 and Cav1.2 channels. A , Currents mediated by Cav2.1 and Cav1.2 in response to voltage steps to potentials from −50 to 20 or 50 mV (10 mV increment) from a holding potential of −80 mV. Current and time scaling is as indicated. B , Average I–V relationships of the two Cav channel subtypes determined from experiments as in A . Data points are the mean ± SEM (n = 9 for Cav2.1; n = 8 for Cav1.2) of peak current amplitudes normalized to the maximal current. Continuous lines are fits of Equation 1 to the data. C , Activation time constants (τactivation) derived from monoexponential fits to the onset of currents through Cav2.1 (open triangles, n = 8) or Cav1.2 channels (filled squares, n = 8). D , Overlay of the I–V relationships of Cav2.1 (red) and Cav1.2 (blue) channels from B with the activation curves of BKCa channels in complex with the respective Cav subtype (from Fig. 1 C).

The I–V relationships of the two Cav subtypes closely resembled the activation curve of complexed BKCa channels providing explanation for both its shape and position on the voltage axis (Fig. 3 D). Thus, onset and ascending phase of the BKCa currents are determined by the voltage-dependent activation of the associated Cav channels, whereas the descending phase is attributable to the decrease in Ca2+ currents; the latter was quite distinct for Cav2.1 and Cav1.2 (see Discussion), as was the decrease of BKCa currents observed for the respective BKCa–Cav complex (Fig. 3 D). Together, these results indicated that the Cav channel mainly shapes the current output of BKCa–Cav complexes when voltage steps were used as command inputs.

Native BKCa–Cav complexes are operated by APs. Therefore, we next investigated the responses of Cav channels as well as BKCa–Cav complexes to AP-like voltage commands. Starting from a holding potential of −80 mV, these commands depolarized the membrane potential by a 0.5 ms voltage-ramp to 40 mV, before a subsequent ramp with durations varying between 0.5 and 24 ms generated repolarization (Fig. 4 A, top). Figure 4 A shows families of respective current responses recorded with Cav2.1 (middle) and Cav1.2 channels (bottom). With either channel, Ca2+ currents were only measured during repolarization, whereas at the upstroke or the peak of the AP the Ca2+ current was almost zero, similar to reports of Cav channels in CNS neurons (Raman and Bean, 1999; Bischofberger et al., 2002). Otherwise, the responses of the two Cav subtypes were greatly different, predominantly with respect to their time course as well as the time point of peak current amplitudes during a given AP. Thus, Cav2.1 channels exhibited the largest Ca2+ currents for APs with half-widths ≤1 ms, whereas at longer APs the peak amplitudes successively decreased (Figs. 4 A,B). In contrast, Cav1.2-mediated currents displayed maximal amplitudes at APs with half-widths ≥6.3 ms, whereas only approximately one-third of this peak level was obtained at short APs (half-widths ≤1) (Fig. 4 A,B). Comparison of the time courses showed that AP-generated currents through Cav2.1 lasted longer and reached their peak amplitude significantly later than the currents mediated by Cav1.2 (Fig. 4 C). Both observations are directly related to the different I–V relationships of Cav2.1 and Cav1.2 channels, as the time domain of the AP waveform command translates into progressive hyperpolarization of the membrane potential (Fig. 3 B).

Figure 4.
Figure 4.

Current responses of Cav2.1 and Cav1.2 channels to AP stimuli. A , Representative current traces evoked in Cav2.1 (middle) and Cav1.2 (bottom) channels by AP-like waveforms (top) comprising a 0.52 ms depolarizing ramp from −80 to 40 mV and a repolarizing ramp from 40 to −80 mV with variable duration of 0.76, 1.52, 3.04, 6.08, 12.16, or 24.32 ms; these durations correspond to AP half durations of 0.64, 1.02, 1.78, 3.3, 6.34, and 12.42 ms. Note the distinct current profiles generated by the two Cav subtypes. B , Peak Ca2+ currents elicited by AP-like waveforms normalized to the maximal current amplitude obtained with the AP series indicated in A . Data points are the mean ± SEM of six experiments for each Cav channel. C , Period between peak current and start of the AP command (Δt peak current) as a function of the AP half duration. Data points are mean ± SEM of six experiments for each Cav channel. Note the distinct increase in Δt peak current for both Cav subtypes.

Current responses of distinct BKCa–Cav complexes to AP waveform commands

Next, we investigated the K+ current output of the two different BKCa–Cav complexes on AP waveform commands with a tail-current protocol scanning the time course of sole BKCa-mediated currents during the repolarization phase of the AP waveform (Fig. 5 A, top). Figure 5 A (middle) shows a typical family of K+ current traces recorded from BKCa–Cav2.1 complexes during repolarization of an AP waveform with 6.34 ms half-duration. Thus, after a short lag phase, the K+ currents (measured as instantaneous current amplitude at the tail step) increased to peak level and subsequently declined to zero toward the end of the AP waveform (Fig. 5 A, bottom). Similar bell-shaped profiles were observed in all K+ current responses of BKCa–Cav2.1 complexes to our set of AP waveform commands (half-width ranging from 1.02 to 12.24 ms), although both the current amplitude and the time-to-peak interval increased with increasing AP duration (Fig. 5 B). Remarkably, the time-to-peak interval determined for the K+ currents of BKCa–Cav2.1 complexes almost perfectly coincided with the respective interval obtained for Ca2+ currents through Cav2.1 channels (Figs. 4 C, 5 B).

Figure 5.
Figure 5.

Differential responses of BKCa–Cav2.1 and BKCa–Cav1.2 complexes to AP stimuli. A , Top, Middle, Family of K+ current traces recorded from BKCa–Cav2.1 complexes in an individual (i–o) patch in response to the scanning tail protocol indicated. Activation of the BKCa channels along the AP repolarization was determined by equally spaced tail steps to 60 mV. Time and current scaling as indicated. Bottom, I tail plotted over time of AP (origin is the start of the AP). B , Plots of I tail obtained with a series of APs of variable half-width (as given in Fig. 4) applied to BKCa–Cav2.1 (top) and BKCa–Cav1.2 channels (bottom); currents were normalized to the peak amplitude obtained with an AP of 12.4 ms half-width. Data points are mean ± SEM of five and seven experiments, respectively. C , Summary of normalized peak K+ current amplitudes determined for BKCa–Cav2.1 (triangles) and BKCa–Cav1.2 (squares) complexes in the experiments in B as a function of AP half duration. Color coding is as in D . D , I tail plots of BKCa–Cav2.1 (lines and triangles in red) and BKCa–Cav1.2 (lines and squares in blue) complexes determined with short APs of 1.02 and 1.78 ms half-durations.

The current responses of BKCa–Cav1.2 channels to AP waveform commands were similar in their basic pattern to those of their BKCa–Cav2.1 counterparts including the bell-shaped time course as well as the dependence of the time-to-peak interval and current amplitude on AP waveform duration (Fig. 5 B). However, the distinct characteristics described above for the AP waveform responses of the two Cav channels alone (Fig. 4) were well preserved in the K+ current responses of the two BKCa–Cav complexes. Thus, K+ currents through BKCa–Cav2.1 complexes lasted longer and reached their peak amplitude considerably later than the K+ currents mediated by BKCa–Cav1.2 complexes (Figs. 4 A,C, 5 B). In addition, pronounced differences between the two complexes were observed for K+ currents generated by short AP waveforms with half-durations of 1.02 and 1.78 ms. Although BKCa channels were effectively activated by these APs when associated with Cav2.1 channels, they either remained silent or were significantly less activated by the same AP waveforms when integrated into complexes with Cav1.2 (Fig. 5 C,D).

Together, these results indicated that the K+ current responses to AP waveform stimuli are distinct for the two different BKCa–Cav complexes mainly as a result of the distinct properties inherent to the respective Cav channel subtype.

Discussion

The central finding of the present work is that K+ current responses of macromolecular BKCa–Cav complexes are mostly determined in both their voltage dependence and their time course by the Ca2+ currents through the Cav subunit. The distinct gating properties of Cav2.1 (or P/Q-type channels) and Cav1.2 (or L-type channels) generated BKCa currents with distinct profiles, particularly evident when complexes were operated by AP-like voltage commands. Whereas BKCa–Cav2.1 complexes responded robustly to short APs and provided repolarizing currents over almost the entire AP waveform, activation of BKCa channels associated with Cav1.2 channels required longer APs and their currents declined far before the end of the AP waveform. These results establish the critical importance of the molecular composition of BKCa–Cav complexes and show how repolarizing responses of BKCa channels can be adapted to distinct cellular requirements by association with distinct Cav subunits.

Isolation of the K+ current response of BKCa–Cav complexes

Functional analysis of BKCa–Cav complexes is hampered by the fact that any voltage command in the physiological range evokes combined Ca2+ inward and K+ outward currents. Because of their opposite direction, both currents distort or cancel each other when their amplitudes are similar in size, which occurs in both heterologous expression systems and native cells at voltages around the activation threshold of the BKCa channels or in responses to AP waveform commands (Figs. 2, 5). Because membrane depolarization elicits Ca2+ currents in Cav channels forming complexes with BKCa, but also in complex-free channels, the Ca2+ current amplitude may equal that of BKCa currents, despite the markedly smaller conductance of the Cav channels.

For characterization of isolated BKCa-mediated K+ currents we, therefore, used scanning tail-current protocols that circumvent the problem of interfering current components: by tail steps to the Ca2+ reversal potential, these protocols cancel the Ca2+ currents and, at the same time, enhance the K+ currents to be investigated (because of the increased driving force for K+ ions) (Prakriya and Lingle, 1999; Yazejian et al., 2000). Thus, this strategy offers two major advantages: (1) it detects BKCa channel activity even at the low levels observed around their activation threshold or in response to the brief Ca2+ currents generated by APs; (2) this detection is not affected by the superimposing Ca2+ currents.

Consequently, the scanning tail-current protocols enabled the precise determination of BKCa channel activity with voltage steps as well as with dynamic AP waveform commands.

Operation of BKCa–Cav complexes: control of K+ channel activity by the Cav partner

The functional properties of BKCa channels were greatly different when integrated into bimolecular complexes with either Cav2.1 or Cav1.2 channels, the Cav channels encoding P/Q- and L-type channels, respectively (Fig. 1, 2). Most prominently, the steady-state activation curve of BKCa channels fueled by coassembled Cav2.1 rose and declined at markedly more negative potentials (Fig. 1 C), and the onset of their K+ currents was faster by approximately a factor of twofold (Figs. 2 C–E). Both of these differences are paralleled by the differences observed for the activation time course and I–V relationship of the Ca2+ currents mediated by the two different Cav channel subtypes. Thus, the bell-shaped I–V relationship of Cav2.1 channels was positioned negative to that of Cav1.2 on the voltage axis (Fig. 3 B), and the τactivation for Cav2.1 exhibited approximately half the value of the τactivation for Cav1.2 (Fig. 3 C). These findings establish the Ca2+ currents through the coassembled Cav partner as the major determinant of BKCa channel gating in BKCa–Cav complexes under steady-state conditions.

However, BKCa channel activity is not exclusively shaped by the Ca2+ current as visualized by the deviations of the BKCa activation curves from the Cav channel I–V relationships (Fig. 3 D). At voltages in the “rising phase” of the Cav channel I–V relationship, BKCa channels were less active than anticipated from the Ca2+ current amplitude, whereas in its “falling phase” BKCa channel activity exceeded the respective predictions. In either case, the discrepancy most likely results from the gating properties of both BKCa and Cav channels. In particular, the higher BKCa activity in the voltage range of decreasing Ca2+ currents is likely attributable to the unique gating mechanism of BKCa channels in which decreasing [Ca2+]i can be compensated by increasingly positive membrane potentials. Thus, in addition to the Ca2+ currents, the gating characteristics of both Cav and BKCa channels participate in shaping the K+ current output of BKCa–Cav complexes under steady-state conditions.

Considering BKCa as an attached sniffer for Ca2+ ions (Berkefeld et al., 2006), the overlays in Figure 3 D emphasize an interesting difference in the I–V relationships of the two Cav channel subtypes used. Cav2.1-mediated Ca2+ currents steeply decrease at voltages >0 mV (as do BKCa-mediated K+ currents), although the open probabilities of the channels are maximal in this voltage range and the driving force for Ca2+ ions is sufficiently high to promote robust currents, as evident from the contrasting results obtained with Cav1.2 channels and BKCa–Cav1.2 complexes. How this phenomenon is brought about is unclear.

Tuning repolarizing AP responses by distinct Cav subunits

When subjected to dynamic voltage commands with AP-like waveforms, the distinct gating properties of Cav1.2 and Cav2.1 (Fig. 3) shaped Ca2+ currents with particular properties (Fig. 4). Whereas Cav1.2 promoted rapidly peaking currents that require long-lasting AP waveforms to reach their maximal amplitude, Cav2.1 channels reliably responded even to submillisecond AP waveforms and were active over almost the entire length of the AP waveform. In complexes with BKCa channels, the distinct characteristics of the two Cav subtypes translated into K+ currents that almost perfectly mirrored the respective Ca2+ current input (Fig. 5). Thus, responses to short AP waveforms were only observed with BKCa channels coassembled with Cav2.1, whereas longer AP waveforms evoked robust currents in both BKCa–Cav2.1 and BKCa–Cav1.2 complexes, although with mostly distinct time courses (Fig. 5 B–D).

These findings for BKCa–Cav complexes with different subunit compositions should impact native cells in several ways. First, they explain why BKCa-mediated K+ currents can be quite different among different types of cell (Edgerton and Reinhart, 2003; Jackson et al., 2004). Second, they illustrate how BKCa-mediated repolarizations may be adapted in both time course and amplitude to the distinct cellular requirements via the expression pattern of Cav channels. Accordingly, in cells with fast APs and/or millisecond fAHPs, BKCa channels should be preferentially associated with Cav2.1 channels, whereas in cells with longer-lasting APs, Cav1.2 channels or both Cav subtypes would be expected as partners of BKCa. This has indeed been observed. Thus, in cerebellar Purkinje cells where narrow spikes are found together with pronounced fAHPs, BKCa channels are predominantly fueled by P/Q-type channels (Edgerton and Reinhart, 2003; Womack et al., 2004), whereas in chromaffin or smooth muscle cells, where APs are typically as long as a few milliseconds, BKCa channels were found to partner with both Cav1.2 and Ca2.1 channels (Prakriya and Lingle, 1999; Berkefeld et al., 2006).

Footnotes

  • This work was supported by Deutsche Forschungsgemeinschaft Grant Fa 332/5-3 (B.F.). We thank J. P. Adelman for his advice and critical reading of this manuscript.

  • Correspondence should be addressed to Bernd Fakler, Institute of Physiology, University of Freiburg, Hermann-Herder-Strasse 7, 79104 Freiburg, Germany. bernd.fakler{at}physiologie.uni-freiburg.de

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Repolarizing Responses of BKCa–Cav Complexes Are Distinctly Shaped by Their Cav Subunits
Henrike Berkefeld, Bernd Fakler
Journal of Neuroscience 13 August 2008, 28 (33) 8238-8245; DOI: 10.1523/JNEUROSCI.2274-08.2008
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