Sparse But Highly Efficient K_v3 Outpace BK_Ca Channels in Action Potential Repolarization at Hippocampal Mossy Fiber Boutons

Abstract

Presynaptic elements of axons, in which action potentials (APs) cause release of neurotransmitter, are sites of high densities and complex interactions of proteins. We report that the presence of K_v3 channels in addition to K_v1 at glutamatergic mossy fiber boutons (MFBs) in rat hippocampal slices considerably limits the number of fast, voltage-activated potassium channels necessary to achieve basal presynaptic AP repolarization. The ∼10-fold higher repolarization efficacy per K_v3 channel compared with presynaptic K_v1 results from a higher steady-state availability at rest, a better recruitment by the presynaptic AP as a result of faster activation kinetics, and a larger single-channel conductance. Large-conductance calcium- and voltage-activated potassium channels (BK_Ca) at MFBs give rise to a fast activating/fast inactivating and a slowly activating/sustained K⁺ current component during long depolarizations. However, BK_Ca contribute to MFB–AP repolarization only after presynaptic K_v3 have been disabled. The calcium chelators EGTA and BAPTA are equally effective in preventing BK_Ca activation, suggesting that BK_Ca are not organized in nanodomain complexes with presynaptic voltage-gated calcium channels. Thus, the functional properties of K_v3 channels at MFBs are tuned to both promote brevity of presynaptic APs limiting glutamate release and at the same time keep surface protein density of potassium channels low. Presynaptic BK_Ca channels are restricted to limit additional increases of the AP half-duration in case of K_v3 hypofunction, because rapid membrane repolarization by K_v3 combined with distant calcium sources prevent BK_Ca activation during basal APs.

Introduction

During the initial step of information transmission at chemical synapses, the presynaptic action potential (AP) governs calcium influx and therefore is one key determinant of synaptic strength (Augustine, 1990; Ma and Koester, 1996; Sabatini and Regehr, 1997; Borst and Sakmann, 1999; Geiger and Jonas, 2000). We have shown recently that the currents underlying the depolarization–repolarization sequence of the AP at glutamatergic hippocampal mossy fiber boutons (MFBs) are optimized with regard to immediate functional costs (Alle et al., 2009). Simulations predicted that the separation of sodium (Na⁺) and potassium (K⁺) currents is paralleled by comparatively low K⁺ conductance densities (G_K), suggesting a minimized K⁺ channel protein density for any channel type. Individual K⁺ channel types and families, however, differ widely with respect to single-channel conductance, maximal open probability, steady-state availability (Coetzee et al., 1999; Dodson and Forsythe, 2004; Gutman et al., 2005), and recruitment in response to brief depolarizations (Baranauskas et al., 2003; Martina et al., 2007); thus, the protein density for a given conductance density could vary considerably.

According to anatomical data, candidate fast-activating K⁺ channels contributing to the repolarization of presynaptic MFB–APs are of the families K_v1, K_v3, and large-conductance calcium- and voltage-activated potassium channels (BK_Ca) (Kues and Wunder, 1992; Weiser et al., 1994; Veh et al., 1995; Knaus et al., 1996; Riazanski et al., 2001). Functional K_v1 channels were identified at MFBs, presumably containing K_v1.1 and K_v1.4 subunits and/or β-subunits (Geiger and Jonas, 2000). In heterologous expression systems, K_v1 exhibit small single-channel conductances compared with K_v3 and BK_Ca (Gutman et al., 2005; Wei et al., 2005). Functional properties and roles of K_v3, in turn, have not yet been described at MFBs or other hippocampal glutamatergic synapses; however, these channels play a unique role in AP repolarization at the glutamatergic calyx of Held (Ishikawa et al., 2003). In heterologous expression systems, K_v3 exhibit medium-sized single-channel conductances (Gutman et al., 2005), and, at the calyx, they have faster activation kinetics than K_v1 channels (Nakamura and Takahashi, 2007). BK_Ca exhibit the largest single-channel conductance of the candidate channel families (Wei et al., 2005). Their activation requires a sufficient and coincident elevation of membrane potential and intracellular calcium concentration, a requirement that could be met by close spatial interactions with voltage-gated calcium channels (Ca_v) (Fakler and Adelman, 2008).

Pharmacologically, it has been observed that 4-aminopyridine (4-AP), a broad-spectrum K_v channel blocker, can substantially increase EPSCs at the MFB–CA3 pyramidal neuron synapse at concentrations of 5–10 μm (Rutecki et al., 1987). Reported half-maximal inhibitory concentration values (IC₅₀) for 4-AP of homomeric K_v1.1 [147 μm (Stephens et al., 1994)] and homomeric K_v1.4 [647 μm(Judge et al., 1999)] channels and the wide insensitivity of BK_Ca to 4-AP (Wei et al., 2005) raise the question whether K⁺ channel families other than K_v1 (Geiger and Jonas, 2000) and BK_Ca contribute to fast MFB–AP repolarization. Therefore, we set out to analyze fast-activating K⁺ channel families at the MFB and the relationships of their biophysical adaptations and protein densities required to perform basal presynaptic AP repolarization.

Materials and Methods

Slice preparation.

Three- to 4-week-old Wistar rats of either sex were anesthetized with isoflurane and decapitated according to institutional guidelines. Transverse 300-μm-thick hippocampal slices were cut using a custom-built vibratome. For dissection and storage of slices, a solution containing 80 mm NaCl, 25 mm NaHCO₃, 1.25 mm NaH₂PO₄, 2.5 mm KCl, 25 mm glucose, 85 mm sucrose, 0.5 mm CaCl₂, and 3 mm MgCl₂, equilibrated with 95% O₂ and 5% CO₂, was used. Slices were incubated at 34°C for ∼30 min and subsequently stored at room temperature.

Recording solutions.

Slices were superfused with a physiological extracellular solution [artificial CSF (ACSF)] containing the following (in mm): 125 NaCl, 25 NaHCO₃, 1.25 NaH₂PO₄, 2.5 KCl, 25 glucose, 2 CaCl₂, and 1 MgCl₂ (equilibrated with 95% O₂ and 5% CO₂). For standard whole-cell MFB (whole-MFB) recordings and standard outside-out patch recordings from MFBs, pipettes were filled with a solution containing the following (in mm) 155 KCl, 2 MgCl₂, 10 HEPES, 0.2 EGTA, 2 Na₂-ATP, 5 Na₂-phosphocreatine, and 0.5 Na₂-GTP. For outside-out patch recordings of Figure 6A (BK_Ca single-channel recordings), the internal solution contained no calcium chelator but added calcium of 1, 10, or 100 μm. The internal solution used in experiments of Figure 7, C and D, contained the following (in mm): 135 KCl, 2 MgCl₂, 10 HEPES, 10 K₄BAPTA or K₄EGTA, 2 Na₂-ATP, 5 Na₂-phosphocreatine, and 0.5 Na₂-GTP. That used in experiments of Figure 7F recording BK_Ca-mediated currents contained the following (in mm): 155 KCl, 10 HEPES, 10 NaCl, 0.1 EGTA, and 0.2 CaCl₂, resulting in 0.1 mm steady-state free Ca²⁺. For postsynaptic CA3 pyramidal neuron recordings of Figure 1B, a solution containing 135 mm K-gluconate, 5 mm KCl, 2 mm MgCl₂, 10 mm HEPES, 10 mm EGTA, and 2 mm Na₂-ATP was used. The pH of intracellular solutions was set to 7.2. For MFB voltage-clamp recordings, ZD7288 (4-ethylphenylamino-1,2-dimethyl-6-methylaminopyrimidinium chloride) (10 μm; Tocris Bioscience) and XE991 [10,10-bis(4-pyridinylmethyl)-9(10H)-anthracenone dihydrochloride] (10 μm; Tocris Bioscience) (only experiments including long pulses) were added to the ACSF, XE991 was not present in experiments of Figure 6B–E, and TTX (1 μm; Tocris Bioscience) and 4-AP (varying concentrations, maximally 1 mm; Tocris Bioscience) were present when indicated. For CA3 pyramidal neuron recordings, 50 μm d-AP-5 (Tocris Bioscience) and 3 μm gabazine (Tocris Bioscience) were present in the ACSF. The HEPES-buffered ACSF for local drug application contained the following (in mm): 5 HEPES, 145 NaCl, 1.25 NaH₂PO₄, 2.5 KCl, 25 glucose, 1.8 CaCl₂, and 1 MgCl₂, pH adjusted to 7.35 with NaOH. From stocks made of distilled water, blood-depressing substance I (BDS-I) (final concentration in HEPES–ACSF, 3 μm), α-dendrotoxin (DTX) (0.2 or 1 μm), iberiotoxin (IBTX) (3 μm), ω-agatoxin (1 μm), ω-conotoxin (3 μm) (all from Alomone Labs), paxilline (Pax) (10 μm; Tocris Bioscience), and NiCl₂ (200 μm) were added to the HEPES–ACSF, and DTX was added to the ACSF in bath-application experiments. The calcium current shown in Figure 6F was recorded according to Li et al. (2007), albeit at 34°C.

Figure 6.

Two populations of presynaptic BK_Ca channels can be distinguished electrophysiologically at MFBs. A, Outside-out patches drawn from MFBs (steady state at +20 mV) exhibit large single-channel openings that are sensitive to Pax but not to IBTX. Ctrl, Control. B, Long depolarizing voltage steps to 0 mV in the whole-cell recording configuration reveal Pax- and IBTX-sensitive BK_Ca channel-mediated currents (green) composed of two kinetically distinct components. C, Summary of Pax (n = 4), IBTX (n = 7), and cadmium (Cd²⁺, n = 7) sensitivity for recordings as shown in B (filled bars, first, fast activating/fast inactivating component; open bars, second, slowly activating/persistent component; see also filled dots and open squares in B). D, ω-Agatoxin, ω-conotoxin, and Ni²⁺ sensitivity (each n = 6) of currents as recorded in B (bar code as in C). E, Voltage dependence of first, fast activating/fast inactivating component (open squares), and of second, slowly activating/persistent component (filled circles; n = 3). F, Left, Comparison of example K_v-mediated current kinetics (black trace) and BK_Ca-mediated current kinetics (green trace: Cd²⁺-sensitive current); traces scaled to the same peak amplitude. Note the extremely fast inactivation of I_BKCa. Right, Same traces at higher temporal resolution. Blue trace represents example Ca_v-mediated current recorded in a separate MFB. Vertical scale bar applies to BK_Ca- and Ca_v-mediated currents.

Figure 7.

BK_Ca channels do not activate during basal MFB–APs, and their calcium source is remote. A, Focal application of IBTX (1 μm), BDS-I (3 μm) + IBTX, or DTX (0.2 μm) + IBTX to different (diff.) MFBs while recording APs generated by local current injection. Black traces, Baseline; red traces, during drug application. B, Collected data for changes of MFB–AP half-durations (AP-hd) during focal application of IBTX (n = 4), Pax (n = 4), BDS-I + IBTX (n = 3), DTX + IBTX (n = 4), and all three drugs simultaneously (n = 3) (in black), including data from Figure 1C (in gray) for comparison. C, Recordings of locally elicited MFB–APs in ACSF (top) or in ACSF containing 10 μm 4-AP (middle and bottom) with 10 mm BAPTA (top and middle) or 10 mm EGTA (bottom) in the intracellular solution. Black traces represent APs recorded immediately after establishing the whole-cell configuration; red traces represent APs recorded after 6 min in the whole-cell configuration. D, Changes in MFB–AP half-durations after 6 min recording time relative to those determined initially for experiments in ACSF and 10 mm BAPTA (left; n = 3), in ACSF containing 10 μm 4-AP and intracellularly 10 mm BAPTA (middle; n = 4) or 10 mm EGTA (right; n = 4). E, Top traces, MFB–AP-wave commands with different half-durations (left, basal MFB–AP; right, AP-hd, ∼700 μs), applied in TTX (1 μm), 4-AP (1 mm), and with 0.2 mm EGTA in the intracellular recording solution. Both waveforms were applied to an individual MFB. Bottom, Corresponding current traces in control and after focal application of IBTX (1 μm; red traces). Subtracted traces (control-IBTX) shown in green (I_BKCa, BK_Ca-mediated current). I_Cav, Voltage-gated calcium channel-mediated current. Inset shows overlay of both AP waveforms and the BK_Ca-mediated current in response to the broad AP. Arrow indicates membrane potential of broad AP, at which I_BKCa sets on. F, Top, Wave command of basal MFB–AP. Bottom, BK_Ca-mediated current (I_BKCa, green) recorded in the presence of TTX, Cd²⁺, and 1 mm 4-AP, using an intracellular solution containing 0.1 mm free Ca²⁺. Black traces represent recordings from a different MFB in TTX and Cd²⁺ using an intracellular solution containing 0.2 mm EGTA, before (I_Kv, K_v-mediated current) and after bath application of 1 mm 4-AP. Arrows indicate respective membrane potentials, at which currents set on.

Figure 1.

Presynaptic AP repolarization at hippocampal MFBs is performed by both K_v1 and K_v3 channels. A, Experimental configurations for experiments in B and C. Top, Extracellular stimulation (Stim.) of mossy fibers, recording from CA3 pyramidal neuron. Bottom, Recording from MFB, focal application of drug. Ba, CA3 pyramidal neuron EPSC peak amplitudes plotted over time. Bar indicates application of 10 μm 4-AP. Bb, Respective averaged current traces taken from baseline and versus end of drug application. Bar graph, Change in paired-pulse ratio (PPR) for this experiment. Left bar, Baseline; right bar, during drug application. Ca, Focal application of BDS-I (3 μm), DTX (0.2 μm), and both drugs simultaneously to different MFBs while recording APs generated by local current injection (baseline, black traces; during drug application, red traces). Cb, Collected data for changes of MFB–AP half-duration during focal application of the carrier solution alone (HEPES-buffered ACSF; n = 5), BDS-I (n = 4), DTX (n = 4), both drugs simultaneously (n = 3), and 4-AP (30 μm; n = 4). D, Changes in MFB–AP half-duration after bath application of 4-AP (10 and 30 μm; each n = 4).

Electrophysiology.

Patch pipettes for MFB and CA3 pyramidal neuron recordings were pulled from borosilicate glass tubing having an outer diameter of 2 mm and inner diameters of 0.6 and 1 mm, respectively, resulting in open tip resistances of 5–7 and 2–4 MΩ. Recordings were made at either 33–34°C (Figs. 1B–D, 2, 3A, 6, 7) or 36–37°C (Figs. 3B, 4, 5). Voltage-clamp recordings were made using an Axopatch 200 B (Molecular Devices), for experiments at 33–34°C, the recorded signal was filtered at 10 kHz (internal filter) and sampled at 40 kHz. For recordings at 36–37°C, the internal filter was bypassed, and an external eight-pole Bessel low-pass filter was set to 25 kHz and sampled at 100 kHz; the delay introduced by the external filter was 20 μs. For the P/4 protocols recording AP-wave-evoked currents, a previously recorded MFB–AP was used (at respective temperature). For long voltage steps, the respective protocols are indicated in the figures. In outside-out patch recordings from MFBs except that of Figure 6A, a 50 ms prepulse to −120 mV was applied. In whole-MFB recordings, initial uncompensated series resistance ranged from 20 to 30 MΩ, series resistance compensation was set to 95%, and lag was 7–10 μs; no prepulse was applied before the AP waveform. The holding potential in the voltage-clamp mode was set to −80 mV for MFB recordings and to −60 mV for CA3 pyramidal neuron recordings.

Figure 2.

K_v3 channel-mediated currents dominate during the MFB–AP waveform. A, Top, MFB–AP voltage waveform applied to MFBs. Second to last line of traces, K⁺ currents before (black) and during (red) application of drugs as indicated. Concentrations used as in Figure 1C. B, Collected data for the AP-wave-evoked K⁺ current peak amplitude during drug application normalized to baseline (each n = 4). C, Collected data for the AP-wave-evoked K⁺ current peak amplitude during bath application of 4-AP at concentrations as indicated above the bars, normalized to baseline (n = 3–12). D, Left, Top, Representative average trace of AP-wave-evoked K⁺ currents in the presence of DTX (bath; 0.2 μm); left, bottom, different MFB recording in the presence of DTX and 30 μm 4-AP (bath). Right, Summary of peak current amplitudes in the two conditions (different sets of experiments; n = 16 and 9). E, Left, K⁺ currents in DTX (bath; 0.2 μm) in response to the voltage step protocols indicated. Right, Relative K⁺ conductance as function of test potential (open circles; n = 9) and of conditioning prepulse potential (filled circles; n = 5) in DTX.

Figure 3.

K_v3 are better recruited during MFB–AP waves than K_v1. A, Top, MFB–AP-wave command and square-pulse command applied in the whole-MFB configuration (w). Bottom, Left, K⁺ current traces from recordings in DTX; right, K⁺ current traces from recordings without DTX. Responses to the MFB–AP wave on the respective left side of the two drug conditions (blue traces). The 1 ms timescale applies to MFB–AP wave experiments and 80 ms timescale to square-pulse experiments. B, Top, MFB–AP-wave command and square-pulse command applied to outside-out patches (p) from MFBs. Bottom, Left, K⁺ current traces from recordings in DTX; right, K⁺ current traces from recordings without DTX. Responses to the MFB–AP wave (blue traces) were overlaid on the responses to the square pulse and aligned to the rising phase. C, Collected data from experiments as in A and B, displaying the ratio of peak current amplitudes obtained in response to the MFB–AP wave and to the square pulse (p, outside-out patch; w, whole-MFB). D, Time constants of a simple exponential function fitted to the rising phase (15–85% of peak amplitude) of current responses to the square pulse as in B from five (in DTX) and four (without DTX) experiments. E, Left, Time course of MFB–AP-wave-evoked K⁺ currents [I_wave(t), circles] and derived K⁺ conductances [G_wave(t), squares] from experiments in DTX (red symbols; n = 5) and without DTX (black symbols; n = 8), using the outside-out patch configuration. AP superimposed. Right, Ratio of the maximal K⁺ conductance during the MFB–AP (G_max,wave) and the conductance at the peak of the K⁺ current [G(I_max,wave)] during the MFB–AP. Filled bar, Experiments in DTX; open bar, experiments without DTX. F, Fractions of conductances at resting membrane potential that become activated during an MFB–AP. s.p., Square pulse; filled bar, experiments in DTX; open bar, experiments without DTX.

Figure 4.

K_v3 exhibit a higher single-channel conductance than K_v1 at MFBs. A, Left, Top, Square-pulse protocol to elicit K⁺ currents in MFB outside-out patches. Middle, Superimposed individual current responses. Average current trace superimposed in white. Small residual capacitance compensation artifacts were blanked. Left, Bottom, Traces obtained by subtracting the average trace from the individual traces. Right, Nonstationary fluctuation analysis performed on K⁺ currents displayed on the left. N, Number of channels per patch; i, single-channel current; γ, single-channel conductance; P_o.m., maximal open probability. Continuous line indicates parabolic fit to data points. B, Collected data for parameters obtained from nonstationary fluctuation analysis (filled bars, experiments in DTX, n = 12; open bars, experiments without DTX, n = 9). C, Approximate ratios of steady-state availability at resting membrane potential, recruitment by the MFB–AP, single-channel conductance γ, and maximal open probability P_o.m. for K_v3 and K_v1 channels at MFBs.

Figure 5.

Absence of Na⁺-activated K⁺ channel-mediated currents at MFBs. A, Top, Voltage protocol applied to an MFB. Bottom, Current traces before (black) and after (red) wash-in of 1 μm TTX. Right, Same traces at higher temporal resolution. B, Collected data for current rise time and peak amplitude of four experiments of the kind in A, normalized to baseline. C, Top, MFB–AP wave applied to outside-out patches of MFBs. Middle, Averaged compound K_v current, isolated in TTX and Cd²⁺. Bottom, Composite current consisting of an inwardly directed Na⁺ and an outwardly directed K⁺ current component. Different MFB. D, Collected data of each eight recordings of isolated and composite currents as in C displaying rise times, peak current amplitudes, and half-durations.

For current-clamp recordings, a Multiclamp 700A was used, the internal filter was set to 20 kHz (33–34°C) or 30 kHz (36–37°C), and sampling frequency was 40 kHz (33–34°C) or 100 kHz (36–37°C); bridge-balance series resistance compensation was used. Data acquisition was performed using ClampEx 8.2 (Molecular Devices).

MFBs in the stratum lucidum of hippocampal CA3 were identified as reported previously (Geiger and Jonas, 2000; Alle et al., 2006). For extracellular stimulation of mossy fibers in the hilus, a patch pipette containing HEPES-buffered ACSF was used.

In experiments shown in Figure 5C (composite I_Na and I_K currents), the total duration of the recording (going whole-MFB, establishing the outside-out patch configuration and recording currents) was <3 min to preserve possible sodium-activated K⁺ channel (K_Na)-mediated currents in outside-out patches (Egan et al., 1992).

Data analysis.

All analyses of traces were done with ClampFit 10 (Molecular Devices). The paired-pulse ratio (see Fig. 1B) was calculated as ratio of the peak amplitudes of EPSC₂ and EPSC₁, with the peak amplitude measured from the current level immediately preceding the respective EPSC. The MFB–AP half-duration was determined as the time between rise and decay of the AP at 50% of the AP amplitude from a baseline of −80 mV and amounted to 312 ± 6 μs (n = 32) in control conditions (basal APs) at 33–34°C (at 36–37°C, the AP half-duration is ∼250 μs) (Alle et al., 2009). The baseline peak amplitude of the K_v-mediated currents in experiments bath applying 4-AP (see Fig. 2C) was multiplied by a factor of 0.86 to correct for a decrease over the standardized wash-in time (reduction to 0.86 ± 0.06 of initial amplitude; n = 4).

The data points obtained in activation and steady-state inactivation experiments (see Fig. 2E) were fitted using a Boltzmann sigmoidal function of the form y = bottom + (top − bottom)/(1 + exp((V_1/2 − x)/slope)), where y is G/G_max, x is the test potential or conditioning potential, and V_1/2 is the membrane potential at which half of the conductance was activated or inactivated. For inactivation curves, the slope value resulting from a fit to this equation is negative; however, in text the absolute value is given.

The recruitment of the DTX-sensitive current component from whole-MFB data was calculated as (I_max,wave − I_{max,wave DTX})/(I_max,s.p. − I_{max,s.p. DTX}) = 0.41 ± 0.15 nA/3.4 ± 0.54 nA ≈ 0.11 ± 0.01, where s.p. was the square pulse [given: means A, B with errors a, b; to be calculated: C with error c of the difference A − B and of the ratio A/B; C = A − B and c = Sqrt(a² + b²); and C = A/B and c = C Sqrt((a/A)² + (b/B)²), where Sqrt is “square root of”)]. To calculate the conductance recruitment MFB–AP wave versus square pulse, the current traces recorded from outside-out patches at 36–37°C in response to the MFB–AP wave were at first shifted 20 μs to the left, correcting the time lag introduced by the external filter. Then, the mean ± SEM of averaged current traces, which were normalized to the maximal amplitude, was determined for each time (i.e., sampling) point across experiments (see Fig. 3E, left). The MFB–AP wave-activated conductance at a time point t was calculated as G_wave(t) = I_wave(t)/(U(t) − U_rev), with U_rev = −100 mV.

The maximally recruited conductance (i.e., the fraction of available conductance activated or the channels opened by the AP compared with the square pulse) (see Fig. 3F) was calculated as wave/s.p. = f G(I_max,wave)/G_max,s.p., with f = G_max,wave/G(I_max,wave) (see Fig. 3E, bars on the right) to correct for the difference of time points of maximal conductance and current (see Fig. 3E, left), and with G(I_max,wave) = I_max,wave/(U(I_max,wave) − U_rev). The maximal square-pulse-activated conductance was taken as G_s.p. = I_max,s.p./(U(I_max,s.p.) − U_rev), with U(I_max,s.p.) = +40 mV. The value for the K_v1-mediated conductance was estimated as K_v1-mediated current recruitment of ∼0.11, multiplied with 0.43/0.26 (G_recruitment/I_recruitment of mixed K_v) or 0.78/0.45 (G_recruitment/I_recruitment of the K_v3 component) ≈ 0.18 or 0.19, indicating a low recruitment of K_v1 channels during the MFB–AP.

The fit function used to describe the data in Figure 4A–C for the nonstationary fluctuation analysis was σ²(I) = iI − I²/N + σ₀². σ represents variance, I the mean current, i the single-channel current, N the number of channels in the patch, and σ₀² the variance of the baseline noise (Sigworth, 1980; Li and Jonas, 2007). The analysis was performed for the first millisecond after the onset of the current in response to the square pulse.

For channels composed of K_v1.1 subunits, γ was reported to be 10 pS (Ji et al., 2002) and 5 pS for K_v1.4 (Stühmer et al., 1989). In contrast, for channels composed of K_v3.1, γ was reported to be 27 pS (Grissmer et al., 1992) and 14 pS for K_v3.4 (Schröter et al., 1991; Rettig et al., 1992). These values were determined at room temperature; assuming a Q₁₀ of ∼1.4 for open channel conductances (Hille, 2001) and a ΔT of 14 K, they would amount to 16 and 8 pS for K_v1 and 43 and 22 pS for K_v3 at 37°C, the latter values being consistent with 34 pS obtained from recordings in DTX isolating the K_v3 component. The extrapolated values for γ of K_v1 channels may justify the assumption of 17 pS in our calculations on the relative repolarization efficacy. The considerable difference in single-channel conductances of K_v1 and K_v3 channels might reflect adaptations to their respective membrane potential working ranges.

The number of channels per MFB was estimated from the relationships G_max,wave = f I_max,wave/(U(I_max,wave) − U_rev); thus, the respective AP-wave-activated peak conductances are ∼7 and 11 nS for K_v1 and K_v3, respectively. Furthermore, the number of channels n per MFB amounts to G_max,wave/(availability recruitment γ P_o.m.), where P_o.m. is maximal open probability; thus, for n(K_v1)/MFB = 7 nS/(0.75 * 0.2 * 17 pS * 0.7) ∼ 3922/MFB, for n(K_v3)/MFB = 11 nS/(0.94 * 0.79 * 34 pS * 0.7) ∼ 609/MFB, and in case of a single population consisting only of K_v1 channels, n(K_v1 only)/MFB = 17.3 nS/(0.75 * 0.2 * 17 pS * 0.7) ∼ 9692/MFB; 17.3 nS = 1.15 * 1.46 nA/(0.097 V).

The homogeneously distributed, AP-wave-activated peak K⁺ conductance density was determined from the respective current recorded in the whole-MFB configuration: G_max,wave = f I_wave,max/((U(I_max,wave) − U_rev) * Area) = 1.15 * 1.46 nA/(0.097 V * 180 μm²) = 96 pS/μm² = 9.6 mS/cm², assuming the average entire surface of MFBs (including filopodial extensions), which were selected for presynaptic voltage-clamp recordings to be ∼180 μm² (i.e., large boutons) (Alle and Geiger, 2006; Alle et al., 2009).

In the experiments of Figure 7, C and D, APs were recorded for 6 min starting from break-in and immediate switch to current clamp (AP half-duration stabilized after 3–5 min in the conditions of 10 μm 4-AP and 10 mm calcium chelator). The onset of BK_Ca- and K_v-mediated currents (I_BK and I_Kv) (see Fig. 7F and text) was determined by visual inspection of the respective average traces.

Membrane potential values for MFB recordings are given without correction for the error introduced by the liquid junction potential of the KCl-based internal solutions (∼3 mV). Statistical tests and fits to data points in Figure 2E were performed using Prism 4 (GraphPad Software). To assess statistical significance, the Mann–Whitney U test was used. Data in the text are given as mean ± SEM, and error bars in the figures also represent SEM.

Results

K_v1 and K_v3 contribute to fast MFB–AP repolarization

The experiment shown in Figure 1B replicates the observation of Rutecki et al. (1987) with respect to the effect of 10 μm 4-AP on CA3 pyramidal neuron EPSCs evoked by mossy fiber stimulation (Fig. 1A). In five experiments, the peak amplitude of EPSCs increased by a factor of 2.16 ± 0.37, whereas the paired-pulse ratio (EPSC₂/EPSC₁) decreased from 1.86 ± 0.26 to 1.45 ± 0.11, consistent with a presynaptic effect, as it was found for 10 μm 4-AP-induced increases of synaptic transmission at the CA3–CA1 pyramidal neuron synapse (Qian and Saggau, 1999).

To study the potential contribution of K_v3 and K_v1 channels to basal AP repolarization, we applied BDS-I, a K_v3 channel-selective gating modifier (Yeung et al., 2005), and/or DTX, a K_v1-selective channel blocker, focally to the MFB while recording APs evoked by local current injection (Fig. 1A,C). BDS-I (3 μm) increased the MFB–AP half-duration to 1.37 ± 0.04 (n = 4), DTX (0.2 μm) to 1.38 ± 0.03 (n = 4), and BDS-I (3 μm) plus DTX (0.2 μm) to 1.97 ± 0.15 (n = 3) of control (Fig. 1C). BDS-I at 0.5 μm prolonged the AP half-duration to 1.22 ± 0.02 (n = 4) and DTX at a concentration of 1 μm to 1.40 ± 0.07 (n = 4) of control (data not shown). Application of the HEPES-buffered ACSF carrier solution without toxins prolonged the AP half-duration to 1.06 ± 0.02 of control (n = 5) (Fig. 1C). Furthermore, we tested the 4-AP sensitivity of AP repolarization in MFBs. Focal application of 30 μm 4-AP increased the AP half-duration to 1.41 ± 0.03 (n = 4) (Fig. 1C). Bath application of 10 μm 4-AP broadened the MFB–AP to 1.28 ± 0.06 (n = 4) and 30 μm 4-AP to 1.46 ± 0.07 (n = 4) of control (Fig. 1D). In summary, BDS-I, DTX, and 10–30 μm 4-AP prolonged the half-duration of MFB–APs. Consistent with the BDS-I effect on the MFB–AP repolarization, we found that BDS-I (1 μm bath application) increased CA3 pyramidal neuron EPSCs evoked by mossy fiber stimulation to 1.89 ± 0.05 of control (n = 2) (data not shown).

K_v3 dominate the AP-wave-evoked presynaptic K⁺ current

To evaluate the relative current contributions by these two different K⁺ channel types during presynaptic APs at MFBs, we applied a previously recorded AP to MFBs in the whole-cell voltage-clamp recording configuration (Fig. 2A) (Alle et al., 2009). Focal application of BDS-I (3 μm) to the recorded MFB reduced the compound K⁺ current to 0.39 ± 0.04 (n = 4) and DTX (0.2 μm) to 0.55 ± 0.03 (n = 4) of control (Fig. 2A,B). Simultaneous focal application of both toxins at the above concentrations blocked the K⁺ current to 0.015 ± 0.015 (n = 4) (Fig. 2A,B) of control, indicating that the AP-wave-evoked current consisted of an entirely DTX-sensitive K_v1 and a larger K_v3 current component contributing to AP repolarization. AP-wave-evoked K⁺ currents in ACSF had peak amplitudes of 1.46 ± 0.13 nA (n = 16) and in separate experiments in ACSF containing 0.2 or 1 μm DTX of 1.05 ± 0.08 nA (n = 16; group comparison DTX vs ACSF: p < 0.001; block by 0.2 μm DTX, n = 7, vs 1 μm DTX, n = 9, was not different: p > 0.1, group comparison).

In contrast, presynaptic K_v3-mediated currents evoked by square-pulse depolarizations (≥30 ms to +40 mV) in DTX (0.2 μm) amounted to only ∼40% of the compound K_v current peak amplitudes in ACSF (2.5 ± 0.2 nA in DTX vs 5.9 ± 0.5 nA in ACSF, each n = 16; group comparison DTX vs ACSF: p < 0.001; block by 0.2 μm DTX, n = 7, vs 1 μm DTX, n = 9, was not different: p > 0.5, group comparison). Together, the contribution of K_v3 channels to the AP-wave-evoked K⁺ current at MFBs is larger than that of K_v1 channels, in contrast to their respective maximal conductances as indicated by the peak current amplitudes evoked by long depolarizing pulses.

The observed high 4-AP sensitivity of the AP repolarization at MFBs (Fig. 1C,D) raised the question on the 4-AP sensitivity of AP-wave-evoked currents. Bath application of 10 μm 4-AP reduced the peak amplitude of the AP-wave-evoked compound K⁺ current to 0.74 ± 0.05 (n = 4), 30 μm to 0.59 ± 0.01 (n = 4), and 100 μm to 0.29 ± 0.03 (n = 3) of control (Fig. 2C) (see Materials and Methods). 4-AP at 1 mm completely blocked the AP-wave-evoked compound K⁺ current (0.0 ± 0.0, n = 12) (Fig. 2C) (Alle et al., 2009). Experiments performed in ACSF containing 0.2 μm DTX and 30 μm 4-AP revealed a mean AP-wave-evoked K⁺ current peak amplitude of 0.29 ± 0.04 nA (n = 9) (Fig. 2D); thus, additional 30 μm 4-AP reduced the isolated K_v3-mediated current component by >50% compared with currents recorded in ACSF containing only DTX (see above; group comparison: p < 0.001). This indicates that 30 μm 4-AP is an upper boundary for the IC₅₀ value of the K_v3-mediated component.

Isolated K_v3-mediated currents at MFBs inactivated markedly (Fig. 2E, left) (in ACSF containing 1 μm DTX). The respective activation and steady-state inactivation properties of the K_v3 channels are shown in Figure 2E. The midpoint potentials and slope factors were −21.6 ± 0.8 mV and 12.3 ± 0.7 (n = 9) for activation, very similar to data for Kv3.1/3.4 subunit containing channels expressed in HEK293 cells (Baranauskas et al., 2003), and −54 ± 0.5 mV and 8 ± 0.5 (n = 5) for inactivation, respectively.

The percentage of available channels at a membrane potential of −80 mV was 94 ± 2% for K_v3 (Fig. 2E), whereas that for the mixed population of K_v1 and K_v3 amounted to ∼75% in a previous study (Geiger and Jonas, 2000), indicating an even lower fraction of available K_v1 channels. In addition, experiments in ACSF containing DTX (0.2 μm) revealed that the membrane potential of MFBs was found to be between −65 and −70 mV (measured immediately after establishing the whole-cell recording configuration) (our unpublished observations) compared with between −75 and −80 mV in ACSF (Geiger and Jonas, 2000; Alle and Geiger, 2006); in experiments using ACSF that contained 10 μm 4-AP, the resting membrane potential determined immediately after break-in was unaffected, consistent with the steady-state activation properties of K_v3 channels (Fig. 2E).

Highly different K_v1 and K_v3 channel recruitment during the MFB–AP

Having identified K_v1 and K_v3 channels as fast-activating voltage-gated K⁺ channel types becoming opened during basal presynaptic MFB–APs, we analyzed the fraction of the respective conductances at resting membrane potential that are activated by the AP, i.e., the recruitment of available channels. Recently, a surrogate parameter for this functional property has been determined, the current recruitment, calculated as the ratio of AP-wave-evoked versus square-pulse-evoked K⁺ current peak amplitude (Baranauskas et al., 2003; Martina et al. 2007). The current recruitment for the K_v3-mediated component amounted to 0.43 ± 0.02 in whole-MFB recordings (n = 16) and to 0.45 ± 0.04 in outside-out patch recordings (n = 4) (Fig. 3A–C), very similar to data for K_v3.1/3.4 heteromeric channels expressed in HEK293 cells (Baranauskas et al., 2003). The K_v1 current component at MFBs cannot be isolated at present, because there is no pharmacological tool available to selectively block K_v3 channels. BDS-I slows gating (Yeung et al., 2005) but in fact is not “blocking” K_v3 channels, thus having negligible effects on the peak amplitude of currents in response to long depolarizing pulses (Martina et al., 2007). Therefore, we compared the results of the isolated K_v3 component with that of the compound K⁺ channel population (K_v1 and K_v3). The recruitment of the compound K⁺ current by the MFB–AP amounted to 0.25 ± 0.02 in the whole-MFB configuration (n = 16) and to 0.26 ± 0.01 in the MFB outside-out patch configuration (n = 4; compared with K_v3 whole-MFB: p < 0.0001) (Fig. 3A–C). The current recruitment related specifically to K_v1 channels can be estimated from whole-MFB data by subtracting average peak current amplitudes obtained in DTX and ACSF (different sets of experiments; see Materials and Methods); this revealed a recruitment ratio of 0.11 ± 0.01.

To link the current recruitment to the conductance recruitment, information about the exact K⁺ current time course as function of the membrane potential during the AP is needed. Therefore, we analyzed K⁺ currents obtained in response to an MFB–AP waveform in DTX and without DTX in the outside-out patch recording configuration (Fig. 3E, left). In DTX, the membrane potential at the peak of the K⁺ current was −10 ± 2 mV (n = 5), and the calculated ratio of AP-wave-activated peak conductance and conductance at the peak of the K⁺ current G_max,wave/G(I_max,wave) amounted to f = 1.13 ± 0.04 (Fig. 3E, right). Similar experiments without DTX (n = 8) (data from Alle et al., 2009) yielded results of −3 ± 2 mV and f = 1.15 ± 0.07 (Fig. 3E, right). From these data, we calculated the fraction of available channels opened by the MFB–AP (see Materials and Methods). For the K_v3 channels, the recruitment amounted to 0.79 ± 0.07 (n = 5) (Fig. 3F) and for the compound K_v to 0.43 ± 0.02 (n = 4) (Fig. 3F). An estimate performed similarly to the K_v1 current recruitment resulted in conductance recruitment values for K_v1 channels at the MFB of 0.18–0.19, thus being four times less than that for the K_v3 component.

One likely explanation for this very different recruitment behavior of K_v1 and K_v3 channels at hippocampal MFBs is the different activation kinetics of the two channel types as observed, e.g., at the calyx of Held (Nakamura and Takahashi, 2007). Probably as a result of limited resolution at the small current amplitudes in outside-out patch recordings when applying the MFB–AP waveform, there was no appreciable difference in the current or conductance kinetics between the condition in DTX and without DTX (Fig. 3B,E). A clear difference, however, was observed for the rising phase of the currents in response to long depolarizing square pulses (Fig. 3B). The time constant of an exponential function fitted to the rising phase between 15 and 85% of maximal current amplitude was 198 ± 12 μs (n = 5) for the K_v3-mediated current and 400 ± 33 μs (n = 4; p < 0.02) (Fig. 3D) for the compound K⁺ current. Together, K_v3 channels are recruited four times better than K_v1 channels at hippocampal MFBs in response to an AP-wave-like depolarization.

Single-channel conductance of K_v3 channels keeps total presynaptic K_v density low

To address whether single-channel properties of K_v3 channels at MFBs are adapted to high membrane conductance states during APs in addition to their high availability at negative membrane potentials and good recruitment by the AP, we performed nonstationary fluctuation analysis of K_v3-mediated currents in DTX (Fig. 4A). Again, we compared these results with experiments done without DTX, being aware that this is only a tentative indication of K_v1 channel properties (Fig. 4B). The single-channel conductance γ of the K_v3 component amounted to 34 ± 5 pS and the maximal open probability P_o.m. to 0.70 ± 0.04 (n = 12) (Fig. 4A,B). The apparent single-channel conductance γ of the mixed population of K_v3 and K_v1 channels was 20 ± 2 pS, and the apparent maximal open probability P_o.m. was 0.70 ± 0.02 (n = 9). Both the single-channel conductance and the number of channels per outside-out patch were significantly different (p < 0.05), whereas the maximal open probability was not different (p > 0.8) (Fig. 4B). The comparatively large number of K_v3 channels per patch in DTX (Fig. 4 B) could have resulted from selection of patches with large peak current amplitudes in these experiments or channel clustering with clusters of different subfamilies having different localizations at and around the MFB (for K_v1.4-subunit-containing channels in mossy fibers, see Cooper et al., 1998). Taking availability, recruitment, single-channel conductance, and maximal open probability into account, we estimate that K_v3 channels at MFBs have an ∼10 times higher AP repolarization efficacy per channel than K_v1 channels (Fig. 4C).

How do these highly different repolarization efficacies translate into physical densities of channels necessary to perform the AP repolarization? Taking the contribution of K_v1 and K_v3 channels to the MFB–AP-wave-evoked peak current amplitude as ∼0.4 and ∼0.6 (Fig. 2A) and the K⁺ current peak amplitude in MFBs as 1.46 nA, then the absolute contributions are 0.58 and 0.88 nA, respectively. Accordingly, the AP-wave-activated peak conductances G_AP are ∼7 and 11 nS. The number of channels per MFB necessary to achieve these AP-wave-activated conductances amounts to ∼3900/MFB or ∼22/μm² for K_v1 channels, assuming the single-channel conductance to be half of that of K_v3 channels (see Materials and Methods), and for K_v3 to ∼600/MFB or 3.3/μm² (for densities in channels per square micrometer, a homogeneous channel distribution across the MFB surface was assumed). What if the repolarization of the MFB–AP as observed in control conditions would have to be performed solely by K_v1 channels? Ignoring the slower activation kinetics and longer activation delay of the K_v1 channels at MFBs compared with the K_v3 channels (Fig. 3B), at least ∼9700 K_v1 channels per MFB or 54/μm² ought to be present, comparable with the number of 41 Na_v channels/μm² at MFBs (Engel and Jonas, 2005). In consequence, the additional expression of K_v3 channels at hippocampal MFBs reduces the number of fast-activating K⁺ channels necessary to achieve the maximal MFB–AP-wave-activated K⁺ conductance density for fast repolarization by ∼50%.

Lack of evidence for Na⁺-activated K⁺ channels at MFBs

The experimental conditions used to evaluate the contribution of both K_v1 and K_v3 to the AP-wave-evoked compound K⁺ current (TTX, cadmium) (Fig. 2A,B) might have obscured the contribution of other fast-activating K⁺ channels, especially K_Na or BK_Ca. This could also explain the modest increase of the MFB–AP half-duration in current-clamp recordings in response to focal application of DTX or BDS-I (Fig. 1C).

To test for the presence of K_Na channels, we used a protocol to maximally activate currents mediated by these channels (Hess et al., 2007) in whole-MFB recordings (Fig. 5A). However, the K⁺ outward current did not differ from that in TTX (Fig. 5 A,B) (n = 4). In addition, we compared K⁺ currents obtained in TTX and cadmium (data from Alle et al., 2009) with the outward current component of currents obtained in experiments without TTX (Fig. 5C), both recorded in response to the same MFB–AP waveform in outside-out patches drawn from MFBs. The kinetic parameters and the peak current amplitudes did not differ between the two conditions (each condition n = 8; p > 0.15 for each parameter) (Fig. 5D). The isolated sodium current and the inward current component of the combined Na⁺ and K⁺ current did not differ, either (p > 0.15 for half-duration, rise time, and peak current amplitude). The lack of electrophysiological evidence for K_Na-mediated currents in rat hippocampal MFBs is consistent with immunohistochemical studies on Slick (Slo2.1) and Slack (Slo2.2) (Bhattacharjee et al., 2002, 2005), which did not show positive antibody staining in the mossy fiber tract of rat hippocampus.

MFBs contain at least two populations of presynaptic BK_Ca channels

In contrast to the absence of anatomical and functional evidences for a K_Na in MFBs, there is clear antibody staining against the α-subunit of BK_Ca (Slo1) in the mossy fiber system (Knaus et al., 1996). When drawing outside-out patches from MFBs, large single-channel openings can readily be seen at steady-state membrane potentials of ∼0 mV (Fig. 6A). They were sensitive to 10 μm Pax (n = 7 patches), a broad-spectrum BK_Ca channel blocker, but not to 1 μm IBTX (n = 7) (Fig. 6A), which blocks BK_Ca channels containing β-subunits other than β4 (Meera et al., 2000). The single-channel conductance γ of BK_Ca channels found in outside-out patches amounted to 83 ± 2 pS (n = 13; reversal potential assumed to be −100 mV). Recordings performed in the whole-MFB configuration in the presence of TTX (1 μm) and 4-AP (1 mm) revealed Pax-sensitive (10 μm; n = 4), IBTX-sensitive (1 μm; n = 7), and cadmium-sensitive (200 μm; n = 7) currents (Fig. 6 B,C) (focal application of blockers). The first current component exhibited fast activation and fast inactivation (mean peak amplitude, 2.7 ± 0.3 nA; n = 18), indicative of BK_Ca channels containing β2- and/or β3-subunits (Wallner et al., 1999; Xia et al., 2000); the second component was slowly activating, persistent, and only partially IBTX sensitive (mean peak amplitude, 1.8 ± 0.2 nA; n = 18) (Fig. 6B,C,F), indicating the contribution of channels containing β4 subunits, but not β-subunits conferring fast inactivation. The first BK_Ca-mediated current component represents 30% of the total K_v1 + K_v3 + BK_Ca-mediated presynaptic K⁺ current peak amplitude, approximately three times more than at the calyx of Held (Ishikawa et al., 2003).

From the mean peak amplitudes of the two components, the single-channel conductance (assuming a similar γ for the respective channels), and an MFB surface of 180 μm² (see Materials and Methods), a lower density boundary of homogeneously distributed BK_Ca channels of ∼3/μm² can be estimated.

The compound BK_Ca channel-mediated currents were sensitive to the P/Q-type Ca_v blocker ω-agatoxin (1 μm), to the N-type Ca_v blocker ω-conotoxin (3 μm), and to nickel (200 μm; each blocker n = 6; focal application) (Fig. 6D). The sensitivity distribution of the BK_Ca-mediated currents was shifted to the P/Q-type Ca_v blocker for the fast activating/fast inactivating component (0.84 ± 0.04) compared with that of Ca_v-mediated currents found at the MFB (P/Q-type Ca_v blocker, 0.61 ± 0.03) (Li et al., 2007), in contrast to the slowly activating BK_Ca current component (P/Q-type, 0.68 ± 0.06). This is likely attributable to the different activation kinetics of the Ca_v subtypes (Li et al., 2007), with the P/Q-type being the fastest. The fast and slowly activating BK_Ca channel-mediated current components activated at approximately −20 and −30 mV, respectively (n = 3) (Fig. 6E), and decreased in amplitude above 0 mV, consistent with Ca_v current–voltage relationships recorded at MFBs (Li et al., 2007). Compared with the compound K_v1- and K_v3-mediated current at the MFB, the fast activating/fast inactivating BK_Ca channel-mediated current component inactivated faster (Fig. 6F); for a comparison regarding Ca_v-mediated current amplitude and kinetics, an example average trace (square-pulse depolarization to 0 mV) is indicated in Figure 6F. Together, MFBs contain at least two populations of BK_Ca channels, one containing β2- and/or β3-subunits, another one containing β4-subunits.

Presynaptic BK_Ca become activated when K_v3 channels are disabled

Focal application of IBTX (1 μ)m (Fig. 7A) or Pax (10 μm) to MFBs did not increase the half-duration of presynaptic APs (Fig. 7B) (1.09 ± 0.04 of control for IBTX, n = 4; 1.04 ± 0.01 of control for Pax, n = 4; both results were not different from application of the HEPES-buffered ACSF carrier solution alone, p > 0.5). IBTX + BDS-I (3 μm) led to an increase of the MFB–AP half-duration to 1.95 ± 0.09 (n = 3), but IBTX + DTX (0.2 μm) to only 1.41 ± 0.03 (n = 4) of control, and IBTX + DTX + BDS-I to only 1.91 ± 0.12 (n = 3) (Fig. 7B), similar to respective results with DTX and DTX + BDS-I (Fig. 1C), respectively. Because this differential dependence of the IBTX effect on K_v3 versus K_v1 was not expected, we focally applied DTX to MFBs in the presence of 0.2 mm cadmium in the ACSF and 10 mm of the calcium chelator BAPTA in the intracellular recording solution to exclude unspecific inhibition of IBTX action in the presence of DTX; again, the increase of the MFB–AP half-duration amounted to only 1.43 ± 0.03 of basal MFB–AP half-duration (n = 3) (data not shown). In contrast, focal application of IBTX + 10 μm 4-AP to MFBs broadened the AP to 1.59 ± 0.04 (n = 4; data not shown) relative to the AP half-duration in 10 μm (corresponding to an AP half-duration increase by a factor of ∼2 relative to the basal MFB–AP half-duration without drugs), corroborating the results when coapplying the BK_Ca- and K_v3-specific toxins.

In line with the lack of an IBTX-effect on the MFB–AP half-duration, 10 mm BAPTA in the intracellular solution did not increase the half-duration (1.03 ± 0.04 after 6 min whole-MFB compared with the initial half-duration; n = 3) (Fig. 7C,D). However, 10 μm 4-AP in the ACSF and 10 mm BAPTA in the intracellular recording solution increased the half-duration after 6 min whole-MFB to 1.67 ± 0.11 (n = 4) (Fig. 7C,D) relative to the initial half-duration in 10 μm 4-AP (corresponding to an AP half-duration increase by a factor of ∼2 relative to the basal MFB–AP half-duration without drugs). The calcium chelator EGTA at 10 mm in the intracellular solution increased the half-duration to a similar extent as did BAPTA (to 1.73 ± 0.09 after 6 min whole-MFB relative to the initial half-duration in 10 μm 4-AP; n = 4) (Fig. 7C,D).

In accordance with above results, we found no IBTX-sensitive outward K⁺ current component in 4-AP (1 mm) and TTX (1 μm) when applying a basal MFB–AP waveform in the voltage-clamp configuration (Fig. 7E, left) (n = 3 for IBTX; n = 3 for Pax; all six experiments gave a mean peak current amplitude of −0.04 ± 0.03 nA) (data not shown). Increasing the half-duration of the AP waveform to ∼700 μs in the respective same MFB recordings revealed a prominent IBTX-sensitive outward current (Fig. 7E, right) (n = 3 for IBTX; n = 3 for Pax; all six experiments gave a mean peak current amplitude of 0.27 ± 0.04 nA) (data not shown). The onset of the BK_Ca channel-mediated current in response to the broader AP waveform of Figure 7E occurred at considerably depolarized membrane potentials, at a time when the basal AP was already more or less completely repolarized (Fig. 7E, inset). In the continuous presence of a nonphysiologically high intra-MFB free calcium concentration (100 μm), BK_Ca channels activated already during the rising phase of the basal MFB–AP wave, considerably earlier than delayed-rectifier K_v channels during the same waveform (50 ± 6 μs earlier at 33–34°C, n = 3) (Fig. 7F, traces scaled to the same amplitude), indicating that the activation delay of BK_Ca in these conditions is shorter than that of presynaptic K_v. In summary, we found no evidence that BK_Ca become activated during basal MFB–APs, which seem to be too brief for sufficient coincident membrane depolarization and intracellular calcium rise at the BK_Ca. EGTA was as effective as BAPTA in preventing BK_Ca activation during broader-than-basal APs, indicating that BK_Ca and Ca_v are not organized in nanodomain protein complexes (Fakler and Adelman, 2008) at MFBs.

Discussion

Our results reveal that the properties of presynaptic K_v3 are adapted to perform brief APs at low channel densities, extending the observation of efficiency in MFB–AP generation from the immediate electrical signaling (Alle et al., 2009) to infrastructure. The brevity of MFB–APs and the apparent lack of close spatial Ca_v–BK_Ca interactions may explain why presynaptic BK_Ca are not activated during basal MFB–APs, resulting in minimized interferences of a presumed rescue-type channel with presynaptic calcium signaling and transmitter release.

K_v conductance densities underlying the AP at MFBs

The immediate AP costs close to the theoretical minimum were shown previously to depend on a fast Na⁺ conductance decay compared with the repolarization kinetics of the MFB–AP, which results from a considerable and precisely matched onset delay of the repolarizing K⁺ conductance. Simulations using experimentally derived conductance time courses predicted AP-wave-activated peak conductance densities necessary to achieve the experimentally observed MFB–AP repolarization kinetics (Alle et al., 2009). Calculations using the average AP-wave-evoked compound K⁺ current peak amplitude (see Results) and surface area of MFBs selected for presynaptic voltage-clamp recordings (Alle and Geiger, 2006; Alle et al., 2009) yielded an AP-wave-activated G_K,max of 9.6 mS/cm² (this study; see Materials and Methods), in close agreement with a predicted value of 7.8 mS/cm² (Alle et al., 2009).

K_v3 at MFBs and other (glutamatergic) presynaptic elements

APs leading to transmitter release at glutamatergic terminals have half-durations in the order of 190–250 μs (Taschenberger and von Gersdorff, 2000; Alle et al., 2009). In the non-myelinated part of layer 5 pyramidal cell axons, half-durations of 250 μs have been recorded (Kole et al., 2007), suggesting comparable presynaptic AP half-durations. Thus, glutamatergic boutons require distinct K⁺ channel densities and/or expression patterns compared with other cellular compartments, such as somata and dendrites, in which APs decay slower. Studies on the calyx of Held (Ishikawa et al., 2003) and CA3 pyramidal cell axons (Qian and Saggau, 1999; Hu et al., 2001) together with our findings suggest that glutamatergic presynaptic elements contain fast-activating channels of the families K_v1, K_v3, and BK_Ca. The same constellation of K⁺ channel families is found at GABAergic synapses of fast-spiking interneurons onto glutamatergic layer II/III neocortical pyramidal cells (Goldberg et al., 2005), but the functional roles of the K⁺ channel families differ from that at the MFB.

Which K_v3 subunits might constitute the K_v3 channels mediating the BDS-I-sensitive current in response to MFB–AP waveforms? K_v3-mediated currents recorded at MFBs exhibit inactivation in response to long depolarizing voltage steps, which is consistent with the presence of K_v3.3 and K_v3.4 subunits detected immunohistochemically in hippocampal granule cells (Veh et al., 1995; Coetzee et al., 1999; Chang et al., 2007); furthermore, these currents activate at approximately −50 mV, which is consistent with the presence of K_v3.4 subunits, because they lead to a substantial membrane potential left-shift of channel activation in K_v3.1/3.4 heteromers compared with channels lacking K_v3.4 (Baranauskas et al., 2003).

A noteworthy property of K_v3-mediated currents in response to MFB–AP waveform is their high sensitivity to 4-AP, which is consistent with the observation of a high 4-AP sensitivity of transmitter release at the MFB–CA3 pyramidal neuron synapse. Sensitivity of presynaptic K⁺ currents to 4-AP in the low micromolar concentration range was also found at the calyx of Held for a major current component [IC₅₀ of 17 μm (Ishikawa et al., 2003)] and at cerebellar GABAergic basket cell terminals [IC₅₀ of 8 μm (Southan and Robertson, 2000)].

The properties of native K_v3-mediated currents at MFBs discussed so far are compatible with K_v3.3/K_v3.4 subunits, but the 4-AP sensitivities of currents mediated by K_v3.3 and K_v3.4 homomers expressed in heterologous systems are substantially higher (Gutman et al., 2005). One explanation might be that K_v3.3 and/or K_v3.4 subunits in native K_v3 channels at MFBs are more sensitive to 4-AP than homomeric channels expressed in heterologous expression systems. Alternatively, the high 4-AP sensitivity of K⁺ currents that are evoked by the MFB–AP waveform might suggest the presence and contribution of K_v3.1 subunits, because K_v3.1 homomeric channels have been reported to exhibit an IC₅₀ of 29 μm for 4-AP (Grissmer et al., 1994). In hippocampal granule cells, in situ hybridization signals for K_v3.1 mRNA are documented (Kues and Wunder, 1992; Weiser et al., 1994). However, K_v3.1 protein has stayed undetected in immunohistological experiments (Sekirnjak et al., 1997; Chang et al., 2007), which might be attributable to either very low levels of protein expression or the inability by the antibody to detect K_v3.1 protein in native heteromeric K_v3 channel complexes.

Candidates of cortical neurons expressing a similar pattern of K_v3 subunits are layer 2/3 glutamatergic cells of the entorhinal cortex giving rise to the perforant path (Veh et al., 1995; Zahn et al., 2008) and neurons in the piriform cortex (Weiser et al., 1994). In contrast, neocortical glutamatergic neurons in rodents seem to lack K_v3 expression (Weiser et al., 1994; Rudy and McBain, 2001). However, our results suggest that the K_v3 channel density at the presynaptic site, which is required to keep APs brief, is very low (three to four K_v3 channels/μm²). Because the surface of single neocortical en passant boutons is only ∼0.5–1 μm² (Braitenberg and Schüz, 1998) and GABAergic interneurons contain high amounts of K_v3 mRNA and protein (Rudy and McBain, 2001), respective small signals in glutamatergic neurons might have stayed undetected in anatomical studies.

Alternatively, these glutamatergic neurons may have found different solutions for the fast repolarization of their presynaptic APs, e.g., by high densities of K_v1 channels (see axonal recordings in the study by Kole et al., 2007). However, apart from being activated during APs, K_v1 channels also contribute to the resting membrane potential at the axon and presynaptic elements, controlling excitability (Smart et al., 1998; Dodson et al., 2003; Ishikawa et al., 2003) and the steady-state availability of Na_v channels. Thus, K_v1 with their comparatively high density act as multipurpose channels, whereas K_v3 and BK_Ca at considerably lower densities seem to have more restricted functions.

BK_Ca at MFBs and other (glutamatergic) presynaptic elements

Our observation that presynaptic BK_Ca do not become activated during MFB–APs under basal conditions is consistent with results in acute brain slices at the hippocampal CA3–CA1 pyramidal neuron synapse (Hu et al., 2001) and at the calyx of Held–medial nucleus of trapezoid body principal cell synapse (Ishikawa et al., 2003) (but for the CA3–CA3 pyramidal neuron synapse in hippocampal slice cultures, see Raffaelli et al., 2004). A minor contribution of BK_Ca to presynaptic AP repolarization at GABAergic synapses of fast-spiking interneurons onto layer II/III neocortical pyramidal neurons can be inferred from an effect of BK_Ca blocker on synaptic transmission (Goldberg et al., 2005).

The lack of BK_Ca activation during basal MFB–APs likely results from the brevity of the AP caused by powerful K_v channels and the lack of nanodomain complexes of Ca_v and BK_Ca, concluded from the equally effective action of EGTA and BAPTA (Fakler and Adelman, 2008) on pre-broadened APs. At the hippocampal granule cell soma, a close spatial arrangement was suggested for Ca_v and BK_Ca (Müller et al., 2007), possibly ensuring BK_Ca activation in a cellular compartment with low Ca_v and/or BK_Ca densities. At MFBs, BK_Ca become activated in response to reduced K_v3 channel availability, as could occur during certain modulatory states (Rudy and McBain, 2001) or hypoxia (Patel and Honoré, 2001). In line with this, the compound K_v peak current amplitude at MFBs in response to an MFB–AP wave is reduced to 0.50 ± 0.02 of baseline when transiently exposing the tissue slice to 95% argon/5% CO₂ (n = 4) (our unpublished observations). The differential effect of IBTX + BDS-I and IBTX + DTX on AP half-duration increase (Fig. 7) is not understood at present, but it might suggest subtle, unresolved differences in AP shape alteration during K_v3 and K_v1 block or a functional coupling of presynaptic K_v3 and BK_Ca beyond a simple interplay via an altered voltage waveform; this, however, remains to be studied.

Thus, presynaptic BK_Ca channels at glutamatergic synapses studied so far may act indeed as “emergency brakes” regarding neurotransmitter release (Hu et al., 2001), in contrast to GABAergic synapses onto neocortical pyramidal neurons (Goldberg et al., 2005), suggesting that basal presynaptic AP half-duration at these synapses might be slightly longer than at the MFB and calyx of Held and/or that there is a spatially close coupling of calcium sources and BK_Ca channels. The presumed lack of Ca_v–BK_Ca nanodomain complexes at the MFB could result from the need of close spatial interactions between Ca_v and calcium sensors of the vesicle release machinery (although this varies across presynaptic elements of different synapses) (Augustine et al., 2003). Concerning the hippocampal MFB, this interpretation is consistent with the observation that 10 mm EGTA do not prevent glutamate release (Alle and Geiger, 2006).

Together, the results of this study show that specialized voltage-activated K⁺ channels of the K_v3 family at a low channel density efficiently contribute to the presynaptic AP repolarization at hippocampal MFBs and that the AP repolarization is backed up by BK_Ca channels also at low densities, which are designed and arranged not to interfere with basal AP repolarization.