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Department of Physiology, David Geffen School of Medicine at UCLA, Los Angeles, CA 90095, USA

	Abstract

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Single channel properties of Ca²⁺-activated K⁺ (BK or Maxi-K) channels have been investigated in presynaptic membranes in Xenopus motoneurone–muscle cell cultures. The occurrence and density of BK channels increased with maturation/synaptogenesis and was not uniform: highest at the release face of bouton-like synaptic varicosities in contact with muscle cells, and lowest in varicosities that did not contact muscle cells. The Ca²⁺ affinity of the channel (K_d= 7.7 µM at a membrane potential of +20 mV) was lower than those of BK channels that have been characterized in other terminals. Hill coefficients varied between 1.5 and 2.8 at different potentials and open probability increased e-fold per 16 mV change in membrane potential over a range of [Ca²⁺]_i from 1 µM to 1 mM. The maximal activation rate of ensembled single BK channel currents was in the submillisecond range at +20 mV. The activation rate increased 10-fold in response to a [Ca²⁺]_i increase from 1 to 100 µM, but increased only 2-fold with a voltage change from +20 to +130 mV. The fastest activation kinetics of BK channels in cell-attached patches resembled that in inside-out patches with [Ca²⁺]_i of 100 µM or more, suggesting that many BK channels are located very close to calcium channels. Given the low Ca²⁺ affinity and rapid Ca²⁺ binding/unbinding properties, we conclude that BK channels in this preparation are adapted to play an important role in regulation of neurotransmitter release, and they are ideal reporters of local [Ca²⁺] at the inner membrane surface.

(Received 8 January 2004; accepted after revision 25 March 2004; first published online 26 March 2004)
Corresponding author A. D. Grinnell: Department of Physiology, David Geffen School of Medicine at UCLA, Los Angles, CA 90095-1751, USA.  Email: adg{at}ucla.edu

	Introduction

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Large conductance calcium-activated potassium (BK or Maxi-K) channels are found in many nerve terminal membranes, where they are associated with voltage-gated Ca²⁺ channels and help modulate release (Mallart, 1985; Lindgren & Moore, 1989; Roberts et al. 1990; Sivaramakrishnan et al. 1991; Robitaille & Charlton, 1992; Wang et al. 1992; Robitaille et al. 1993; Wangemann & Takeuchi, 1993; Bielefeldt & Jackson, 1993; Issa & Hudspeth, 1994; Blundon et al. 1995; Knaus et al. 1996; Katz et al. 1997; Sakaba et al. 1997; Sun et al. 1999; Womack & Khodakhah, 2002). Blockage of BK channels with the peptide toxins charybdotoxin (CTX) or iberiotoxin (IBTX) causes broadening of action potentials and usually increases transmitter release (Morita & Barrett, 1990; Pattillo et al. 1999; Faber & Sah, 2003), suggesting that a prominent role is the truncation of action potentials and reduction in Ca²⁺ entry and release. In other cases, BK channels may cause more subtle changes in action potential shape that enhance release (Pattillo et al. 1999, 2001; Van Goor et al. 2001).

BK channels are also functionally coupled with Ca²⁺ channels in the bouton-like synapses formed by Xenopus motoneurone neurite varicosities on muscle cells in culture; this preparation is one of the very few in which it is possible to patch clamp both pre- and postsynaptic membranes simultaneously to correlate presynaptic currents with neurotransmitter release during synaptic activity (Yazejian et al. 1997, 2000; Sand et al. 2001; Pattillo et al. 2001). The Xenopus BK channel gene has recently been cloned. It is expressed in five alternatively spliced variants, including one, xSlo59, which is neurone-specific, is not expressed until late in neuronal differentiation, and differs from other variants in being activated at lower voltages (Kukuljan et al. 2003). It is probably this neurone-specific BK channel that is found in the Xenopus motoneurone terminals in culture.

For these channels to be useful in modulation of release in fast-releasing synapses, it is necessary that they be closely colocalized with Ca²⁺ channels, have activation kinetics in the submillisecond range and have a Ca²⁺ affinity appropriate to concentrations that might occur in or near microdomains at active zones, without saturating. BK channels that have been characterized biophysically tend to vary greatly in their kinetics and Ca²⁺ and voltage sensitivity (for reviews see McManus, 1991; Vergara et al. 1998; Gribkoff et al. 2001; Sah & Faber, 2002). Those found in non-neuronal membranes, or even in neuronal cell bodies, tend to exhibit activation kinetics too slow and/or Ca²⁺ affinity too high (Ikemoto et al. 1989; Wang et al. 1992; Bielefeldt & Jackson, 1993; Safronov & Vogel, 1998) to respond to changes in [Ca²⁺] on the submillisecond time scale between 1 µM and >100 µM. In contrast, the few BK currents (I_BK) that have been studied in ‘fast’-transmitting nerve terminals (Sakaba et al. 1997; Sun et al. 1999) or terminal-like cells (e.g. hair cells, Art et al. 1995) have shown significantly faster kinetics and often lower Ca²⁺ affinity. Interestingly, cloned Slo-1 channels expressed in Xenopus oocytes exhibit Ca²⁺ affinity close to that of nerve terminals (Xia et al. 2002). BK channels in the Xenopus varicosity synapses are activated so rapidly by the combination of depolarization and Ca²⁺ influx that whole-terminal BK currents can track changes in local Ca²⁺ concentration during action potentials or Ca²⁺ tail currents with delays of no more than a few tens or hundreds of microseconds (Yazejian et al. 2000).

The reported close functional coupling between Ca²⁺ channels and BK channels has also led to use of the latter to quantify the [Ca²⁺] achieved during depolarization in frog and turtle hair cells (Roberts et al. 1990; Art et al. 1995), bipolar cell terminals (Sakaba et al. 1997), and rat chromaffin cells (Prakriya & Lingle, 1999). This method, in principle, has many advantages over using Ca²⁺-sensitive dyes to measure [Ca²⁺], since BK channels are endogenous molecules located in the membrane near Ca²⁺ channels and their level of activation can be studied without introduction of extrinsic Ca²⁺ binding molecules or washout of endogenous buffers or essential cofactors.

To clarify the kinetic basis of BK channel activity during action potential invasion and to validate the usefulness of BK channels in reporting, quantitatively, changes in [Ca²⁺] near release sites in nerve terminals, we have investigated single channel sensitivity to [Ca²⁺]_i and to voltage, the degree of coupling to Ca²⁺ channels and the responses of BK channels to transient membrane potential changes at different [Ca²⁺]. We find that these nerve terminal BK channels exhibit lower Ca²⁺ affinity and faster activation kinetics than most others studied to date, that the time constant of BK channel activation is a more accurate reflection of [Ca²⁺] than is channel open probability, and that differences in activation time constant can distinguish [Ca²⁺] over a range from 0.2 µM to >100 µM. The data support the conclusions that BK channels are closely colocalized with Ca²⁺ channels, that they can be used to accurately measure [Ca²⁺] during Ca²⁺ transients, and that they can contribute importantly to shaping the terminal action potential.

	Methods

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Preparation of motor neurone and muscle cell coculture

Motoneurone–muscle cell cocultures were prepared as previously described (Spitzer & Lamborghini, 1976). Stage 19–22 Xenopus laevis embryos (Niewkoop & Faber, 1967) were rinsed in sterile 10% normal frog Ringer solution (NFR) (mM: 116 NaCl, 1 NaHCO₃, 2 KC1, 1.8 CaCl₂, 1 MgCl₂, 3 D-glucose, pH 7.3), and the spinal cord and associated myotomes were dissected away and allowed to dissociate in a Ca²⁺- and Mg²⁺-free Ringer solution for 30–60 min. Dispersed cells were then plated onto glass coverslip-bottomed Petri dishes (Falcon 1008) and incubated at room temperature for 24–28 h in a medium composed of 49% NFR and 51% L-15 (Life Technologies, Gaithesberg, MD, USA) supplemented with 3 mg ml^–1 glutamine, 0.1 mg ml^–1 insulin, 0.7 mg ml^–1 selenite, 0.6 mg ml^–1 transferrin, 1 mg ml^–1 sodium pyruvate and, in some preparations, 35 ng ml^–1 brain-derived neurotropic factor (BDNF, kindly provided by AMGEN, Thousand Oaks, CA, USA or Neurogen, Branford, CT, USA). BDNF enhances neuronal survival and neurite growth, but did not alter the properties of varicosity synapses in any perceptible way.

Neurone–muscle cell cultures were normally studied approximately 24 and 48 h after plating, although some cultures were also studied after 3 or 4 days. Neurites begin to grow out within 12 h in culture and continue to grow for several days. Hence synapses can form at any time. Since it was not feasible to know when any given neurite–muscle cell contact was first made, varicosities were characterized simply by the age of the culture: 1, 2, 3, or 4 days old. Varicosity synapses on muscle cells in the cultures develop at approximately the same rate they would in vivo (Kullberg et al. 1977), and exhibit pre- and postsynaptic morphological and physiological specializations characteristic of vertebrate neuromuscular junctions (Weldon & Cohen, 1979; Cohen & Weldon, 1980; Kidokoro et al. 1980; Brehm et al. 1984; Buchanan et al. 1989; Evers et al. 1989; Takahashi et al. 1996). The specializations for synaptic transmission develop over a period of many hours. Some apparent synaptic contacts were inevitably so recently formed that they would be expected to show only low levels of evoked neurotransmitter release and limited expression of synapse-enriched proteins, hence little or no presynaptic I_BK. Moreover, some neurones in the cultures are not motoneurones. There is a gradation in neuronal sizes, and frequently synaptic currents are observed in neurones, presumably reflecting innervation by interneurones. Neurites also frequently appear to avoid contact with muscle cells. When muscle cells are manipulated into contact with neuronal processes, it has been found that about 70% release ACh, and hence are presumptive motoneurones (Cohen & Weldon, 1980; Chow & Poo, 1985). In our experiments, we selectively patched varicosities formed by large, spherical neurones. In approximately 74% (n= 78) of the cases where both pre- and postsynaptic cells were patched, curare-blockable postsynaptic currents could be evoked. Thus we feel confident that most of our presynaptic recordings were from motoneurones.

Three types of varicosities are accessible for patch pipette recording: (1) ‘isolated varicosities’ that are not in contact with muscle cells; (2) the exposed surfaces of varicosities in contact with muscle cells (‘synaptic varicosities’); and (3) the inner face (‘release face’) of synaptic varicosities, apposed to a muscle cell. The release face can sometimes be accessed by gently pushing the muscle cell away with the patch pipette. Within 1–2 days in culture, varicosities in contact with muscle cells exhibit pre- and postsynaptic morphological specializations and release properties similar in most respects to mature neuromuscular synapses (Weldon & Cohen, 1979; Buchanan et al. 1989; Yazejian et al. 1997) as well as physiological evidence of presynaptic active zones (DiGregorio et al. 1999).

Recording

Voltage-clamp experiments were performed with Axopatch 200B (Axon Instruments, Union City, CA) or EPC-7C patch-clamp amplifiers at room temperature. Ionic currents were studied using perforated whole-terminal and single channel voltage clamp techniques. Patch electrodes were fabricated from borosilicate glass (1.5 mm outer diameter) with a Flaming-Brown horizontal puller (P-87, Sutter Instrument Co, Novato, CA, USA). Electrodes were fire-polished and coated with beeswax to reduce pipette capacitance. After filling, electrodes had a final resistance of 2–5 M for perforated whole-terminal and 7–15 M for single channel recording.

Whole-terminal current recording from varicosities

Perforated whole-terminal recording was done as previously described (Yazejian et al. 2000). Briefly, amphotericin B (Sigma, St Louis, MO, USA) was freshly dissolved in dimethyl sulfoxide (DMSO; 60 mg ml^–1) and further diluted with internal solution containing (mM): 52 K₂SO₄, 38 KCl, 1 EGTA and 5 Hepes, pH 7.3, to yield a final amphotericin B concentration of 240 µg ml^–1. A series resistance of <15 M was reached within 10 min after the formation of a gigaohm seal (seal resistance, >2 G) and remained stable for up to 0.5–1 h. Series resistance compensation was optimized. All current recordings were corrected for linear leakage resistance and capacitance by using a P/–N procedure. The average varicosity membrane capacitance (C_m) was 4.6 ± 0.2 pF. The bath solution was glucose-free NFR, containing 1 mM 3,4-dihydropyridine (DAP) and 300 nM TTX (pH 7.3). Pulse generation, data acquisition and analysis were done with a PC equipped with a Digidata 1200 or 1322A analog-to-digital (A/D) interface in conjunction with Clampex 6–9.0 programs (Axon Instruments). Currents were filtered with a 4-pole Bessel Filter at 10 kHz. Unless otherwise specified, the holding potentials were –70 mV (varicosity) and –80 mV (muscle cell).

Single channel recording

Single-channel currents were recorded in both cell-attached and inside-out patch configurations. Inside-out patches were obtained by withdrawing patch pipettes from varicosities after formation of a gigaohm seal. To prevent the formation of a vesicle, patches were usually exposed to air for a short period of time (1–5 s). The pipette solution was (mM): 116 KCl or potassium gluconate, 1 MgCl₂, 1 DAP and 5 Hepes. The bath solution contained 116 mM KCl, 5 mM Hepes, with variable amounts of CaCl₂ and 5 mM Ca²⁺ buffers. Three Ca²⁺ buffers with different dissociation properties were used under different free [Ca²⁺] conditions to ensure the buffering capacity of the solution: ethylene glycol-bis(ß-aminoethyl ether)-N,N,N',N'-tetraacetic acid (EGTA) was used with [Ca²⁺] < 1 µM, N-(2-hydroxyethyl)-ethylenediamine triacetic acid (HEDTA) with [Ca²⁺] of 1–20 µM, and nitrilotriacetic acid (NTA) with [Ca²⁺] > 20 µM. The amount of CaCl₂ added was first estimated by on-line software, Slider 1.0 (created by C. Patton, Stanford University, CA, USA). The final desired [Ca²⁺] was finely adjusted by adding Ca²⁺ buffer or CaCl₂ while monitoring with a Ca²⁺-sensitive electrode (Accumet, Hudson, MA, USA) which had been calibrated with a standard Ca²⁺ calibration kit (World Precision Instruments, Sarasota, FL, USA).

In cell-attached patches, single channel recordings were made with NFR containing 1 mM DAP and 0.3 mM TTX in the patch pipette and the cell bathed either in Ca²⁺-free NFR, in Ringer of the same composition but with the Ca²⁺ replaced by Mg²⁺, or in a Ca²⁺-free bath solution containing 116 potassium aspartate, as specified in the text.

Single-channel currents were filtered at 5–10 kHz with an Axopatch 200B amplifier and sampled at 50–200 kHz using pCLAMP 6.02 software (Axon Instruments). Data were stored on the hard drive of a computer and later transferred to recordable compact disks for future analysis.

Data analysis

Non-stationary noise analysis was carried out in perforated whole-terminal clamp configuration following the standard procedure (Heinemann, 1995). Fifty to 100 current traces were recorded when the membrane potential was stepped from –60 mV to +20 mV and averaged to obtain mean current (I). The variances (s²) in I for individual traces were ensembled and plotted against I. The unitary single current (i_K), open probability (P_o) and number of channels (N_K) were estimated according to the following equations:

(1)

(2)

(3)

Open probability (P_o) was calculated by fitting an all-points histogram with a sum of Gaussian functions. P_o was determined by calculating the relative area under the peak corresponding to the open channel. For multichannel patches in which the number of channel was known, the open probability was calculated using the equation:

(4)

where N was the number of channels in the patch and P_i the probability that i channels open simultaneously.

Ensembled single channel current traces were obtained by summing 50–200 single-channel sweeps. During each sweep, single channels were activated by square pulses stepped from a holding potential of –60 mV to potentials up to +80 mV. The capacitive and minor leak currents were corrected by subtraction of a trace averaged from null traces. Ensembled single channel currents were stored for activation and deactivation kinetic analysis.

Data are expressed as means ± standard error of the mean (S.E.M.) with n indicating the number of recordings. Quantitative data were judged to be significant if P < 0.05 by Student's t test or the ² (Chi-square) test.

Chemicals

3,4-Diaminopyridine (DAP), HEDTA and NTA were obtained from Sigma (St Louis, MO, USA). Iberiotoxin (IBTX) was from Bachem Americas (Torrance, CA, USA). Paxilline, also a selective BK channel blocker (Sanchez & McManus, 1996), was from Sigma. Tetrodotoxin (TTX) and -conotoxin GVIA (-CTX GVIA) were obtained from CalBiochem (San Diego, CA, USA).

	Results

Top Abstract Introduction Methods Results Discussion References

 
Using Xenopus embryonic motoneurone–muscle cell cocultures, in which bouton-like nerve terminal varicosities are accessible to patch clamping, we have investigated presynaptic BK channels at whole-terminal and single channel levels. Below we summarize some of the dynamic properties of the whole-terminal BK currents, then describe the number, density, distribution, conductance, and voltage and Ca²⁺ sensitivity of single BK channels in the terminal membrane, and finally describe the kinetic properties of these channels in response to step depolarizations in cell attached configuration and in inside-out patches exposed to known [Ca²⁺]_i.

Relationship of presynaptic Ca²⁺ to BK currents and postsynaptic EPSCs in whole-cell recordings

In many motoneurone varicosities, a large proportion of the outward current (up to one half) is Ca²⁺ dependent and blocked by charybdotoxin or iberiotoxin (Yazejian et al. 1997). Figure 1 illustrates the whole-cell BK presynaptic currents and excitatory postsynaptic currents (EPSCs) recorded simultaneously on presynaptic and postsynaptic membranes during a series of 20 ms depolarizations from a rest level of –60 mV to –40 to +100 mV in 10 mV steps in a representative experiment. (Voltage-sensitive Na⁺ and most K⁺ currents had already been blocked by TTX and DAP.) Subtraction of current evoked by similar stimuli after block of the Ca²⁺-sensitive BK channels with IBTX yielded the I_BK. Further subtraction after block of Ca²⁺ channels by -CgTX GVIA yielded the I_Ca. The relationship between these currents is shown in Fig. 1B. The I_BK and EPSC in response to the depolarizing pulses closely mirrored the I_Ca. The DAP and Ca²⁺-insensitive K⁺ current could be blocked by TEA or replacement of intracellular K⁺ by Cs⁺. DAP (1 mM) does not significantly block BK channels in this preparation (Yazejian et al. 1997).

View larger version (27K): [in this window] [in a new window]   Figure 1.  Voltage dependence of component currents in a representative synapse A, presynaptic currents (control) after block of voltage-sensitive INa and most IK by 1 µM TTX and 1 mM DAP. Voltage steps of 20 ms were from a rest level of –60 mV to –40 to +100 mV. Below are EPSCs recorded to the same voltage steps. Subtraction of presynaptic current responses after block of BK channels with 0.3 µM IBTX yielded the IBK. Further subtraction after block of most Ca2+ channels by 1 µM-CTX GVIA yielded the ICa. Current traces recorded at +20 mV (thick line) and +60 mV (intermediate thickness line) are distinguished for comparative purposes. B, relationship between the different current components. Note the different pre- and postsynaptic amplitude scales. Current amplitudes were taken at 3 ms after starting membrane depolarization from a holding potential of –60 mV.

In order to characterize single channel properties of the BK channels in the varicosities, we examined single BK channels under three conditions: (i) cell-attached patches, (ii) inside-out patches with asymmetrical K⁺ concentrations ([K⁺]_o : [K⁺]_i= 2 : 120), and (iii) inside-out patches with symmetrical 120 mM K⁺. In cell-attached patches, the single channel slope conductance, obtained by fitting a straight line to the linear part of I–V curves, was 78.3 ± 6.2 pS (n= 35) between –20 and +80 mV (Fig. 2A, left, and 2C). These channels were blocked by 300 nM IBTX in the outside-out patch configuration (data not shown), confirming that they are K_Ca channels of the BK or maxi-K variety, as concluded from the whole-terminal experiments (Yazejian et al. 1997, 2000).

View larger version (26K): [in this window] [in a new window]   Figure 2.  Single BK channel current–voltage relationships, and the selectivity for K+over Na+ A, single-channel traces recorded at different membrane potentials from representative cell-attached and inside-out patches with 100 µM internal [Ca2+] and 120 mM K+. In both cases the patch electrode contained NFR with 2 mM K+ (asymmetrical condition). B, single channel recordings from an inside-out patch with 120 mM[K+] on both sides of the membrane. [Ca2+]= 100 µM. C, I–V curves for inside-out patches with different ratios of [K+]o : [K+]i (mM): 2 : 120 (); 120 : 120 (), Erev= 0.44 mV; 120: 80 (), Erev= 10.0 mV; 120 : 50 (•), Erev= 21.1 mV, and 120 : 20 (), Erev= 44.4 mV. Reductions in [K+] were effected by replacement with equimolar Na+. Smooth lines were generated according to eqn (5). A PK of 4.16 x 10–13 cm s–1 was calculated based on a simplified eqn (5), when [K]o=[K]I, IBK=PK(EF/RT)[K+]. Each point represents the mean of 7–8 experiments.

(5)

where P_K is the permeability to K⁺ of a single channel in cm s^–1, E is the membrane potential, and F, R and T have their usual meanings. The slope conductance was 120.7 ± 8.8 pS (n= 26) in the range from –20 mV to +80 mV (Fig. 2A, right and 2C). The difference between the single channel conductance in cell attached and inside-out configurations is probably the result of partial block of the cell-attached channels by internal Na⁺ (Yellen, 1984).

Finally, the conductance of BK channels was studied in inside-out patches with symmetrical potassium concentration. Figure 2B shows a typical recording of single-channel currents examined in symmetrical 120 mM K⁺. The current amplitudes varied linearly over the range –80 to +80 mV (Fig. 2C). The P_K value, calculated from eqn (5), was 4.3 x 10^–13 cm s^–1, similar to that in the asymmetrical condition. The mean slope conductance was 243.5 ± 5.3 pS (n= 41). Others have observed comparable differences in single channel conductance under symmetrical versus asymmetrical recording conditions (Benham et al. 1986; Berweck et al. 1994).

The selectivity of channels for K⁺ over Na⁺ was investigated at different concentrations of Na⁺ and K⁺ in the solution bathing the intracellular surface of the patch. The K⁺ concentration in the pipette was held constant at 120 mM. By substituting the P_K value (4.3 x 10^–13 cm s^–1) in symmetrical K⁺ conditions into eqn (5), curves were generated according to their [K⁺]_o : [K⁺]_i ratios (Fig. 2C). The good fit between experimental data and the predicted curves, based on the assumption that K⁺ is the only permeant ion, indicates that the channels are highly selective for K⁺ over Na⁺. The permeability ratio of K⁺versus Na⁺ (P_K/P_Na), calculated from the modified Goldman–Hodgkin–Katz equation (Benham et al. 1986), was 44 ± 15 from three tested [K⁺]_o/[K⁺]_i ratios.

The number of BK channels in a varicosity

Consistent with the hypothesis that BK channels are an integral part of specialized presynaptic structures, the probability of finding BK channels in comparable patches under similar recording conditions increased significantly (P < 0.005) with time in culture. They were found in 35.4% of patches (n= 396) in 1-day cultures, 56.2%(n= 386) after 2 days and 90.9%(n= 11) after 4 days.

The large single-channel conductance of BK channels (mean 78.3 pS in cell-attached patches) facilitates the estimation of the total number of channels in the varicosity using non-stationary noise analysis techniques. Figure 3 shows the ensembled mean current and variance generated during 100 stimuli from –60 mV to +20 mV in a representative varicosity in a 2-day culture, with Na⁺ and most voltage-sensitive K⁺ channels blocked by TTX and DAP. Only the data collected within the first 3 ms from the beginning of depolarization were used for analysis of the variance–mean current relationship. A parabolic relationship (Fig. 3B) between variance and mean current was verified by fitting the data to eqn (3) described in Methods. The number of channels (N_K), single channel current (i_K) and open probability (P_o) were computed from maximum-likelihood fits. From several such experiments, the mean values of i_K, P_o and N_K were 5.0 ± 1.2 pA, 0.91 ± 0.026 and 121 ± 42 for 0 mV (n= 5), 6.9 ± 1.0 pA, 0.93 ± 0.07 and 197 ± 41 (range 102–352) for +20 mV (n= 7) and 8.7 ± 0.7 pA, 0.83 ± 0.07 and 159 ± 27 for +40 mV (n= 8). The slope conductance was 93 ± 1.4 pS, determined by linearly fitting the current values above. This is comparable to the value derived from single channel recordings. The number of channels per varicosity seems not to depend on voltage, at least in the testing range of 0 to +40 mV. The P_o values at all three test potentials were 0.83, suggesting that most or all BK channels were activated during the first 3 ms after onset of depolarization at all depolarizations of 0 mV or higher.

View larger version (15K): [in this window] [in a new window]   Figure 3.  Number of BK channels in a varicosity, estimated by noise analysis A, mean (middle trace) and variance (bottom trace) of membrane currents evoked by depolarizing the membrane from –60 mV to +20 mV (upper trace) in the perforated whole-terminal clamp configuration. The recording pipette was filled with a solution containing 120 mM KCl and 0.5 mM EGTA, and the bath solution was NFR containing 300 nM TTX and 1 mM DAP. The mean current was the average of 100 traces. B, relationship between the mean and variance of IBK. Variance values for the first 3 ms of the pulse were obtained by subtracting each individual current from the mean current. Data are fitted by a parabolic function (continuous line) as described in Methods. In this varicosity, the single channel conductance, open probability, and number of channels were estimated to be 60 pS, 0.95 and 102, respectively.

View larger version (18K): [in this window] [in a new window]   Figure 4.  BK channels are distributed heterogeneously on varicosities and differ in degree of coupling to Ca2+channels Percentage of cell-attached and inside-out patches containing one or more BK channels, for three different categories of varicosity. The number of patches in each category is shown within each column. Mean values were significantly different (P < 0.01) for the same type of patches in each of the three types of varicosities, and between cell-attached and inside-out patches in isolated and synaptic varicosities.

View larger version (19K): [in this window] [in a new window]   Figure 12.  Comparison of BK channel activation kinetics in cell-attached and inside-out configurations A, ensembled current traces constructed from recordings of a single BK channel in a cell-attached patch with step depolarizations to –20, 0, +20 and +60 mV. Differences in amplitude reflect predominately differences in driving force. The time constants of activation were 4.5, 3.1, 2.3 and 1.4 ms, respectively. B, plot of activation time constants from individual cell-attached patches (open triangles and circles) at various membrane potentials. Values for the half of the population with the fastest activation time constants (open circles) were averaged and the mean indicated by the filled stars and line. C, time constants derived from fitting the activation phase of the ensemble currents in inside-out patches after excision with NFR in the patch pipette and exposed to different [Ca2+] (dashed curves) as a function of membrane potential. Each data point expresses the mean ±S.E.M. with n= 7–24. The filled symbols and line are a plot of the activation time constant of the fastest half population of channels in the cell-attached patch configuration (as in B) after subtraction of the activation time constant for the ICa at that potential (1.5 ms at –20 mV, 0.92 ms at 0 mV, 0.46 ms at +20 mV, 0.3 ms at +40 mV and 0.25 ms at +60 mV; unpublished data from B.-M. Chen & A. D. Grinnell). The patch pipette was filled with NFR solution. The bath solution was 116 mM potassium aspartate, 5 mM KCl and 0 mM Ca2+ to equalize the membrane potential before excision. After excision, the bath solution was changed to 121 KCl with 1, 5, 10 and 100 µM Ca2+.

View larger version (37K): [in this window] [in a new window]   Figure 6.  Effect of [Ca2+]i on BK channel open probability A, representative single-channel currents recorded at +60 mV in symmetrical K+ at different [Ca2+]i in an inside-out patch (dashed line indicates the closed level). B, dose–response curve showing the effect of [Ca2+]i on open probability (Po) of BK channels at +20 mV. Each data point represents an average of 5–11 experiments at a holding potential of +20 mV. The maximum Po was 0.85. The data are fitted to the Hill function and give K= 7.7 ± 0.3 µM(n= 7) and Hill coefficient (nH) = 2.6. C, straight lines were fitted to plots of Poversus[Ca2+] on a logarithmic [Ca2+] scale in a range from 1 to 200 µM at a holding potential of –20 (), +20 (), +40 () and +60 mV (•). Numbers indicate the Hill coefficient (slope) for each line. Values for K were 10.0 ± 0.4, 7.7 ± 0.3, 5.1 ± 0.2 and 2.7 ± 0.3 µM, respectively. Each point represents the mean value averaged from 6 to 9 experiments.

The number of BK channels calculated from the measured whole-terminal I_BK values (e.g. Fig. 1B) is consistent with the noise analysis values. With a whole terminal I_BK of 1.0–1.5 nA and a single channel current of 7 pA at +20 mV, the number of BK channels is in the range of 140–210. The difference between this range and that obtained from noise analysis is probably not significant. If one accepts that the representative varicosity has 150–200 BK channels, the ratio of Ca²⁺ : BK channels is 5 : 1.

BK channels distribute heterogeneously in the varicosities

If BK channels are present in the soma membrane, they are apparently not coupled functionally to Ca²⁺ channels. Little or no I_BK can be recorded in whole-cell mode from the motoneurone soma, and block of BK channels by paxilline has no effect on current traces (data not shown). Even in the varicosity membrane, where the I_BK can represent a large fraction of the total I_K, BK channel expression/insertion depends on location within the varicosity and whether or not the varicosity is in contact with a muscle cell. Figure 4 summarizes the results of single channel recordings from several hundred patches on varicosities that were not in contact with a muscle cell (‘isolated varicosities’) or on varicosities in contact with muscle cells (‘synaptic’). BK channels were found in 54.2 ± 2.3% (248/457) of inside-out patches made from isolated, non-synaptic varicosities treated with 5 µM or higher [Ca²⁺]_i, compared with 86.3 ± 3.2% (101/117) of inside-out patches from the exposed surface of synaptic varicosities. Thus contact with a muscle cell increases the probability of finding BK channels by about 30%. There was a similar difference in detectability of BK channels in cell-attached patches. Of patches from isolated varicosities, 43.4 ± 3.0% (122/281) showed BK channels, compared to 69.6 ± 4.8% (64/92) of cell attached patches from non-synaptic varicosities. Both of these differences are highly significant (P < 0.001). Although differences in the state of maturation independent of contact with a target cell might account for some of these differences, it seems more likely, in view of earlier evidence for the effects of muscle cell contact on transmitter release (Xie & Poo, 1986; Chow et al. 1988; Evers et al. 1989; Liou et al. 1999; Martin-Caraballo & Dryer, 2002), that it is contact with target cells that causes the increase in expression of BK channels and greater coupling to Ca²⁺ channels. Additional evidence for this is the finding that all (11/11) patches from the ‘release face’ of synaptic varicosities (the surface exposed to the muscle cell, achieved by gently pushing the muscle cell away before patching) showed BK channels, usually at high density (see Fig. 4 and below).

Interestingly, as Fig. 4 illustrates, in both isolated varicosities and non-synaptic membranes of synaptic varicosities, BK channels were found with greater probability in detached patches exposed to [Ca²⁺] of 5 µM than in cell-attached patches with Ca²⁺ entering through open Ca²⁺ channels. Thus a significant fraction (10–20%, P < 0.01) of the BK channels were apparently not closely enough coupled, functionally, to Ca²⁺ channels to open in the cell-attached configuration. Non-coupled BK channels would not be expected to contribute to either the whole-terminal I_BK or to noise analysis measurements. Hence the total number of BK channels is probably larger than indicated by either measurement. Moreover, considering only those patches that had BK channels, the number of channels per patch was clearly much higher in the synaptic than in the isolated varicosities. Figure 5A shows representative channel traces from varicosities of the three types in response to depolarizations to +60 mV. Figure 5B summarizes these differences graphically for non-synaptic and synaptic (non-release face) varicosity membrane patches. The mean number of BK channels per patch that had one or more channels was 2.39 ± 0.16 (n= 74) in non-release regions of synaptic varicosities, compared with 1.81 ± 0.06 (n= 203) in isolated varicosities (P < 0.002). Again, the release face patches had even greater densities (mean 3.82 ± 0.54, n= 11), with one patch having eight channels.

View larger version (27K): [in this window] [in a new window]   Figure 5.  Number of BK channels per patch differs in different types of varicosities Aa, single channel traces in inside-out patches excised from isolated-, synaptic-, and release face of synaptic varicosities. Patches were depolarized from –60 mV to +60 mV. About 100 single channel traces were used to create the all-point amplitude histograms shown in b. The number of channels in these representative patches was 2, 3 and 5. Data are binned at 1.0 pA. The patch pipettes were filled with 120 mM KCl and the same concentration of K+ with free calcium of 5.0 µM was used in the bath solution facing the inner membrane surface. B, percentage of patches from isolated and synaptic varicosities that showed the indicated numbers of BK channels per patch in inside-out configuration of +60 mV membrane potential. Data for release face patches are included with synaptic varicosities.

Channel open probability exhibited a clear Ca²⁺ dependence. Figure 6A shows representative records of BK channel activity in an inside-out patch at +60 mV transmembrane potential with different Ca²⁺ concentrations on the cytosolic surface of the patch. Figure 6B plots the mean open probability as a function of [Ca²⁺]_i at a membrane potential of +20 mV, which is reached during an action potential (Yazejian et al. 2000). At +20 mV, there was no channel activity when Ca²⁺ was below 1 µM and the maximal open probability (P_o), 0.85, was achieved above approximately 100 µM[Ca²⁺]_i. The dissociation constant (K) values varied between 2.7 and 10 µM in the range of the membrane potentials from –20 to +60 mV. Channel activity rose steeply with increasing calcium concentration, suggesting cooperative effects of calcium gating. To see this effect, dose–response curves were fitted to the Hill equation by fitting straight lines to plots of log [P_o/(1 –P_o)]. From these results, Hill coefficients varied from 1.5 (+60 mV) to 2.8 (–20 mV), suggesting that channel activation involves the binding of up to three Ca²⁺ ions and that there is interaction between Ca²⁺ binding and voltage gating mechanisms. The observation that the Hill coefficients declined at high voltage can be attributed to the decreased P_o seen at high voltage and high [Ca²⁺] (see Fig. 7C and Discussion).

View larger version (34K): [in this window] [in a new window]   Figure 7.  Voltage dependence of BK channels at different [Ca2+]i A, BK channel currents recorded over a range of membrane potentials in symmetrical K+ at 100 µM[Ca2+]i. B, mean Po as a function of potential at [Ca2+]i of (µM) 1 (), 2 (), 5 (), 10 (•), 20 (), 100 (), 200 () and 1000 (). Inset: normalized Po (relative to Pmax) plotted against voltages for a representative patch. Smooth lines are fitted with a single Boltzmann function. Pmax= 0.94. C, the half-maximal activation voltages (V, left) and slope factors (Vslope, right) derived from the Boltzmann fits to the data in B. V values declined continuously as [Ca2+] increased. Vslope was essentially independent of [Ca2+]. Each point represents data from 4 to 11 experiments.

Nerve terminal BK channel open probability exhibited characteristic voltage dependence. Figure 7A shows sample traces of channel openings as a function of transmembrane potential with [Ca²⁺]_i= 100 µM, and Fig. 7B shows mean values for P_o over this voltage range at [Ca²⁺]_i from 1 µM to 1 mM. At low Ca²⁺ concentrations ( 5 µM) channel activity was not found at hyperpolarizing potentials of –60 mV or lower, i.e. at the normal resting membrane potential of the terminal. The P_o rose steeply with depolarization (increasing e-fold per 16.3 ± 0.72 mV) at 100 µM[Ca²⁺]_i and the activation curves were sigmoid in shape (Fig. 7B, inset). Elevation of intracellular Ca²⁺ concentration caused a shift of the activation curves in the hyperpolarizing direction along the voltage axis. At high [Ca²⁺]_i values, channel open probability dropped slightly after reaching a maximum, as the membrane was further depolarized. This may be attributable to block of channels by divalent cations – either Ca²⁺ or trace amounts of Ba²⁺ (Morales et al. 1995; Rothberg et al. 1996; Bello & Magleby, 1998).

To quantify the results further, the data from Fig. 7B were fitted to a single Boltzmann equation:

(6)

where V, the voltage for half-maximal activation (P_o= 1/2), determines the left–right position of each curve, and V_slope determines the steepness of each curve. The results are shown in Fig. 7C and are similar to those reported for other BK channels (see Discussion).

The activation kinetics of BK channels in inside-out patches

Study of BK currents in whole-terminal recordings is complicated by the presence of the slowly activating, Ca²⁺-insensitive K⁺ current. To study the kinetics of the I_BK without interference from this DAP-insensitive K⁺ current, it is necessary to subtract currents after iberiotoxin or paxilline block from those before (Fig. 1 and Yazejian et al. 2000). At the single channel level, we could minimize this complication by selectively using data from patches that contained only large conductance BK channels. The activation and deactivation kinetics were analysed on the basis of their ensemble currents.

Figure 8 shows families of single BK channel traces recorded in an inside-out patch and their ensembled currents in response to depolarizing voltage steps at various intracellular [Ca²⁺]. The membrane potential was first hyperpolarized to –60 mV, causing almost all BK channels to close. The potential was then stepped to a depolarized level (+20, +40 and +60 mV shown). To evaluate the channel activation kinetics, only current traces in which the BK channel had closed before the depolarizing pulse was imposed were ensembled and fitted by a single exponential function. The activation times decreased with increase in voltage and in [Ca²⁺] (Fig. 8, lower panels). In contrast, at any given potential, the amplitude of the ensembled currents, reflecting the channel open probability, were comparable when [Ca²⁺] was 5 µM or more. The result is consistent with whole-terminal recordings (Yazejian et al. 2000) in showing that the activation kinetics of BK channels is a more sensitive criterion than P_o for quantifying changes of internal [Ca²⁺] in response to transient depolarization.

View larger version (27K): [in this window] [in a new window]   Figure 8.  The activation of BK channels depends on voltage and [Ca2+] Single BK channel current traces (four middle traces) recorded at +20 mV (A), +40 mV (B) and +60 mV (C) in different [Ca2+] (as indicated) in response to the presynaptic voltage waveform shown at the top. Ensemble currents were obtained by averaging 70–120 single channel traces in which the channel was closed at –60 mV at the time depolarizing test pulses were applied (bottom traces for each panel). The activation phase for each ensembled current trace is fitted to a single exponential function and indicated by continuous lines. This patch had only a single channel with a conductance of 215 pS. All data were recorded in symmetrical 120 mM K+ solution and filtered at 3 kHz.

As an alternative test, we performed a cumulative first-latency analysis to confirm that the time constants obtained in this way were similar to those derived from ensembles made on the same set of data. A cumulative distribution histogram of the time delay between membrane depolarization and the first observable channel opening event in a large number of trials specifies the probability that the ion channel will have opened at any time after the voltage step. Figure 9 plots the cumulative first latency of opening and ensemble currents for a representative channel at two voltages and Ca²⁺ concentrations. Correspondence of the ensemble activation time constants was very close. Tests again showed that the time constants obtained by fitting these curves with single or double exponential functions were similar.

View larger version (27K): [in this window] [in a new window]   Figure 9.  Activation kinetics modes derived from direct fitting to the ensemble current trace and first opening latency Normalized BK current traces (noisy continuous lines) ensembled from single channel recordings are plotted with the cumulative distribution of first channel opening latency (dashed lines), obtained from the same data, at different [Ca2+] (10 and 100 µM) and membrane potentials (+40 and +60 mV). A single exponential function was used to fit the ensembled current traces (smooth continuous lines) and first latency. The time constants derived from fitting the ensemble currents (ens) and first latency (lat) are indicated in the figure. Only one BK channel was present during the entire experimental period. Bin width for constructing first latency histograms varied between 0.2 and 1 ms (n= 100–200 for ensembled traces, n= 74–143 for first latency traces). Noise in ensembled current traces, especially at lower [Ca2+]i levels, was due to the low frequency of channel closing–opening fluctuations.

View larger version (17K): [in this window] [in a new window]   Figure 10.  Time constants of BK channel activation are dependent mainly on [Ca2+] Activation time constants are plotted against voltage at [Ca2+] of (µM) 0.2 (), 1 (•), 5 (), 10 () and 100 (). Time constants were obtained by fitting the rising phase of ensembled single channel currents with single exponential functions. Each data point represents the mean ±S.E.M., n= 3–15. All measurements were made in symmetrical 120 mM K+.

Deactivation kinetics of BK channels

Figure 11 shows the effect of intracellular [Ca²⁺] on the deactivation kinetics of a representative varicosity BK channel. The tail currents were ensembled from single channel traces activated by a test pulse to +60 mV for 20 ms and repolarization to a holding potential of –60 mV in an inside-out patch (Fig. 11A, inset). The traces were well fitted by a single exponential function. The time constants were extremely fast at low [Ca²⁺] and became progressively slower with elevation of [Ca²⁺] (Fig. 11B), but not with larger depolarizing voltages (data not shown). The deactivation time constants were linearly related to log of [Ca²⁺]_i. The degree of calcium dependence of deactivation kinetics was less than that of the activation kinetics. The time constant of deactivation increased <2-fold (from 0.33 ms to 0.57 ms) for a change in [Ca²⁺] from 1 µM to 100 µM.

View larger version (18K): [in this window] [in a new window]   Figure 11.  Deactivation kinetics of BK channels is dependent on [Ca2+] A, expanded deactivation phase of ensemble currents (inset) at Ca2+ concentrations of 1–100 µM following repolarization from +60 mV to –60 mV. The dotted lines show the results fitted with a single exponential function. B, deactivation time constants (deact) plotted against the log [Ca2+]. Each point represents the mean ±S.E.M., n= 3–15.

Under normal conditions, BK channels are exposed to Ca²⁺ that has entered through Ca²⁺ channels. To test our expectation that most BK channels are in or close to the Ca²⁺ microdomains created by Ca²⁺ influx through open Ca²⁺ channels, we recorded BK channel activity in cell-attached patches. Although the whole-terminal membrane potentials were no longer controlled, the problem of loss of potentially important intracellular constituents was avoided. Moreover, since responses occur too quickly to be mediated by release of Ca²⁺ from internal stores, the elevation of [Ca²⁺]_i above rest level was due only to influx through open Ca²⁺ channels.

Usually, no BK channel activity was seen when patches were formed on varicosities in the cell-attached configuration until the membrane was depolarized. To ensure that BK channels under the patch pipette would be activated by Ca²⁺ influx only through nearby Ca²⁺ channels within the patch, the bath solution was changed to zero Ca²⁺ Ringer after the patch was made. Coupling of BK channel opening to changes in membrane potential in the cell-attached patch membrane requires close physical proximity between Ca²⁺ channels and Ca²⁺-activated K⁺ channels, and the activation kinetics of the BK channels reflect the magnitude of local changes in [Ca²⁺].

The ensembled currents of Fig. 12A were constructed from a representative single channel activated by depolarizing pulses in the cell-attached configuration. Figure 12B plots the activation time constants of single BK channels for all BK channels studied in cell-attached configuration showing kinetics faster than 8 ms. As is evident from this figure, there was great diversity in the rate of activation. This is not surprising in view of the observation that, in 10–20% of patches, BK channels were not observed until the patch was detached and exposed to 5 µM[Ca²⁺] (Fig. 4). Our interpretation of these results therefore is that the variability in activation rate reflects diversity in the degree of coupling of BK and Ca²⁺ channels in the patches studied (which were both on isolated varicosities and on the non-synaptic surface of synaptic varicosities). It seems probable therefore that a better criterion for the maximal [Ca²⁺] near active zones would be that reported by the subpopulation of channels showing the fastest activation times in the cell-attached configuration. If one takes the average of values for that half of the population of individual channels showing the fastest kinetics at each potential, the activation time constant was 2.79 ± 0.25 ms at –20 mV, 1.8 ± 0.13 ms at 0 mV, 1.22 ± 0.09 ms at +20 mV, 0.92 ± 0.1 ms at +40 mV, and 1.04 ± 0.1 ms at +60 mV. These means are plotted in the continuous curve of Fig. 12B.

The same pulse protocol was also used to compare these values with the activation kinetics after cell-attached patches were excised and exposed to an internal solution containing 120 mM KCl and known [Ca²⁺] from 1 to 100 µM. The activation phases of the ensemble currents in both patch configurations were fitted by single exponential functions. Figure 12C shows collected data from many such experiments. The dashed curves show mean values for inside-out patches exposed to [Ca²⁺] of 1, 5, 10 and 100 µM.

Calibrations obtained with inside-out patches exposed to known [Ca²⁺]_i (Fig. 12C) are not directly comparable to the time constants in cell-attached patches (Fig. 12B) since the cell-attached values also include the time necessary for voltage activation of the Ca²⁺ channel(s) to which the BK channel was coupled. Although the time of opening of the Ca²⁺ channel(s) in any given patch could not be determined, it is possible to estimate the magnitude of this delay by consideration of a simple model. If one treats each BK channel as part of a three state system in which (a) a BK channel and coupled Ca²⁺ channel(s) are all closed at the time of a step depolarization, (b) the Ca²⁺ channel(s) open with a probability described by the macroscopic time course of the I_Ca at that potential, (c) the BK channel then opens with a time constant appropriate to the resulting [Ca²⁺] and that potential (derived from the detached patch calibrations of Fig. 10 or 12C), and (d) if the calcium channel and BK channel time constants are not grossly dissimilar, then simple simulations show that the resulting overall time constant of BK channel opening in the cell-attached configuration is closely approximated by the sum of the time constants of the I_Ca and of BK channels in detached patches. When we subtracted the time constants of the whole terminal I_Ca at each potential (see Fig. 12 legend) from the observed BK channel activation time in each patch (Fig. 12B), we obtained the BK channel activation time constants shown by the continuous line in Fig. 12C. Except at +60 mV, where the reduced driving force for Ca²⁺ reduces [Ca²⁺]_i and decreases the rate of BK channel activation, these data indicate that the half of the BK channels on the non-synaptic surface of the varicosity that showed the fastest activation time were sensing 100 µM Ca²⁺– a level expected only in or very close to Ca²⁺ microdomains (see Discussion).

This correction for Ca²⁺ activation time makes the assumption that the opening of any given BK channel in the cell attached configuration is dependent either on the opening of a single coupled Ca²⁺ channel or on the opening of all of a population of surrounding Ca²⁺ channels. If instead some BK channels can be activated by the opening of only one or a small subset of a large number of nearby Ca²⁺ channels (Marrion & Tavalin, 1998), the expected delay due to Ca²⁺ channel activation could be much shorter, and our subtraction of the mean I_Ca value would lead to an overestimate of the BK channel activation rate and of [Ca²⁺]. The fact that several of the cell attached BK channels had mean activation times much shorter than the average for the fastest half of the population (down to 0.58 ms at +40 mV, Fig. 12B) may reflect instances in which opening of one or a few of a larger number of nearby Ca²⁺ channels was sufficient to trigger opening of the BK channel.

Controlled titration of active zone [Ca²⁺]

Given the calibrations described above, it is now feasible to interpret whole-terminal BK currents in terms of [Ca²⁺] at the site of the BK channels responsible for the rising phase of the I_BK. For reasons outlined below (see Discussion), these values are likely to be those achieved in the Ca²⁺ domains at active zones ([Ca²⁺]_AZ). In contrast to the I_BK, the release sensor integrates [Ca²⁺] over at least several hundred microseconds (Yazejian et al. 2000). Hence, in order to quantify accurately the dependence of release on [Ca²⁺]_AZ, it will be necessary to analyse release under conditions in which the active zone is exposed to [Ca²⁺]_AZ of different known magnitudes for different periods of time. This can be done in the Xenopus varicosity preparations.

Using the stimulus waveform shown in Fig. 13A, it is possible to expose the active zone to a wide range of [Ca²⁺] for different lengths of time in a controlled, quantifiable way for correlation with neurotransmitter release. In this experimental protocol, the varicosity potential is stepped to +80 mV or higher (+130 mV in the experiment shown) to open Ca²⁺ channels without Ca²⁺ influx. The potential is then stepped back to intermediate potentials between 0 and +80 mV, allowing Ca²⁺ entry through the already opened Ca²⁺ channels. Thus, the Ca²⁺ entry starts immediately at the beginning of the intermediate potentials and is not dependent on the time course of Ca²⁺ channel opening. The lower the potential, the greater the Ca²⁺ driving force, so Ca²⁺ entry occurs at different rates at different intermediate potentials. In practice, only intermediate steps to +10 mV and above are useful for quantification of [Ca²⁺], since at potentials below this, Ca²⁺ channels close significantly during the intermediate step, compromising the measure of I_BK activation time. Differences in driving force for K⁺ also affect the I_BK amplitude and activation rate. Figure 13B shows the BK currents obtained in such an experiment by subtracting responses after IBTX block from those before, and the responses during the intermediate step are shown expanded in Fig. 13C. The traces in Fig. 13B and C were obtained in NFR, with 1.8 mM[Ca²⁺]_o. In addition, in the experiment shown, the external [Ca²⁺]_o was changed between 0.5 and 5 mM. The I_BK activation time constants at each intermediate potential in each [Ca²⁺]_o, calibrated by the data in Fig. 10, yielded the [Ca²⁺]_i values shown (Fig. 13D). Note that in this experiment, with 1.8 mM[Ca²⁺]_o, the [Ca²⁺]_i could be varied over approximately a 1000-fold range, from about 150 nM to 150 µM, and maintained at that level for many milliseconds. (The shift toward higher voltage ranges at higher [Ca²⁺]_o is expected for two reasons: the change in E_Ca from 0.5 to 5 mM[Ca²⁺]_o would account for a shift of 30 mV, and the well-known dependence of ion channel activation threshold on membrane surface charge (Hille, 2001) is probably responsible for the rest of the effect.)

View larger version (21K): [in this window] [in a new window]   Figure 13.  Titration of [Ca2+]AZ by changes in [Ca2+]O and Ca2+ driving force A, at four different [Ca2+]o concentrations: 0.5, 1.8, 3.0 and 5.0 mM, a varicosity synapse was depolarized from rest level of –70 mV to +130 mV, opening Ca2+ channels without permitting Ca2+ entry. After 20 ms, the potential was stepped to an intermediate value of 0 to +80 mV, allowing Ca2+ entry with different driving forces. After 8 ms, the potential was returned to +130 mV, terminating Ca2+ entry, for 10 ms. B, IBK in response to this waveform in 1.8 mM Ca2+, obtained by subtraction of records after IBTX block from those before. C, expanded traces showing the IBK during the intermediate step at several potentials. D, graph showing the [Ca2+]AZ at each potential and [Ca2+]o, based upon activation time constants obtained by fitting a single exponential to the IBK traces such as those shown for 1.8 mM[Ca2+]o and the calibration curves of Figs 10 and 12C. All traces were the average of 3 responses to a given waveform.

	Discussion

Top Abstract Introduction Methods Results Discussion References

Spatial colocalization of BK and Ca²⁺ channels

BK channels were distributed non-uniformly in the motoneurone membrane, consistent with a specialized role in neurotransmitter release. No evidence could be seen for BK channels in the motoneurone soma based upon whole-cell currents. On the other hand, most varicosities exhibited a prominent I_BK. The occurrence of BK channels varied with culture maturity, type of varicosity, and patch location on the varicosity. BK channels were found in larger numbers and at higher density in synaptic-type varicosities than in isolated ones, and the density was particularly high where patches were made directly on the ‘releasing face’ of the varicosity (that in contact with the muscle cell and possibly containing one or more active zones). Given the small percentage of the synaptic contact surface that actually includes an active zone (DiGregorio et al. 1999), however, it is probable that few if any of the ‘release face’ patches actually included an active zone. The higher density of BK channels on this surface may reflect a tendency for these channels to cluster th