Voltage-dependent potassium currents during fast spikes of rat cerebellar Purkinje neurons:...

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JN-00269-2006, 2nd revision, October 12, 2006

Voltage-dependent potassium currents during fast spikes of rat cerebellar Purkinje neurons:inhibition by BDS-I toxin

Marco Martina1, Alexia E. Metz1, and Bruce P. Bean2

1Department of Physiology, Feinberg School of Medicine, Institute forNeuroscience, Northwestern University, Chicago, Illinois 60611, USA and 2Department of Neurobiology, Harvard Medical School, 220 Longwood Avenue, Boston, MA 02115, USA.

Running head: Fast K+ currents in Purkinje neuronsTotal pages: 24Abstract: 208/250

Correspondence: Marco MartinaDepartment of PhysiologyNorthwestern UniversityFeinberg School of Medicine 303 East Chicago AvenueChicago, Illinois 60611312-503-4654 (phone)617-503-5101 (fax)[email protected]

of 32 Articles in PresS. J Neurophysiol (October 25, 2006). doi:10.1152/jn.00269.2006

Copyright © 2006 by the American Physiological Society.

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Abstract

We characterized the kinetics and pharmacological properties of voltage-activated potassium

currents in rat cerebellar Purkinje neurons using recordings from nucleated patches, which

allowed high resolution of activation and deactivation kinetics. Activation was exceptionally

rapid, with 10-90% activation in ~ 400 microseconds at +30 mV, near the peak of the spike.

Deactivation was also extremely rapid, with a decay time constant of about 300 microseconds

near -80 mV. These rapid activation and deactivation kinetics are consistent with mediation by

Kv3-family channel but are even faster than reported for Kv3-family channels in other neurons.

The peptide toxin BDS-I had very little blocking effect on potassium currents elicited by 100-ms

depolarizing steps, but the potassium current evoked by action potential waveforms was

inhibited nearly completely. The mechanism of inhibition by BDS-I involves slowing of

activation rather than total channel block, consistent with the effects described in cloned Kv3-

family channels, and this explains the dramatically different effects on currents evoked by short

spikes versus voltage steps. As predicted from this mechanism, the effects of toxin on spike

width were relatively modest (broadening by ~25%). These results show that BDS-I -sensitive

channels with ultra-fast activation and deactivation kinetics carry virtually all of the voltage-

dependent potassium current underlying repolarization during normal Purkinje cell spikes.

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Introduction

Cerebellar Purkinje cells are capable of firing at frequencies as high as 200-400 Hz

(Armstrong and Rawson, 1979; McKay and Turner, 2004; Khaliq and Raman, 2005; Monsivais

et al., 2005), and intracellular recordings show that they have unusually narrow action potentials,

with a half-width of ~300-500 microseconds at room temperature (Stuart and Häusser, 1994;

Martina et al., 2003; McMahon et al., 2004) and as little as 220 microseconds at 35 °C (McKay

and Turner, 2004). These properties are broadly similar to a number of other "fast-spiking"

neurons, where narrow action potentials have been related to expression of Kv3-family

potassium channels, whose unusually rapid kinetics of activation and deactivation seem well-

suited for generating narrow action potentials and short refractory periods (Du et al., 1996;

Massengill et al., 1997; Martina et al., 1998; Erisir et al., 1999; Lien at et al., 2002; Baranauskas

et al., 2003; Lien and Jonas, 2003; reviewed by Rudy and McBain, 2001). Consistent with a

major role of Kv3-family channels, the predominant potassium current of Purkinje neurons is

highly sensitive to both TEA and 4-aminopyridine and displays rapid activation and deactivation

kinetics (Raman and Bean, 1999; Southan and Robertson, 2000; Martina et al., 2003; McKay and

Turner, 2004). In situ hybridization and immunocytochemistry have shown expression of Kv3.3,

Kv3.4, and Kv3.1 subunits in Purkinje neurons, with different subunits showing different

patterns of expression during development and different patterns of expression between somatic

and dendritic compartments (Goldman-Wohl et al., 1994; Weiser et al., 1994; Rashid et al.,

2001; Martina et al., 2003; McMahon et al., 2004). Kv3.1 appears to have minor expression in

mature Purkinje neurons (Weiser et al., 1994), and there is little change in spike width in mice

lacking Kv3.1 subunits (McMahon et al., 2004). In contrast, Kv3.3 is strongly expressed in

Purkinje cell somata (Martina et al., 2003; McMahon et al., 2004) and spike width is nearly

doubled in mice lacking Kv3.3 subunits (McMahon et al., 2004), suggesting a major role. Kv3.4

is also expressed in Purkinje neurons, both in dendrites and cell bodies (Martina et al., 2003)

although there is so far no information on how much Kv3.4 subunits may contribute to overall

potassium current.

Pharmacological tools are crucial for understanding how firing properties depend on

expression of particular channel types. In the case of Kv3-family channels, an increasingly

widely-used tool is the sea anemone toxin BDS-I. BDS-I was introduced as a selective blocker of

Kv3.4 channels (Diochot et al., 1998), and subsequent studies on a variety of central neurons

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showed the presence of components of potassium current sensitive to the toxin (Riazanski et al.,

2001; Baranauskas et al., 2003; Shevchenko et al., 2004). In these studies, the BDS-I-sensitive

component of current evoked by large depolarizations was found to be transient, consistent with

rapid inactivation of Kv3.4 homomeric channels seen in heterologous expression systems

(Diochot et al., 1998; Baranauskas et al., 2003). However, Baranauskas and colleagues (2003)

showed that BDS-I affects Kv3.4/Kv3.1 heteromeric channels, which show little inactivation, by

slowing their activation kinetics; thus, BDS-I-sensitive current shows apparent decay that in fact

results from unblock of BDS-I toxin during large depolarizations. Moreover, a recent study of

BDS-I action on multiple types of heterologously-expressed Kv3 channels has shown that the

toxin affects not only Kv3.4-containing channels but also affects homomeric channels composed

of Kv3.1 and Kv3.2 channels by slowing channel activation (Yeung et al., 2005). Here, we have

characterized the potassium currents from the cell bodies of cerebellar Purkinje neurons using

recordings from nucleated patches, which allowed high resolution of activation and deactivation

kinetics and their alteration by BDS-I toxin. Activation and deactivation of the predominant

potassium currents were exceptionally rapid, even in comparison to other central neurons

expressing Kv3-family channels. BDS-I had very little blocking effect on peak potassium

currents elicited by 100-ms depolarizing steps, but the potassium current evoked by action

potential waveforms was inhibited nearly completely. Further experiments showed that the

mechanism of inhibition involves slowing of activation rather than total channel block,

consistent with the effects described in cloned Kv3-family channels. These results show that

BDS-sensitive channels underlie virtually all of the voltage-dependent potassium current flowing

during normal Purkinje cell spikes and that BDS acts on the native channels in Purkinje neurons

by slowing activation rather than completely blocking a component of potassium current.

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Materials and MethodsParasagittal slices of 300 µm thickness were cut from the cerebella of Long Evans rats using a

vibrating tissue slicer (Dosaka DTK-1000, Ted Pella Inc, Redding, CA, USA). Rats (14 to 21

days old) were anesthetized by methoxyflurane before decapitation and removal of the

cerebellum. After cutting, slices were incubated at 35 °C for 20 minutes and then stored at room

temperature. During recording, slices were continuously superfused with physiological

extracellular solution containing (in mM): 125 NaCl, 25 NaHCO3, 2.5 KCl, 1.25 NaH2PO4, 2

CaCl2, 1 MgCl2 and 25 glucose, bubbled with 95% O2 and 5% CO2. Slices were visualized with

an Olympus BX50WI (Olympus Optical Co., Tokyo, Japan) upright microscope using infrared

differential interference contrast videomicroscopy with an immersion 60X objective.

Patch pipettes were pulled from borosilicate glass tubing (1.65 mm outer, 0.75 mm inner

diameter, Dagan Corp., Minneapolis, USA) and heat polished before use. Pipettes were filled

with an internal solution consisting of (in mM): 140 KCl, 2 MgCl2, 10 EGTA, 2 Na2 ATP, 10

HEPES, pH adjusted to 7.3 with KOH. Tip resistances in working solutions were 2-4 MΩ. The

pipettes were brought close to the target while exerting positive pressure (30-60 mbar). This

process helped in cleaning off glial cells that often wrap Purkinje cells. We used outside-out

nucleated patches for characterizing potassium currents because they allow recording of large

currents with ideal voltage-clamp conditions (Martina et al. 1997) and facilitate rapid application

of peptides like BDS-I at a range of well-defined concentrations. Recordings were performed

using an Axopatch 200B amplifier (Axon Instruments, Foster City, CA, USA). Signals were

low-pass filtered at 5 or 10 KHz (4-pole low-pass Bessel filter on amplifier) and digitized (10-20

kHz) using a Digidata 1321A data-acquisition system controlled by pClamp8 software interface

(Axon instruments, Foster City, CA, USA). Patches were held at a steady holding potential of -

80 mV. In some protocols (including those shown in Figure 1 and Figure 2), test pulses were

preceded by a 50-msec prepulse to -100 mV. Currents were corrected for leak current and for

capacity transients remaining after electronic capacity compensation using the scaled capacity

transient recorded in response to the change in voltage from -80 to -100 mV (or in some cases a

P/-4 leak correction protocol). To construct activation curves, chord conductance (G) was

calculated from peak current assuming ohmic behavior and a reversal potential of –88 mV. This

reversal potential was obtained experimentally by polynomial interpolation of data obtained from

the current/voltage relationship of the tail currents elicited by a 1 ms pulse to 50 mV; the reversal

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potentials obtained from each cell were then pooled and the mean of the pooled data was used.

Activation curves were fit with a Boltzmann function raised to the fourth power: 1/1+exp [-(V-

Vh)/k]4, where V is the membrane potential, Vh is the potential at which the value of the

Boltzmann function is 0.5, and k is the slope factor.

Different concentrations of BDS-I (Alomone Labs, Jerusalem) were applied using quartz

microcapillaries in a linear array. Toxin application was performed using a HEPES-buffered

external solution containing: 140 mM NaCl, 4 mM KCl 4, 2 mM CaCl2, 1 mM MgCl2, 25 mM

glucose, 10 mM HEPES, pH adjusted to 7.3 with NaOH. The currents were evoked in the

presence of TTX (300 nM, Sigma Chemicals, St. Louis) and cadmium (50 µM) to block currents

through voltage-dependent sodium and calcium channels.

Current clamp experiments examining the effect of BDS-1 on action potentials were

performed on freshly dissociated Purkinje neurons. These experiments were done with

dissociated neurons rather than neurons in brain slice in order to facilitate rapid solution

exchange of well-defined toxin concentrations. Cells were isolated from the brains of 13- to 16

day-old rats using the following procedure (Swensen and Bean, 2003): the cerebellum was

isolated from the brain and chopped into small chunks which were stored in an ice-cold

dissociation solution containing (in mM): 82 Na2SO4, 30 K2SO4, 10 HEPES, 10 glucose, 5

MgCl2, 0.001% phenol red (pH 7.4 with NaOH). The chunks were then transferred into a tube

containing dissociation solution plus 3 mg/ml Protease XXIII (Sigma) and incubated at 33°C for

7 to 9 minutes. Subsequently, the tissue was transferred into a tube containing dissociation

solution plus trypsin inhibitor (1 mg/ml) and kept for 10 minutes at 4°C. The chunks were

finally transferred into a tube containing ice-cold dissociation solution and stored in this solution

at 4°C. To free cells, 2-3 chunks were mechanically dissociated by gentle trituration through the

tip of three fire-polished Pasteur pipettes of progressively smaller diameter.

Recordings were performed at room temperature (21-24 °C), except those shown in

Figure 7, done at 31 °C. It was not feasible to obtain data at warmer temperatures than this,

because the nucleated patches had dramatically shorter lifetimes as the temperature was raised

above room temperature. Also, even at room temperature, application of BDS-I toxin at

concentrations >1 µM frequently caused loss of the patch, apparently by disrupting the seal.

Data are reported as mean ± SEM, and error bars in figures also represent SEM.

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Results

We first used step depolarizations to characterize the kinetics and voltage-dependence of

potassium currents in Purkinje neurons (Figure 1A). To obtain optimal kinetic resolution, voltage

clamp experiments were carried out using nucleated patches formed from Purkinje neurons in

brain slice. Nucleated patches had large macroscopic potassium currents, with typical amplitudes

of 2-10 nA when elicited by a series of depolarizing voltage steps (-70 mV to +70 mV). The

potassium currents in nucleated patches were larger than in previous recordings from outside-out

patches from Purkinje neurons (Southan and Robertson, 2000; Martina et al., 2003; McKay and

Turner, 2004) but were otherwise similar, with rapid activation, rapid deactivation, and slow and

incomplete inactivation over hundreds of milliseconds. Figure 1B shows the voltage-dependence

of activation of potassium current in collected data from nucleated patches from 7 Purkinje

neurons. Potassium conductance activated with a midpoint of +6 mV.

To characterize voltage-dependent potassium currents during the brief action potential

characteristic of Purkinje neurons, we elicited potassium currents in voltage-clamp mode using

as a waveform a typical action potential (previously recorded in current clamp). Figure 1C shows

the current elicited by an action potential waveform. As expected, the current begins to be

activated near the peak of the spike and is maximal during the falling phase of the spike. What

fraction of the potassium channels present in the patch are activated by the spike? We addressed

this question by comparing (in the same patch) the spike-evoked current to the current activated

by a prolonged step to +30 mV, the approximate voltage reached at the peak of the spike (Figure

1D). Since this step produces nearly complete (~80%) activation, the comparison (Baranauskas

et al., 2003) gives an indication of the fraction of total potassium channels that are activated

during the brief spike. In the experiment shown in Figure 1C-D, the peak current activated by the

spike waveform was about 25% of that activated by a maintained step to +30 mV. In collected

experiments, the potassium current activated by the action potential waveform was 19±3% (n=8)

of the peak voltage-gated potassium current evoked by a square pulse to +30 mV. To examine

how different the kinetics of potassium currents in Purkinje neurons are from potassium currents

in other neurons (when examined on the time scale of a Purkinje spike), we also used the

waveform of a Purkinje neuron action potential as voltage command in nucleated patches from

two other neuronal types, dentate gyrus basket cells (fast-spiking GABAergic neurons) and

hippocampal CA1 pyramidal cells. In both of these cell types, the action potential waveform was

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much less effective at activating potassium current compared to Purkinje neurons (Figure 2). For

potassium currents in dentate gyrus basket cells and hippocampal pyramidal neurons, the fraction

of the total current activated by the Purkinje cell spike waveform was respectively only 5±3%

(n=3) and 4±2% (n=3) of that elicited by a step to +30 mV. The comparison suggests that the

potassium currents of Purkinje neurons have functional properties specialized for rapid activation

during narrow spikes - not only in comparison with relatively slow-spiking glutamatergic

pyramidal neurons, but also when compared with the fast-spiking GABAergic dentate basket

cells. Interestingly, similar experiments have shown even more efficient activation of K

currents (spike/step ratio ~40%) by action potentials in other populations of fast-spiking neurons,

including globus pallidus neurons, subthalamic nucleus neurons, and a different population of

hippocampal interneurons (Baranauskas et al., 2003). The difference probably reflects

differences in the spike shapes as much as kinetics of potassium currents, since the spikes of

Purkinje neurons are exceptionally brief even compared with many other fast-spiking neurons.

Figure 3A shows the kinetics of activation of Purkinje cell potassium currents on a fast

time scale. Activation kinetics were both rapid and steeply voltage dependent, with a rise-time

(measured as the time for current to rise from 10% to 90% of its peak value) that declined from

about 4 msec at 0 mV to an asymptotic value of about 0.3 msec above +60 mV (Figure 3B).

Deactivation kinetics were also very rapid (Figure 3C-D). Deactivation was well fit by a single

exponential with a steep dependence on voltage, reaching a value of about 0.3 msec at -80 mV

and 0.2 msec near -120 mV. Rapid activation and deactivation kinetics are typical of Kv3 family

channels and are consistent with previous reports of the predominant potassium current in

Purkinje neurons (Raman and Bean, 1999; Southan and Robertson, 2000; Martina et al., 2003;

McKay and Turner, 2004).

To explore the pharmacological characteristics of the dominant potassium current in

Purkinje neurons, we tested the effect of BDS-I. Since the major potassium current in cell bodies

of Purkinje neurons has the kinetic characteristics and high TEA-sensitivity typical of Kv3-

mediated current (Southan and Robertson, 2000; Raman and Bean, 1999; Martina et al., 2003;

McKay et al., 2005) and Purkinje neurons express Kv3 subunits (Martina et al., 2003; Rashid et

al., 2001; McKay et al., 2005), it seemed likely that much of the current in nucleated patches

would be blocked by the toxin. Surprisingly, however, the toxin was remarkably ineffective at

reducing the current elicited from Purkinje cell nucleated patches by step depolarizations from -

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80 mV (Figure 4A, B). Concentrations of up to 1 µM BDS-I reduced step depolarization-evoked

current by <5%, and a concentration of 10 µM reduced the current by only 24±5% (n=5; Figure

4B).

We also tested the effect of BDS-I on potassium current elicited by action potential

waveforms, reasoning that such waveforms might preferentially elicit a subpopulation of

channels with particularly high affinity for BDS-I. In fact, the current elicited by an action

potential waveform was highly sensitive to BDS-I. Figure 4C shows the result from a typical

patch, in which 500 nM BDS-I blocked the action potential-evoked current by more than 50%

and 5 uM BDS-I blocked almost completely. These results were typical (Figure 4D), with 500

nM BDS-I blocking action potential-evoked current by 57±3 % (n=4) and 5 uM BDS-I blocking

by 94±3 % (n=3).

One possible interpretation of the contrasting results with step depolarizations and action

potential waveforms is that the toxin is highly-selective for a very rapid component of current

that is not obvious on the time-scale of long depolarizations or is obscured by other components

of current with slower kinetics. Figure 5A shows the result of obtaining the BDS-I-sensitive

current by subtraction of traces before and after application of the toxin. As defined in this way,

the BDS-I-sensitive current appears to be a component of current with rapid activation and rapid

decay. At first glance, the properties of the BDS-sensitive current defined by subtraction appear

consistent with the expected properties of current mediated by K3.4 homomers, which in

heterologous expression systems produce current with rapid inactivation (Schröter et al. 1991;

Diochot et al., 1998). If this interpretation is correct, the rising phase of the total control current

contains a fast-inactivating component of current that is not obvious due to overlapping slower-

activating components of current. In this case, it might be possible to isolate the rapidly

inactivating component of current in another way by using a prepulse to inactivate this

component of current. Figure 5B shows a test with such a protocol (in the same patch as in

Figure 5A). However, no such fast-inactivating component of current was revealed by the

prepulse protocol, even when the recovery interval between the prepulse and subsequent test

pulse was made extremely short (1.5 msec in the experiment in Figure 5B).

Examination of the rising phase of the current with and without BDS-I on a fast time base

(Figure 5C) suggests a different interpretation of the results in Figure 5A. In the presence of

toxin, the current rises more slowly and with an increased delay, as if the effect of the toxin is

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not to completely block the predominant potassium current but rather to slow its activation while

having only a small effect on the steady-state current. This interpretation is strongly supported by

the experiments of Baranauskas and colleagues (2003) and Yeung and colleagues (2005), who

found that when applied to heterologously expressed Kv3.4/Kv3.1 heteromeric channels or to

Kv3.1 or Kv3.2 homomeric channels, BDS-I dramatically slows activation in a very similar

manner to the results in Figure 5C with native Purkinje cell channels. Since the results of Yeung

and colleagues were obtained with homogeneous channels formed by a single type of subunit,

their results are best interpreted as modification of gating of a uniform population of channels

rather than block of a subpopulation. The effects of BDS-I on the potassium currents in Purkinje

neurons can most reasonably be interpreted in exactly the same way.

According to this interpretation, the high sensitivity of the action potential-evoked current

in Purkinje neurons to BDS-I results from the slowing of activation, together with the very

narrow action potentials that are typical of Purkinje neurons. Since the toxin only slows

activation of the predominant potassium current in Purkinje neurons rather than blocking it, the

voltage clamp experiments predict that in current clamp, the effects of the toxin on spike width

might be relatively modest, even if all the channels responsible for the rapid action potential

repolarization are affected by the toxin. Figure 6 shows the effect of the toxin on action potential

firing in current clamp. These experiments were done using acutely dissociated Purkinje neurons,

which greatly facilitated rapid application of toxin. (We could not use nucleated patches for the

current clamp experiments since they generally have only small sodium currents and cannot

generate action potentials with normal parameters.) As previously described, in the absence of

synaptic input Purkinje neurons fired spontaneous action potentials at typical frequencies of 10-

40 Hz (Häusser and Clark, 1997; Raman and Bean, 1997). As predicted from the voltage clamp

results, application of 1 uM BDS-I slowed the rate of spike repolarization and increased spike

width of spontaneous action potentials (Figure 6A). On average, spike width (measured at half-

maximum amplitude) was increased by 29 ± 4 %, from an average of 420±23 to 543±24

microseconds (p<0.01, n=4). In addition, the most negative voltage reached immediately after

the first spike was less negative (by an average of 3.8 ± 0.8 mV, n=4) in the presence of toxin (-

76.2 ± 4.8 mV in control and -72.4 ± 5.0 in BDS-I toxin, p<0.05, n=4), consistent with inhibition

of a spike-activated potassium conductance.

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Figure 6B shows the effect of BDS-I on burst firing elicited by injection of current. In

this experiment, spontaneous firing was stopped by passing steady hyperpolarizing current (100

pA). As previously described (Raman and Bean, 1997), injection of brief depolarizing current

pulses produced all-or-none firing of bursts of action potentials. In the experiment shown in

Figure 6B, a 1.5-msec current pulse elicited a burst of two spikes followed by an after-

depolarization. Application of 1 uM BDS-I produced a substantial broadening of the spike,

increasing spike width from 380 to 420 microseconds. In 5 such experiments, the duration of the

first spike increased by 17 ± 2%, from an average of 392 ± 29 to 549 ± 31 microseconds

(p<0.01). A similar change was observed for the second spike (spike broadening by 16 ± 5%,

p<0.05).

The effects of BDS-I on spike width, though not large, seem consistent with the voltage

clamp experiments suggesting that Kv3 channels underlie the majority of potassium current

flowing during repolarization of normal fast spikes. The relatively modest effects on spike width

might be expected since BDS-I only slows activation rather than completely blocking Kv3-

mediated current. Also, even if Kv3 channels were completely blocked, other types of potassium

channels might become activated as spikes get wider. The experiments described so far were

carried at out at 21-24 °C, where resolution of kinetics is better than at physiological

temperature. Do Kv3 channels also play a dominant role at more physiological temperatures,

where in principle other channel types might activate more rapidly and contribute more to spike

repolarization than at room temperature? To explore this, we carried out experiments at warmer

temperatures. A significant technical problem was that nucleated patches tended to have much

shorter lifetimes at warmer temperatures, especially with applications of BDS-I toxin, which at

concentrations >1 µM often caused loss of the patch, seemingly due to destabilization of the

gigohm seal between membrane and pipette glass. Because of these limitations, it was not

feasible to obtain data with BDS-I at physiological temperatures. However, we were able to

perform experiments at 31 °C using toxin concentrations up to 3 µM (Figure 7). The results were

essentially identical to those at room temperature. Figure 7A shows the effect of increasing

concentrations of BDS-I on current elicited by an action potential waveform previously recorded

from a Purkinje neuron in brain slice at 31 °C, and (in the same application of toxin to the same

patch) on current elicited by a rectangular step from -60 mV to +15 mV (the voltage at which the

action potential peaked). Just as at room temperature, BDS-I was far more effective at reducing

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the current evoked by the action potential than the current activated by the voltage step. The

spike-evoked current was blocked essentially completely by 3 µM BDS-I, while the step-evoked

current was reduced by less than 30%. The slowing of activation kinetics by toxin was evident at

31 °C as well as at room temperature. In collected results, the dose-response relationship for

BDS-I reduction of spike-evoked current was very similar for the experiments carried out at 31

°C as for the more extensive experiments at room temperature (Fig. 7B).

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Discussion

Our results using action potential clamp show that the potassium current flowing through

voltage-dependent potassium channels during the repolarizing phase of action potentials in

Purkinje neurons is entirely inhibited by BDS-I toxin, even though the toxin has relatively little

blocking effect on peak current elicited by voltage steps. Analysis of BDS-I action shows that the

inhibition of spike-evoked potassium current is due to a slowing of activation of the current that

is profound enough to completely abolish activation of the channels on the time-scale of the

action potential. Although most of our experiments were performed at room temperature (22-24

°C), nearly complete inhibition of spike-evoked potassium current by BDS-I was also seen at 31

°C, suggesting that the dominant role of Kv3 currents in spike repolarization is likely to hold for

spikes at physiological temperature as well.

All central neurons so far studied in any detail appear to have multiple types of purely

voltage-dependent potassium channels (in addition to calcium-activated potassium channels).

This includes cerebellar Purkinje neurons, which express currents or channels corresponding to

Kv1, Kv3, and Kv4 families (Chung et al. 2001; Veh et al., 1995; McKay et al., 2005; Serodio

and Rudy, 1998; see also Gruol et al., 1989; Sacco and Tempia, 2002). In glutamatergic neurons

in hippocampus and cortex, which have been studied the most extensively, there is evidence that

at least two different types of voltage-dependent potassium currents play major roles in action

potential repolarization (Locke and Nerbonne, 1997; Kang et al., 2000; Riazanski et al., 2001;

Mitterdorfer and Bean, 2002). It is therefore striking that although Purkinje neurons express

multiple types of potassium currents, Kv3-like currents sensitive to BDS-I accounted for all of

the current flowing during the normal action potential in our experiments. These data are

consistent with previous experiments with action potential clamp showing that all of the voltage-

dependent potassium current flowing during Purkinje neuron spikes is blocked by 1 mM external

TEA (Raman and Bean, 1999). Evidently, the current from Kv3-like channels in Purkinje

neurons is so large and activates so quickly that repolarization is complete before the other

currents present in the neurons are able to activate. This fits with the remarkably narrow action

potentials of Purkinje neurons. We note that our experiments were confined to studying purely

voltage-dependent potassium current, not calcium-activated potassium channels. Since block of

calcium-activated potassium current has no effect on spike width in Purkinje neurons (Womack

and Khodakhah, 2002; Edgerton and Reinhart, 2003) it is unlikely to play a major role in at least

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initial repolarization, although it may flow in late stages of repolarization (Raman and Bean;

1999; Swensen and Bean, 2003) and clearly contributes to the after-hyperpolarization (Womack

and Khodakhah, 2002; Edgerton and Reinhart, 2003).

The effect of BDS-I to produce slowing of activation of native potassium channels in

cerebellar Purkinje neurons (Figure 5) is very similar to that reported for cloned homomeric

Kv3.1 and Kv3.2 channels (Yeung et al., 2005) as well as cloned channels containing Kv3.4a

subunits and native channels in globus pallidus neurons (Baranauskas et al., 2003). Yeung et al.

(2005) found that the interaction site of BDS-I with Kv3.1 and Kv3.2 channels responsible for

producing slowing of activation is conserved in both Kv3.4 and Kv3.3 channels, suggesting that

this effect is likely to be seen with all Kv3-family channels. Thus, the BDS-I sensitivity of the

spike-evoked potassium current in Purkinje neurons is likely not useful in determining the

precise subunit makeup of these channels (other than confirming their identity as Kv3-family

channels).

The finding that BDS-I inhibits almost all spike-evoked current in Purkinje neurons is

consistent with recent data from mice lacking Kv3.3 channels (McMahon et al., 2004; Akemann

and Knöpfel, 2006). The spike width of spontaneous action potentials in Purkinje neurons from

Kv3.3-null mice was increased by 46% compared to wild-type mice (McMahon et al., 2004),

consistent with a dominant role of Kv3-family channels in repolarization. The smaller increase in

spike width we saw with BDS-I (29% for spontaneous spikes) might be expected, since BDS-I

only slows activation rather than totally eliminating the current. Interestingly, Akemann and

Knöpfel (2006) found that the broader spikes in Kv3.3-null mice had, on average, slightly more

negative and significantly longer-lasting afterhyperpolarizations than in wild-type mice,

suggesting that the potassium channels repolarizing the spikes in mutant animals have much

slower deactivation than the Kv3 channels that dominate in wild-type mice. In contrast, the acute

action of BDS-I produced in a change in the opposite direction, shifting the maximum

afterhyperpolarization in the depolarizing direction. The difference raises the possibility that the

changes in mutant animals are partly due to changes in expression levels of other potassium

channels resulting from long-term loss of Kv3.3-containing channels.

The rapid kinetics of activation and deactivation of the potassium current in nucleated

patches from Purkinje neurons are consistent with Kv3-family channels. Interestingly, however,

there are clear quantitative differences from the kinetics described for Kv3-family currents in

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some other fast-spiking central neurons. The kinetics of both activation and deactivation appear

to be even faster for the Kv3-like channels in Purkinje neurons than for the Kv3-like channels in

interneurons so far studied. Thus, the current in Purkinje neurons has a rise-time (10% to 90%)

of about 0.3 msec above +60 mV (Figure 3B), which is three times faster than that basket cells of

the dentate gyrus (0.9 msec; Martina et al., 1998) or oriens-alveus (OA) interneurons (0.8 msec

for 20% to 80% rise; Lien et al., 2002). Clearly, the extremely rapid activation of the channels in

Purkinje neurons will help produce exceptionally narrow action potentials, which in fact are

more narrow in Purkinje neurons (0.3-0.5 msec; Stuart and Häusser, 1994; Martina et al., 2003;

McMahon et al., 2004) than in OA interneurons (~ 1 msec; Lien and Jonas, 2003; all

measurements at room temperature). Similarly, we found a time constant for deactivation of

about 2.5 msec at -40 mV, which can be compared to a time constant of about 11 msec at -40

mV in OA interneurons (Lien et al., 2002) and 5.8 msec in basket cells in the dentate gyrus

(Martina et al., 1998). This suggests that the channels in Purkinje neurons and hippocampal

interneurons, while sharing all the properties expected of Kv3 family channels, are formed of

different combinations of subunits. Most likely, the currents in Purkinje neurons are formed of

some combination of Kv3.3 and Kv3.4 subunits (Martina et al., 2003; McMahon et al., 2004),

while those in dentate gyrus basket cells are formed of Kv3.1 and Kv3.2 subunits (Martina et al.,

1998) and those in OA interneurons are mainly homomeric Kv3.2 channels (Lien et al., 2002).

Lien and Jonas (2003) found that adding Kv3-like currents in OA neurons (using dynamic

clamp, with native currents blocked) produced faster spiking only if the deactivation rate was

tuned to near that of native channels in OA neurons; thus it can be predicted that adding

Purkinje-like currents would not produce faster spiking in OA neurons. It therefore seems likely

that the different kinetics in the two types of neurons are of functional significance.

We measured an average deactivation time constant of 330 microseconds at -80 mV,

similar to that McKay and Turner (2004) reported for outside-out patches from Purkinje neurons

(660 microseconds). Both are comparable to the decay of potassium current in response to action

potential waveforms (time constant of 228 microseconds; Raman and Bean, 1999). The very

rapid decay of voltage-dependent potassium current in Purkinje neurons clearly facilitates high-

frequency firing by limiting the afterhyperpolarization and quickly restoring a high membrane

resistance. The fast decay of voltage-dependent potassium current in Purkinje neurons spikes is

consistent with the afterhyperpolarization (which reaches only about -75 mV, far from the

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potassium equilibrium potential) being mainly due to calcium-activated potassium currents,

primarily through BK channels ((Womack and Khodakhah, 2002; Edgerton and Reinhart, 2003).

Even BK-mediated currents elicited by action potential waveforms decay quickly (Raman and

Bean, 1999) so that the afterhyperpolarization following single spikes in Purkinje neurons is

much less prominent than in many other central neurons. The effects on BDS-I in producing

spike broadening in Purkinje neurons are generally similar as in globus pallidus neurons

(Baranauskas et al., 2003) but with less broadening in Purkinje neurons and with a smaller effect

on the AHP.

The mode of action of BDS-I in slowing activation rather than completely blocking

channels greatly limits its utility for pharmacologically defining specific components of

potassium current. However, this mode of action could actually be a benefit in a broader

pharmacological context. In general, channel-targeted drugs that are most clinically useful do not

completely inhibit their target channels but rather exert more subtle modulating effects. The

effects of BDS-I are naturally delimited in the context of normal firing of action potentials in

central neurons, since even at saturating concentrations of toxin, full activation of channels still

occurs after a delay of less than a msec. Such an action produces only modest broadening of the

action potential, as we saw. 4-Aminopyridine and related compounds (which potently inhibit

Kv3-family channels and have weaker effects on many other potassium channels) have clinical

utility (e.g. Sanders et al., 2000; Maniero et al., 2004; Judge et al., 2006), including actions that

may reflect modification of Purkinje neuron activity (Strupp et al., 2003; 2004), but they also

have serious side effects. If it were possible to develop small molecules with a similar mode of

action as BDS-I, these should have effects on action potential width and excitability with a well-

defined “ceiling” and therefore less necessity to achieve a narrow range of drug concentrations

before production of excessive block and toxicity.

Acknowledgements

Supported by the National Institute of Neurological Diseases and Stroke (NS36855) and the

American Epilepsy Foundation.

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References

Akemann W, Knöpfel T. Interaction of Kv3 potassium channels and resurgent sodium current

influences the rate of spontaneous firing of Purkinje neurons. J Neurosci. 26:4602-4612,

2006.

Armstrong DM, Rawson JA. Activity patterns of cerebellar cortical neurones and climbing fibre

afferents in the awake cat. J Physiol (Lond) 289:425– 448, 1979.

Baranauskas G. Tkatch T. Nagata K. Yeh JZ. Surmeier DJ. Kv3.4 subunits enhance the

repolarizing efficiency of Kv3.1 channels in fast-spiking neurons. Nat Neurosci. 6:258-266,

2003.

Chung YH, Shin C, Kim MJ, Lee BK, Cha CI. Immunohistochemical study on the distribution of

six members of the Kv1 channel subunits in the rat cerebellum. Brain Res. 895:173-177,

2001.

Diochot S, Schweitz H, Béress L, Lazdunski M. Sea anemone peptides with a specific blocking

activity against the fast inactivating potassium channel Kv3.4. J Biol Chem 273:6744-6749,

1998.

Du J, Zhang L, Weiser M, Rudy B, McBain CJ. Developmental expression and functional

characterization of the potassium-channel subunit Kv3.1b in parvalbumin-containing

interneurons of the rat hippocampus. J Neurosci. 16:506-518, 1996.

Edgerton JR, Reinhart PH. Distinct contributions of small and large conductance Ca2+-activated

K+ channels to rat Purkinje neuron function. J Physiol (Lond) 548:53-69, 2003.

Erisir A, Lau D, Rudy B, Leonard CS. Function of specific K(+) channels in sustained high-

frequency firing of fast-spiking neocortical interneurons. J Neurophysiol. 82:2476-2489, ,

1999.

Goldman-Wohl D, Chan E, Baird D, Heintz N. Kv3.3b: a novel shaw type potassium channel

expressed in terminally differentiated cerebellar Purkinje cells and deep cerebellar nuclei. J

Neurosci 14:511-522, 1994.

Gruol DL, Dionne VE, Yool AJ. Multiple voltage-sensitive K+ channels regulate dendritic

excitability in cerebellar Purkinje neurons. Neurosci Lett 97:97-102, 1989.

Häusser M, Clark BA. Tonic synaptic inhibition modulates neuronal output pattern and

spatiotemporal synaptic integration. Neuron 19:665-678, 1997.

of 32

http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&list_uids=10561420&dopt=Abstract


18

Judge SI, Bever CT Jr. Potassium channel blockers in multiple sclerosis: Neuronal K(v) channels

and effects of symptomatic treatment. Pharmacol Ther, in press.

Kang J,Huguenard JR, Prince DA. Voltage-gated potassium channels activated during action

potentials in layer V neocortical pyramidal neurons. J Neurophysiol 83:70-80, 2000.

Khaliq ZM, Gouwens NW, Raman IM. The contribution of resurgent sodium current to high-

frequency firing in Purkinje neurons: an experimental and modeling study. J Neurosci.

23:4899-4912, 2003.

Khaliq ZM, Raman IM. Axonal propagation of simple and complex spikes in cerebellar Purkinje

neurons. J Neurosci. 25:454-463, 2005.

Lien CC, Jonas P. Kv3 potassium conductance is necessary and kinetically optimized for high-

frequency action potential generation in hippocampal interneurons. J Neurosci. 23:2058-2068,

2003.

Lien CC, Martina M, Schultz JH, Ehmke H, Jonas P. Gating, modulation and subunit

composition of voltage-gated K(+) channels in dendritic inhibitory interneurones of rat

hippocampus. J Physiol 538:405-419, 2002.

Llinás R, Sugimori, M. Electrophysiological properties of in vitro Purkinje cell dendrites in

mammalian cerebellar slices. J Physiol (Lond) 305:197-213, 1980.

Locke RE, Nerbonne JM. Role of voltage-gated K+ currents in mediating the regular-spiking

phenotype of callosal-projecting rat visual cortical neurons. J Neurophysiol 78:2321-2335,

1997.

Mainero C, Inghilleri M, Pantano P, Conte A, Lenzi D, Frasca V, Bozzao L, Pozzilli C.

Enhanced brain motor activity in patients with MS after a single dose of 3,4-diaminopyridine.

Neurology 62:2044-2050, 2004.

Martina M and Jonas P. Functional differences in Na+ channel gating between fast-spiking

interneurones and principal neurones of rat hippocampus. J. Physiol (London). 505:593-603,

1997.

Martina M, Schultz JH, Ehmke H, Monyer H and Jonas P. Functional and molecular differences

between voltage-gated K+ channels of fast-spiking interneurons and pyramidal neurons of rat

hippocampus. J. Neurosci. 18:8111-8125, 1998.

of 32

19

Martina M, Yao GL, and Bean, BP. Properties and functional role of voltage-dependent

potassium channels in dendrites of rat cerebellar Purkinje neurons. J Neurosci. 23: 5698-5707,

2003.

Massengill JL, Smith MA, Son DI, O'Dowd DK. Differential expression of K4-AP currents and

Kv3.1 potassium channel transcripts in cortical neurons that develop distinct firing

phenotypes. J Neurosci. 17:3136-3147, 1997.

McKay BE, Turner RW. Kv3 K+ channels enable burst output in rat cerebellar Purkinje cells.

Eur. J. Neurosci. 20: 729-739, 2004.

McKay BE, Molineux ML, Mehaffey WH, Turner RW. Kv1 K+ channels control Purkinje cell

output to facilitate postsynaptic rebound discharge in deep cerebellar neurons. J. Neurosci. 25:

1481-1492, 2005.

McMahon A, Fowler SC, Perney TM, Akemann W, Knöpfel T, Joho RH. Allele-dependent

changes of olivocerebellar circuit properties in the absence of the voltage-gated potassium

channels Kv3.1 and Kv3.3. Eur J Neurosci 19:3317-3327, 2004.

Schröter KH, Ruppersberg JP, Wunder F, Rettig J, Stocker M, Pongs O. Cloning and functional

expression of a TEA-sensitive A-type potassium channel from rat brain. FEBS Lett. 278:211-

216, 1991.

Mitterdorfer J and Bean BP. Potassium currents during the action potential of hippocampal CA3

neurons. J.Neurosci. 22:10106-10115, 2002.

Monsivais P, Clark BA, Roth A, Häusser M. Determinants of action potential propagation in

cerebellar Purkinje cell axons. J Neurosci. 25:464-472, 2005.

Raman IM, Bean BP. Resurgent sodium current and action potential formation in dissociated

cerebellar Purkinje neurons. J Neurosci. 17:4517-4526, 1997.

Raman IM, Bean BP. Ionic currents underlying spontaneous action potentials in isolated

cerebellar Purkinje neurons. J Neurosci. 19:1663-1674, 1999.

Rashid AJ, Dunn RJ, Turner RW. A prominent soma-dendritic distribution of Kv3.3 K+

channels in electrosensory and cerebellar neurons. J Comp Neurol. 441:234-247, 2001.

Riazanski V. Becker A. Chen J. Sochivko D. Lie A. Wiestler OD. Elger CE. Beck H. Functional

and molecular analysis of transient voltage-dependent K+ currents in rat hippocampal granule

cells. J. Physiol. 537:391-406, 2001.

of 32


20

Rudy B. and McBain CJ. Kv3 channels: voltage-gated K+ channels designed for high-frequency

repetitive firing. Trends in Neurosciences. 24:517-526, 2001.

Sacco T, Tempia F. A-type potassium currents active at subthreshold potentials in mouse

cerebellar Purkinje cells. J Physiol (Lond) 543: 505-520, 2002.

Sanders DB, Massey JM, Sanders LL, Edwards LJ. A randomized trial of 3,4-diaminopyridine in

Lambert-Eaton myasthenic syndrome. Neurology. 54:603-607, 2000.

Serodio P, Rudy B. Differential expression of Kv4 K+ channel subunits mediating subthreshold

transient K+ (A-type) currents in rat brain. J Neurophysiol. 79:1081-1091, 1998.

Shevchenko T, Teruyama R, Armstrong WE. High-threshold, Kv3-like potassium currents in

magnocellular neurosecretory neurons and their role in spike repolarization. J Neurophysiol.

92:3043-3055, 2004.

Southan AP, Robertson B. Electrophysiological characterization of voltage-gated K+ currents in

cerebellar basket and Purkinje cells: Kv1 and Kv3 channel subfamilies are present in basket

cell nerve terminals. J Neurosci 20:114-122, 2000.

Strupp M, Schuler O, Krafczyk S, Jahn K, Schautzer F, Buttner U, Brandt T. Treatment of

downbeat nystagmus with 3,4-diaminopyridine: a placebo-controlled study. Neurology

61:165-170, 2003.

Strupp M, Kalla R, Dichgans M, Freilinger T, Glasauer S, Brandt T. Treatment of episodic ataxia

type 2 with the potassium channel blocker 4-aminopyridine. Neurology 62:1623-1625, 2004.

Stuart G, Häusser M. Initiation and spread of sodium action potentials in cerebellar Purkinje

cells. Neuron 13:703-712, 1994.

Swensen AM, Bean BP. Ionic mechanisms of burst firing in dissociated Purkinje neurons. J

Neurosci. 23:9650-9663, 2003.

Veh RW, Lichtinghagen R, Sewing S, Wunder F, Grumbach IM, Pongs O.

Immunohistochemical localization of five members of the Kv1 channel subunits: contrasting

subcellular locations and neuron-specific co-localizations in rat brain. Eur J Neurosci.

7:2189-21205, 1995.

Weiser M, Vega-Saenz de Miera E, Kentros C, Moreno H, Franzen L, Hillmann D, Baker H,

Rudy B. Differential expression of Shaw-related K+ channels in the rat central nervous

system. J Neurosci 14:949-972, 1994.

of 32

21

Womack MD, Khodakhah K Characterization of large conductance Ca2+-activated K+ channels

in cerebellar Purkinje neurons. Eur J Neurosci 16:1214-1222, 2002.

Yeung SY, Thompson D, Wang Z, Fedida D, Robertson B. Modulation of Kv3 subfamily

potassium currents by the sea anemone toxin BDS: significance for CNS and biophysical

studies. J Neurosci. 25:8735-8745, 2005.

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Figure Legends

Figure 1. Efficient activation of voltage-gated potassium channels by the action potential in

cerebellar Purkinje neurons. A, Voltage-activated potassium currents elicited in a nucleated

patch by steps to voltages ranging from -60 mV to +70 mV. B, Voltage-dependence of

activation. Peak conductance was calculated from peak current using a reversal potential of -88

mV and normalized to that elicited by the largest step (to +70 mV) and plotted versus test pulse

voltage. Filled circles show mean values from 7 nucleated patches. Solid curve is given by

[1/(1+exp[-(V-Vh)/k])]4, where V is the test step voltage, Vh is the potential at which the

Boltzmann function is 0.5, and k is the slope factor. The values for Vh and k were obtained from

the average values from fits to individual conductance curves in 7 neurons: Vh = -26 ± 3 mV, and

k = 19 ± 1 mV. This function reaches a midpoint at a value of Vh+1.66*k, or +5.6 mV. C,

Potassium current elicited by an action potential waveform in the same patch as A. Action

potential was previously recorded in a different Purkinje neuron during spontaneous firing, using

the same internal and external solutions except without TTX in the external solution. D, Current

elicited by a maintained step to +30 mV, near the peak of the action potential.

Figure 2. Efficiency of activation of potassium currents by a Purkinje cell action potential

in three neuronal types. A, Voltage-activated potassium currents elicited in a nucleated patch

from a Purkinje neuron (different cell than in Figure 1) by a step to +30 mV (left) and by an

action potential waveform (previously recorded in a different Purkinje neuron during

spontaneous firing). B, Currents elicited in a nucleated patch from a dentate gyrus basket cell by

the same waveforms (step to +30 mV and the same Purkinje cell spike). C, Currents elicited in a

nucleated patch from a CA1 pyramidal neuron by the same waveforms. D, Collected results

(mean ± SEM) for the ratio of peak current elicited by the action potential waveform to peak

current elicited by a maintained step to +30 mV for experiments on 8 Purkinje neurons, 3 basket

cells, and 3 CA1 pyramidal neurons.

Figure 3. Rapid activation and deactivation kinetics of potassium currents in Purkinje

neuron nucleated patches. A, Currents elicited by pulses to test voltages ranging from -70 to 70

mV. B, Time for current to rise from 10% to 90% of its peak value as a function of test voltage.

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23

Markers and error bars show mean ± SEM for measurements in 7 cells. C, Deactivation kinetics.

Channels were activated by a 0.5 ms depolarization to +60 mV, followed by repolarization to

voltages ranging from -130 to -10 mV in 10 mV steps. D, Time constant of deactivation as a

function of test pulse potential (mean ± SEM for measurements in 9 cells).

Figure 4. Effects of BDS-I toxin on the potassium current induced by steps or spike

waveforms. A, Effect of 300 nM, 500 nM, and 1 µM BDS-I toxin on current elicited by a step

from -80 mV to +50 mV. B, Dose-response relationship for BDS-I effect on peak current elicited

by steps from -80 mV to +50 mV (mean ± SEM for measurements in 4-11 cells). C, Effect of

300 nM, 500 nM, 1 µM, and 5 µM BDS-I toxin on current elicited by spike waveform (different

patch). D, Dose-response relationship for BDS-I effect on peak current elicited by the spike

waveform (mean ± SEM for measurements in patches from 3-5 cells at each concentration).

Figure 5. Slowing of activation kinetics by BDS-I. A, Effect of 10 µm BDS-I toxin on

potassium current in response to a pulse to +60 mV from a holding of -90 mV. Right: Toxin-

sensitive current obtained by digital subtraction of the traces before and after toxin application.

The toxin-sensitive current decays rapidly and has a peak amplitude of 57 % of the control

current. B, Prepulse protocol testing for the presence of a rapidly-inactivating component of

current corresponding to the BDS-I-sensitive component. A 15-ms prepulse to +60 mV had little

effect on test current (also elicited by a step to +60 mV), and subtraction of test pulse currents

before and after prepulse (right panel) did not reveal a fast-inactivating component in the control

current. C, Activation of the currents in A shown at a faster time base. BDS-I produces a

slowing of the activation of the channels.

Figure 6. Effects of BDS toxin on action potential properties. A, Current clamp recording of

spontaneous activity in an acutely dissociated Purkinje cell in control conditions and after

application of 1 µM BDS-I. Right: action potentials in control (gray) and in BDS-I (black)

aligned by their rising phases. B, Effect of BDS-I on doublets of action potentials elicited by

brief current injection after spontaneous firing was stopped by passing a steady hyperpolarizing

current. Note increase in interspike interval much greater than accounted for by broadening of

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first spike. Right: action potentials in control (gray) and in BDS-I (black) aligned by their rising

phases.

Figure 7. Effects of BDS-I toxin on spike-evoked and step-evoked current at 31 °C. A,

Effect of 1 µM and 3 µM BDS-I toxin on current elicited by a spike waveform (left) and by a

step from -60 mV to +15 mV (right) in a nucleated patch at 31 °C. The two waveforms were

delivered sequentially to the same patch during the same application of toxin. The action

potential was previously recorded from an intact Purkinje neuron in a cerebellar slice at 31 °C.

B, Dose-response relationship for BDS-I effect on current elicited by spike waveforms at room

temperature and at 31 °C (mean ± SEM for measurements in patches from 3-5 cells at each

concentration). Closed circles: experiments at room temperature (replotted from Figure 4). Open

circles: experiments at 31 °C.

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Figure 1. Efficient activation of voltage-gated potassium channels by the action potential in cerebellar Purkinje neurons. A, Voltage-activated potassium currents elicited in a nucleated patch by steps to voltages ranging from -60 mV to +70 mV. B, Voltage-

dependence of activation. Peak conductance was calculated from peak current using a reversal potential of -88 mV and normalized to that elicited by the largest step (to +70

mV) and plotted versus test pulse voltage. Filled circles show mean values from 7 nucleated patches. Solid curve is given by [1/(1+exp[-(V-Vh)/k])]4, where V is the test

step voltage, Vh is the potential at which the Boltzmann function is 0.5, and k is the slope factor. The values for Vh and k were obtained from the average values from fits to

individual conductance curves in 7 neurons: Vh = -26 ± 3 mV, and k = 19 ± 1 mV. This function reaches a midpoint at a value of Vh+1.66*k, or +5.6 mV. C, Potassium current elicited by an action potential waveform in the same patch as A. Action potential was

previously recorded in a different Purkinje neuron during spontaneous firing, using the same internal and external solutions except without TTX in the external solution. D,

Current elicited by a maintained step to +30 mV, near the peak of the action potential.

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Figure 2. Efficiency of activation of potassium currents by a Purkinje cell action potential in three neuronal types. A, Voltage-activated potassium currents elicited in a nucleated patch from a Purkinje neuron (different cell than in Figure 1) by a step to +30 mV (left) and by an action potential waveform (previously recorded in a different Purkinje neuron

during spontaneous firing). B, Currents elicited in a nucleated patch from a dentate gyrus basket cell by the same waveforms (step to +30 mV and the same Purkinje cell spike). C,

Currents elicited in a nucleated patch from a CA1 pyramidal neuron by the same waveforms. D, Collected results (mean ± SEM) for the ratio of peak current elicited by the

action potential waveform to peak current elicited by a maintained step to +30 mV for experiments on 8 Purkinje neurons, 3 basket cells, and 3 CA1 pyramidal neurons.

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Figure 3. Rapid activation and deactivation kinetics of potassium currents in Purkinje neuron nucleated patches. A, Currents elicited by pulses to test voltages ranging from -70 to 70 mV. B, Time for current to rise from 10% to 90% of its peak value as a function of test voltage. Markers and error bars show mean ± SEM for measurements in 7 cells. C, Deactivation kinetics. Channels were activated by a 0.5 ms depolarization to +60 mV, followed by repolarization to voltages ranging from -130 to -10 mV in 10 mV steps. D,

Time constant of deactivation as a function of test pulse potential (mean ± SEM for measurements in 9 cells).

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Figure 4. Effects of BDS-I toxin on the potassium current induced by steps or spike waveforms. A, Effect of 300 nM, 500 nM, and 1 M BDS-I toxin on current elicited by a

step from -80 mV to +50 mV. B, Dose-response relationship for BDS-I effect on peak current elicited by steps from -80 mV to +50 mV (mean ± SEM for measurements in 4-11 cells). C, Effect of 300 nM, 500 nM, 1 M, and 5 M BDS-I toxin on current elicited by

spike waveform (different patch). D, Dose-response relationship for BDS-I effect on peak current elicited by the spike waveform (mean ± SEM for measurements in patches from

3-5 cells at each concentration).

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Figure 5. Slowing of activation kinetics by BDS-I. A, Effect of 10 m BDS-I toxin on potassium current in response to a pulse to +60 mV from a holding of -90 mV. Right:

Toxin-sensitive current obtained by digital subtraction of the traces before and after toxin application. The toxin-sensitive current decays rapidly and has a peak amplitude of 57 %

of the control current. B, Prepulse protocol testing for the presence of a rapidly-inactivating component of current corresponding to the BDS-I-sensitive component. A 15-

ms prepulse to +60 mV had little effect on test current (also elicited by a step to +60 mV), and subtraction of test pulse currents before and after prepulse (right panel) did not reveal a fast-inactivating component in the control current. C, Activation of the currents

in A shown at a faster time base. BDS-I produces a slowing of the activation of the channels.

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Figure 6. Effects of BDS toxin on action potential properties. A, Current clamp recording of spontaneous activity in an acutely dissociated Purkinje cell in control conditions and after application of 1 M BDS-I. Right: action potentials in control (gray) and in BDS-I (black) aligned by their rising phases. B, Effect of BDS-I on doublets of action potentials elicited

by brief current injection after spontaneous firing was stopped by passing a steady hyperpolarizing current. Note increase in interspike interval much greater than accounted

for by broadening of first spike. Right: action potentials in control (gray) and in BDS-I (black) aligned by their rising phases.

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Figure 7. Effects of BDS-I toxin on spike-evoked and step-evoked current at 31 °C. A, Effect of 1 M and 3 M BDS-I toxin on current elicited by a spike waveform (left) and

by a step from -60 mV to +15 mV (right) in a nucleated patch at 31 °C. The two waveforms were delivered sequentially to the same patch during the same application of toxin. The action potential was previously recorded from an intact Purkinje neuron in a

cerebellar slice at 31 °C. B, Dose-response relationship for BDS-I effect on current elicited by spike waveforms at room temperature and at 31 °C (mean ± SEM for measurements in

patches from 3-5 cells at each concentration). Closed circles: experiments at room temperature (replotted from Figure 4). Open circles: experiments at 31 °C.

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