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1Department of Physiology, Feinberg School of Medicine, Institute for Neuroscience, Northwestern University, Chicago, Illinois; and 2Department of Neurobiology, Harvard Medical School, Boston, Massachusetts
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMETHODSRESULTSDISCUSSIONGRANTSREFERENCES |
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONGRANTSREFERENCES |
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; Khaliq and Raman 2005; McKay and Turner 2004; Monsivais et al. 2005) and intracellular recordings show that they have unusually narrow action potentials, with a half-width of about 300–500 µs at room temperature (Martina et al. 2003; McMahon et al. 2004; Stuart and Häusser 1994) and as little as 220 µs 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 (Baranauskas et al. 2003; Du et al. 1996; Erisir et al. 1999; Lien and Jonas 2003; Lien et al. 2002; Martina et al. 1998; Massengill et al. 1997; 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 (Martina et al. 2003; McKay and Turner 2004; Raman and Bean 1999; Southan and Robertson 2000). In situ hybridization and immunocytochemistry previously showed 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; Martina et al. 2003; McMahon et al. 2004; Rashid et al. 2001; Weiser et al. 1994). 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, which was introduced as a selective blocker of Kv3.4 channels (Diochot et al. 1998). Subsequent studies on a variety of central neurons showed the presence of components of potassium current sensitive to the toxin (Baranauskas et al. 2003; Riazanski et al. 2001; 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 (Baranauskas et al. 2003; Diochot et al. 1998). 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 unblocking 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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METHODS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONGRANTSREFERENCES |
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Patch pipettes were pulled from borosilicate glass tubing (1.65 mm OD, 0.75 mm ID, Dagan, Minneapolis, MN) 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, and 10 HEPES (pH adjusted to 7.3 with KOH). Tip resistances in working solutions were 2–4 M
. 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). Signals were low-pass filtered at 5 or 10 kHz (four-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). Patches were held at a steady holding potential of –80 mV. In some protocols (including those shown in Figs. 1 and 2), test pulses were preceded by a 50-ms 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 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.
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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 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 stored in an ice-cold dissociation solution containing (in mM): 82 Na2SO4, 30 K2SO4, 10 HEPES, 10 glucose, 5 MgCl2, and 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 min. Subsequently, the tissue was transferred into a tube containing dissociation solution plus trypsin inhibitor (1 mg/ml) and kept for 10 min 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, two to three chunks were mechanically dissociated by gentle trituration through the tip of three fire-polished Pasteur pipettes of progressively smaller diameters.
Recordings were performed at room temperature (21–24°C), except those shown in Fig. 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.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONGRANTSREFERENCES |
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; McKay and Turner 2004; Southan and Robertson 2000) 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 seven 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 (Fig. 1D). Because this step produces nearly complete (roughly 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 Fig. 1, C and 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 timescale 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 much less effective at activating potassium current compared with Purkinje neurons (Fig. 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 roughly 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 because 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 timescale. 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 ms at 0 mV to an asymptotic value of about 0.3 ms above +60 mV (Fig. 3B). Deactivation kinetics were also very rapid (Fig. 3, C and D). Deactivation was well fit by a single exponential with a steep dependency on voltage, reaching a value of about 0.3 ms at –80 mV and 0.2 ms 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 (Martina et al. 2003; McKay and Turner 2004; Raman and Bean 1999; Southan and Robertson 2000).
To explore the pharmacological characteristics of the dominant potassium current in Purkinje neurons, we tested the effect of BDS-I. Because the major potassium current in cell bodies of Purkinje neurons has the kinetic characteristics and high TEA-sensitivity typical of Kv3-mediated current (Martina et al. 2003; McKay et al. 2005; Raman and Bean 1999; Southan and Robertson 2000) and Purkinje neurons express Kv3 subunits (Martina et al. 2003; McKay et al. 2005; Rashid et al. 2001), 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 –80 mV (Fig. 4, A and B). Concentrations of 
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; Fig. 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 >50% and 5 µM BDS-I blocked almost completely. These results were typical (Fig. 4D), with 500 nM BDS-I blocking action potential–evoked current by 57 ± 3% (n = 4) and 5 µM 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 timescale 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 (Diochot et al. 1998; Schröter et al. 1991). If this interpretation is correct, the rising phase of the total control current contains a fast-inactivating component of current that is not obvious because of 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 Fig. 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 ms in the experiment in Fig. 5B).
Examination of the rising phase of the current with and without BDS-I on a fast time base (Fig. 5C) suggests a different interpretation of the results in Fig. 5A. In the presence of toxin, the current rises more slowly and with an increased delay, as if the effect of the toxin is 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 Fig. 5C with native Purkinje cell channels. Because 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. Because 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 because 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 µM BDS-I slowed the rate of spike repolarization and increased spike width of spontaneous action potentials (Fig. 6A). On average, spike width (measured at half-maximum amplitude) was increased by 29 ± 4%, from an average of 420 ± 23 to 543 ± 24 µs (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.
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 Fig. 6B, a 1.5-ms current pulse elicited a burst of two spikes followed by an afterdepolarization. Application of 1 µM BDS-I produced a substantial broadening of the spike, increasing spike width from 380 to 420 µs. In five such experiments, the duration of the first spike increased by 17 ± 2%, from an average of 392 ± 29 to 549 ± 31 µs (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, although 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 because 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 that 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 arising from 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 3 µM (Fig. 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 to +15 mV (the voltage at which the action potential peaked). Just as at room temperature, BDS-I was far more effective at reducing 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, whereas the step-evoked current was reduced by <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 |
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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; McKay et al. 2005; Serodio and Rudy 1998; Veh et al. 1995; 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 (Kang et al. 2000; Locke and Nerbonne 1997; Mitterdorfer and Bean 2002; Riazanski et al. 2001). 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. Because block of calcium-activated potassium current has no effect on spike width in Purkinje neurons (Edgerton and Reinhart 2003; Womack and Khodakhah 2002) it is unlikely to play a major role in at least initial repolarization, although it may flow in late stages of repolarization (Raman and Bean; 1999; Swensen and Bean 2003) and clearly contributes to the afterhyperpolarization (Edgerton and Reinhart 2003; Womack and Khodakhah 2002).
The effect of BDS-I to produce slowing of activation of native potassium channels in cerebellar Purkinje neurons (Fig. 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 Kv3.2 channels responsible for producing slowing of activation is conserved in Kv3.1, 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 (Akemann and Knöpfel 2006; McMahon et al. 2004). The spike width of spontaneous action potentials in Purkinje neurons from Kv3.3-null mice was increased by 46% compared with 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 because 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 those 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 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 attributable to changes in expression levels of other potassium channels resulting from long-term loss of Kv3.3-containing channels.
The rapid activation and deactivation kinetics 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 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 (10–90%) of about 0.3 ms above +60 mV (Fig. 3B), which is three times faster than that of basket cells of the dentate gyrus (0.9 ms) (Martina et al. 1998) or oriens-alveus (OA) interneurons (0.8 ms for 20–80% 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 ms; Martina et al. 2003; McMahon et al. 2004; Stuart and Häusser 1994) than in OA interneurons (about 1 ms; Lien and Jonas 2003; all measurements at room temperature). Similarly, we found a time constant for deactivation of about 2.5 ms at –40 mV, which can be compared with a time constant of about 11 ms at –40 mV in OA interneurons (Lien et al. 2002) and 5.8 ms in basket cells in the dentate gyrus (Martina et al. 1998). This suggests that the channels in Purkinje neurons and hippocampal interneurons, although 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), whereas 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 µs at –80 mV, similar to that McKay and Turner (2004) reported for outside-out patches from Purkinje neurons (660 µs). Both are comparable to the decay of potassium current in response to action potential waveforms (time constant of 228 µs; 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 potassium equilibrium potential) arising mainly from calcium-activated potassium currents, primarily through BK channels (Edgerton and Reinhart 2003; Womack and Khodakhah 2002). 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 that in many other central neurons. The effects of BDS-I in producing spike broadening in Purkinje neurons are generally similar to those 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 because even at saturating concentrations of toxin, full activation of channels still occurs after a delay of <1 ms. 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., Judge et al. 2006; Maniero et al. 2004; Sanders et al. 2000), 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 mode of action similar to that of 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.
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FOOTNOTES |
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Address for reprint requests and other correspondence: M. Martina, Department of Physiology, Northwestern University, Feinberg School of Medicine, 303 East Chicago Avenue, Chicago, IL 60611 (E-mail: m-martina{at}northwestern.edu)
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