Properties and expression of Kv3 channels in cerebellar Purkinje cells

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Abstract

In cerebellar Purkinje cells, Kv3 potassium channels are indispensable for firing at high frequencies. In Purkinje cells from young mice (P4–P7), Kv3 currents, recorded in whole-cell in slices, activated at − 30 mV, with rapid activation and deactivation kinetics, and they were partially blocked by blood depressing substance-I (BDS-I, 1 μM). At positive potentials, Kv3 currents were slowly but completely inactivating, while the recovery from inactivation was about eightfold slower, suggesting that a previous firing activity or a small change of the resting potential could in principle accumulate inactivated Kv3 channels, thereby finely tuning Kv3 current availability for subsequent action potentials. Single-cell RT-PCR analysis showed the expression by all Purkinje cells (n = 10 for each subunit) of Kv3.1, Kv3.3 and Kv3.4 mRNA, while Kv3.2 was not expressed. These results add to the framework for interpreting the physiological function and the molecular determinants of Kv3 currents in cerebellar Purkinje cells.

Introduction

The fast spiking neuron (FSN) phenotype requires a brief action potential duration and a quick recovery of Na+ channels from inactivation (Rudy and McBain, 2001). These two goals are attained by the exploitation of two highly specialized ion channels (Akemann and Knopfel, 2006): i) a resurgent Na+ channel with an extremely fast recovery from inactivation, due to an unusual mechanism based on a peptide-mediated channel block, identified as the β4 subunit (Grieco et al., 2005) but also mimicked by an exogenously applied peptide toxin (Schiavon et al., 2006); ii) K+ channels of a subfamily (Kv3 or KCNC), which have very fast activation and deactivation kinetics associated with a high threshold for activation (Rudy and McBain, 2001). These properties of Kv3 channels cause a very fast action potential repolarization followed by a brief afterhyperpolarization, which allows a rapid recovery of Na+ channels from inactivation. Kv3 channels assemble from subunits that in mammals are coded by four genes, named KCNC1–4 (Kv3.1–Kv3.4 subunits). Homomeric Kv3.1 and Kv3.2 channels, in heterologous expression systems, produce sustained currents, while Kv3.3 and Kv3.4 produce currents respectively with a slow and fast rate of inactivation (Rudy and McBain, 2001).

Cerebellar Purkinje cells (PCs) are FSNs, which express both a resurgent Na+ current (Raman et al., 1997) and Kv3 K+ channels (Martina et al., 2003, McKay and Turner, 2004). Kv3 currents have been studied in outside-out somatic and dendritic patches pulled from PCs from rats in a late phase of development, when they express the Kv3.3 and Kv3.4 subunits (Martina et al., 2003, McKay and Turner, 2004). Under these conditions, Kv3 currents were found to inactivate either with a double exponential time course (rapid time constant 10–20 ms; slow time constant 0.2–0.5 s) with no significant difference between soma and dendrites (Martina et al., 2003) or with a single, slow time constant (0.5–1 s; McKay and Turner, 2004). The latter study also described a significant voltage dependence of slow inactivation with a V1/2 (− 51.7 mV) very close to the threshold for action potentials. The time course of recovery from inactivation was not investigated, so that it is uncertain whether channel inactivation can accumulate at subthreshold voltages and affect the amount of Kv3 current available to enable fast spiking.

In this study, we show that mouse PCs, at 7 postnatal days of age, when a dendritic tree with adult branching pattern is already present, although at the beginning of its development, exhibit large Kv3 currents. Such currents show the rapid activation and deactivation properties of Kv3 channels, but they also slowly inactivate at subthreshold potentials and they even more slowly recover from inactivation. Thus, we show for the first time that these currents can accumulate inactivation and that this can occur near the resting membrane potential of Purkinje neurons.

These currents are correlated with an expression in 100% of PCs of Kv3.1, Kv3.3 and Kv3.4 mRNAs. Provided that these mRNAs are indeed translated and the proteic subunits are inserted in the plasma membrane, this result suggests that in these cells Kv3 currents might be due to heteromeric channels composed by a combination of these three subunits, although the presence of homomeric channels in separate cellular regions cannot be excluded. Our single-cell RT-PCR analysis adds new data to the discussion of a subject where contrasting results have been reported. Regarding Kv3.1, a low but detectable expression was shown by Perney et al. (1992) and Weiser et al. (1994), while a lack of expression was reported by Weiser et al., 1995, Martina et al., 2003 and McMahon et al. (2004). Regarding Kv3.4, Weiser et al. (1994) reported a weak expression by in-situ hybridization, while by immunohistochemistry the results depended on the type of antibody used (Veh et al., 1995, Laube et al., 1996, Martina et al., 2003).

Section snippets

Isolation of the Kv3 component from voltage-dependent K+ currents in PCs

In order to study Kv3 currents in cerebellar PCs, voltage-dependent/Ca2+ independent K+ currents activating at high threshold potentials were isolated from other active conductances. The properties of Kv3 currents were studied in whole-cell configuration in patch-clamp recordings from PCs in cerebellar slices from young mice (4–7 postnatal days). Na+ currents were blocked by TTX (0.5 μM), while Ca2+ currents and Ca2+ activated currents were eliminated by omitting this ion and adding EGTA in

Discussion

The firing properties of neurons strictly depend on the type and amount of ion channels present in the plasma membrane. FSNs, including PCs, require channels with special functional features, such as Kv3 voltage-dependent K+ channels (Rudy and McBain, 2001; Akemann and Knopfel, 2006). In agreement with previous studies (Sacco and Tempia, 2002, Martina et al., 2003, McKay and Turner, 2004), we show that PCs express very large voltage-dependent and Ca2+ independent K+ currents sensitive to

Slice preparation and patch-clamp recording

Voltage-clamp recording experiments were performed using the whole-cell configuration of the patch-clamp technique at room temperature (22–25°C) on CD-1 mice of either sex, 4–7 days old (P4–P7). Cerebellar slices were obtained following a previously described technique (Llinas and Sugimori, 1980, Edwards et al., 1989). Briefly, the animals were anesthetized with isoflurane (Isoflurane-Vet, Merial, Italy) and decapitated. The experiments were approved by the University Bioethical Committee of

Acknowledgments

This work was supported by grants from MIUR (PRIN 2001 and 2003), from the University of Torino and Regione Piemonte (Ricerca Sanitaria Finalizzata, bando 2004). The technical assistance of Mrs. Luisella Milano and Dr. Federica Premoselli is acknowledged.

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