Introduction
Cloning of ion channel and receptor cDNAs over the past fifteen years has demonstrated an extensive molecular complexity that underlies the functioning of the central nervous system. Of the classes of genes which have been identified, voltage-gated K+ channels constitute the most diverse ion channel type. Classical voltage-gated K+ channels are activated by membrane depolarization and serve to repolarize the membrane through conduction of an outward K+ current. Specifically, K+ channels typically either repolarize action potentials and synaptic potentials, or limit the rise of these potentials. In mammals over twenty different voltage-gated K+ channel genes have been cloned and have been found to be expressed in the central nervous system in varying cellular and subcellular distributions. The channels encoded by these genes vary in their electrophysiological properties (eg. voltage-dependence, conductance, inactivation), their ability to associate with modulatory proteins and their ability to be regulated by various intracellular factors. This functional heterogeneity suggests that K+ channels play a fundamental role in determining the precise nature of neuronal excitability. In fact, studies have demonstrated that neuronal firing patterns can be determined largely by the types of voltage-gated K+ channels expressed in each cell and their subcellular localization.
The Kv3 Family of Voltage-Gated K+ channels
Four major families of voltage-gated K+ channels have been identified in mammals: Kv1, Kv2, Kv3 and Kv4. Kv3 channels are particularly interesting in that their properties are suited towards enabling neurons to fire at high frequencies or transmit precise timing information. Unlike other voltage-gated K+ channels, Kv3 channels activate at relatively depolarized potentials (at or near 0mV), have relatively large single channel conductances, and open and close very quickly (<3 msec). These properties enable Kv3 channels to rapidly repolarize action potentials, without affecting spike initiation or spike height, and accelerate recovery of Na+ channels from inactivation. Accordingly, members of the Kv3 family are expressed prominently in neurons of the mammalian auditory system as well as other fast spiking neurons.
To date, four different Kv3 genes have been identified (Kv3.1, 3.2, 3.3 and 3.4) as well as alternatively spliced C-terminal variants of each. Most studies have focused on the Kv3.1 channel and have identified a role for this subunit in mediating fast repolarization of action potentials in somatic and axonal membranes of neurons. While it is likely that the other Kv3 subunits (Kv3.2-3.4) also play a role in modulation of action potential waveform, the functional distinction between each of the different Kv3 subunits has not clearly been determined. Differences in the properties of each subunit may underlie key differences between the type of inputs that a neuron can receive or the type of signals it can transmit.
The Apteronotid Electrosensory Lateral Line Lobe: a Model for Sensory Processing
The electrosensory system of the weakly electric fish Apteronotus leptorhynchus provides a useful model to study the roles of ion channels and receptors in neuronal processing. Arguably one of the best understood neural systems, the anatomy and physiology of the electrosensory system has been extensively characterized in vivo and in vitro and has been shown to display remarkable similarity to mammalian visual and auditory processing systems. In fact, the region of primary electrosensory processing, the electrosensory lateral line lobe (ELL), has been proposed to be a homologue of the mammalian dorsal cochlear nucleus. The function of the principal neurons of this lobe have been well characterized and their relatively large size facilitates not only electrophysiological analysis, but molecular analysis as well. An additional advantage of this system is provided by pyramidal cells of the ELL (Fig.1) which are responsible for extracting relevant sensory information and transmitting it to higher brain centers. These cells possess large basilar and apical dendrites which receive afferent and descending inputs respectively. The large size of these dendrites and the clear functional distinction between them provide an ideal model for studying the roles of ion channels and receptors in dendritic physiology.
Figure 1: Two types of pyramiial cells in the ELL
Dr. Ray Turner at the University of Calgary has identified a novel interaction between somatic and apical dendritic currents in ELL pyramidal cells which mediates an oscillatory burst pattern of discharge necessary for feature extraction (Lemon and Turner, 2000). In this mechanism, somatic and apical dendritic spike width are critical parameters in determining the precise nature of bursting. Specifically, consistent spike repolarization at the soma and dendritic spike broadening during repetitive discharge is necessary to trigger burst activity. Given the homologies between the mammalian auditory system and the apteronotid electrosensory system, it is reasonable to hypothesize that Kv3 channels control spike repolarization in ELL pyramidal cells and that differences in Kv3 subunit expression and/or localization underlie critical differences in spike repolarization in somatic and dendritic membranes.
Research
Kv3 Channels in the ELL: Regulation of Bursting
Five different Kv3 genes have been identified by screening an apteronotid brain cDNA library and by in situ hybridization it was determined that two of these genes are expressed in ELL pyramidal cells. These genes are homologues of the mammalian subtypes Kv3.3 and Kv3.1. A combination of immunohistochemical and electrophysiological analysis has been used to show that Kv3.3 is localized throughout the extent of pyramidal cell apical and basilar dendrites and is most likely responsible for action potential repolarization in these compartments (Fig.2). Furthermore, inactivation of Kv3.3 during repetitive discharge likely accounts for the spike broadening necessary for burst activity. The dendritic localization of Kv3.3 is entirely novel for members of the Kv3 family and provides a mechanism by which the threshold for bursting can be controlled in a sensory neuron (Rashid et al., 2001).
Further electrophysiological analysis will be carried out on both Kv3.3 and Kv3.1. The analysis will focus on two aspects as they pertain to burst activity in ELL pyramidal cells: (1) inactivation properties of each of these channels. (2) regulation of these channels by intracellular messengers.
Figure 2: Localization of AptKv3.3 Channels in the Hindbrain
RASHID ET AL. J.NEURSCI 2001
Targeting of Kv3 Channels to Different Subcellular Domains: Studies in mammals and fish
The differential localization of Kv3 channels in ELL pyramidal cells raises the intriguing question of how the subcellular distribution of these channels is controlled. Evidence has suggested that the intracellular carboxyl terminus of Kv3 channels controls this intracellular targeting process. Both fish and mammals express a variety of C-terminal splice variants of Kv3.3 and we hypothesize that each of these variants will be targeted differently in neurons. In order to test this, experiments are underway to express both mammalian and apteronotid Kv3 channels (Kv3.1, Kv3.3a, Kv3.3b, Kv3.3c) in cultured neurons. The long term goal is to identify the molecular determinants of subcellular targeting, with a particular focus on channels that are targeted to dendritic membranes. We have also identified a novel protein that binds to the C-terminal region of Kv3 and which may be responsible for control of intracellular targeting.