Faculty Bibliography

The somato-dendritic subthreshold-activating A-type K+ current (ISA) is a fast K+ current that activates transiently at membrane potentials that are below the threshold for Na+ spike generation, and plays important roles in neuronal excitability. Two key components of the channels underlying most of the ISA have been identified: Kv4 pore-forming subunits (mainly Kv4.2 and Kv4.3) and KChIP associated proteins. We have recently obtained evidence for the presence in rat brain mRNA of transcripts encoding a novel factor (termed KAF), which accelerates the kinetics of Kv4 channels, and could explain the fast kinetics of native ISA in many neurons.) Immunoaffinity purification was used to purify native Kv4 channel complexes solubilized from rat cerebellar membranes. Three specific and prominent bands are observed when the complex is dissociated and separated by SDS-PAGE. Immunoblotting and sequencing has shown that two of these bands correspond to KChIP and Kv4 polypeptides, respectively. A third polypeptide(s) of apprx115 kDa co-purifies specifically with the channel complex and shows an abundance comparable to that of Kv4 and KChIP polypeptides, suggesting it is an important component of native Kv4 channels. Purification and sequencing of this polypeptide will be carried out to explore whether it is responsible for KAF activity. In addition, we are using a functional cloning procedure, suppression cloning, to clone cDNAs encoding KAF

Channels containing Kv2 K+ channel subunits are thought to underlie most of the sustained K+ currents in CNS neurons. Several silent pore-forming subunits interact with Kv2 proteins to produce functional delayed rectifier K+ channels with modified electrophysiological or pharmacological properties. Here we present the cloning and characterization of two novel Kv2 interacting silent pore forming subunits, Kv10.1a and Kv10.1b, from human and rat. These alternatively-spliced variants arise by an alternative splice site in exon 1. The transcripts encode proteins with 436 and 425 amino acids (aa) in human and 433 and 422 aa in rat and mouse. The difference between the two spliced variants consist of a 9 aa insert between the 1st and 2nd transmembrane domains. Expression of Kv10 mRNAs in human was detected by Northern blot analysis in brain, kidney, lung, and pancreas. In human brain Kv10 mRNAs were expressed in cortex, hippocampus, caudate, putamen, amygdala and weakly in substantia nigra. but not in cerebellum, medulla, spinal cord, corpus callosum or thalamus. In rat, Kv10s were detected in brain and adrenal gland. In situ hybridization in rat brain sections demonstrates Kv10 mRNA expression in cortex, hippocampus, caudate-putamen and amygdala. No current was observed in Xenopus oocytes injected with Kv10 cRNAs alone. Co-injection of Kv2.1 and Kv10.1a or b decreased the amount of current expressed, as compared to oocytes injected with Kv2.1 cRNA alone, and reduces the inactivation rate without any appreciable change in other electrophysiological or pharmacological properties. The Kv10 gene maps to human chromosome 2p22.1

Voltage-gated potassium channels containing the K.v.3.2 subunit are expressed in specific neuronal populations such as thalamocortical neurons and fast spiking GABAergic interneurons of the neocortex and hippocampus. These K(+)-channels play a major role in the regulation of firing properties in these neurons. We investigated whether the K.v.3.2 subunit contributes to the generation of the sleep electroencephalogram (EEG). The EEG of a frontal and occipital derivation of K.v.3.2-deficient mice and littermate controls was recorded during a 24-h baseline, 6-h sleep deprivation (SD) and subsequent 18-h recovery to assess also the effects of the K.v.3.2 subunit deficiency under physiological sleep pressure. The K.v.3.2-deficient mice had lower EEG power density in the frequencies between 3.25 and 6 Hz in nonREM (NREM) sleep and 3.25-5 Hz in REM sleep. These differences were more prominent in the frontal derivation than in the occipital derivation. The waking EEG spectrum was not affected by the deletion. In both genotypes SD induced a prominent increase in slow-wave activity in NREM sleep (mean EEG power density between 0.75 and 4.0 Hz), and a concomitant decrease in sleep fragmentation. The effects of SD did not differ significantly between the genotypes. The results indicate that K.v.3.2 channels may be involved in the generation of EEG oscillations in the high delta and low theta range in sleep. They support the notion that GABA-mediated synchronization of cortical activity contributes to the electroencephalogram

In thalamic relay neurons (TRNs) a change in the resting potential (VM) underlies a transition between two modes of firing which is critical for changes in thalamo-cortical transmission associated with physiological and pathological states of brain activity. The change in VM is largely produced by the blockade of a K+ current (IKleak) by neurotransmitters from ascending projections. We seek to identify the channels underlying the subthreshold behavior of TRNs using whole-cell and perforated patch recordings in acute mouse slices combined with molecular analysis of channel products expressed by TRNs. A hyperpolarization-activated cationic current (Ih), a persistent Na+ current and at least two components of K+ current contribute to the resting properties of TRNs. Ih contributes significantly to the VM of TRNs (blockade of Ih produces a apprx10 mV hyperopolarization). At least two inward rectifiers (probably mediated by Kir2.2 and GIRK channels) contribute an inward rectifying K+ current. Blockade of this current by low Ba2+ concentrations depolarizes TRNs by ltoreq 5 mV. The main effect of muscarinic agonists on TRNs is to block a linear K+ 'leak' component, which depolarizes the cell by >10 mV. The effects of volatile anesthetics on K2P channels and on IKleak suggest that K2P channels underlie the thalamic IKleak. The data suggests that TASK-1 channels are not responsible for the thalamic IKleak as they are in other neurons and that different channels mediate the resting permeability of TRNs of distinct thalamic nuclei

The developmental expression of the voltage-gated potassium channel subunit, Kv3.2, and its localization within specific mouse hippocampal inhibitory interneuron populations were determined using immunoblotting and immunohistochemical techniques. Using immunoblotting techniques, the Kv3.2 protein was weakly detected at postnatal age day 7 (P7), and full expression was attained at P21 in tissue extracts from homogenized hippocampal preparations. A similar developmental profile was observed using immunohistochemical techniques in hippocampal tissue sections. Kv3.2 protein expression was clustered on the somata and proximal dendrites of presumed inhibitory interneurons. Using double immunofluorescence, Kv3.2 subunit expression was detected on subpopulations of GABAergic inhibitory interneurons. Kv3.2 was detected in approximately 100% of parvalbumin-positive interneurons, 86% of interneurons expressing nitric oxide synthase, and approximately 50% of somatostatin-immunoreactive cells. Kv3.2 expression was absent from both calbindin- and calretinin-containing interneurons. Using immunoprecipitation, we further demonstrate that Kv3.2 and its related subunit Kv3.1b are coexpressed within the same protein complexes in the hippocampus. These data demonstrate that potassium channel subunit Kv3.2 expression is developmentally regulated in a specific set of interneurons. The vast majority of these interneuron subpopulations possess a 'fast-spiking' phenotype, consistent with a role for currents through Kv3.2 containing channels in determining action potential kinetics in these cells

Voltage-gated K(+) channels containing pore-forming subunits of the Kv3 subfamily have specific roles in the fast repolarization of action potentials and enable neurons to fire repetitively at high frequencies. Each of the four known Kv3 genes encode multiple products by alternative splicing of 3' ends resulting in the expression of K(+) channel subunits differing only in their C-terminal sequence. The alternative splicing does not affect the electrophysiological properties of the channels, and its physiological role is unknown. It has been proposed that one of the functions of the alternative splicing of Kv3 genes is to produce subunit isoforms with differential subcellular membrane localizations in neurons and differential modulation by signaling pathways. We investigated the role of the alternative splicing of Kv3 subunits in subcellular localization by examining the brain distribution of the two alternatively spliced versions of the Kv3.1 gene (Kv3.1a and Kv3.1b) with antibodies specific for the alternative spliced C-termini. Kv3.1b proteins were prominently expressed in the somatic and proximal dendritic membrane of specific neuronal populations in the mouse brain. The axons of most of these neurons also expressed Kv3.1b protein. In contrast, Kv3.1a proteins were prominently expressed in the axons of some of the same neuronal populations, but there was little to no Kv3.1a protein expression in somatodendritic membrane. Exceptions to this pattern were seen in two neuronal populations with unusual targeting of axonal proteins, mitral cells of the olfactory bulb, and mesencephalic trigeminal neurons, which expressed Kv3.1a protein in dendritic and somatic membrane, respectively. The results support the hypothesis that the alternative spliced C-termini of Kv3 subunits regulate their subcellular targeting in neurons

We report the cloning of human KT3.2 and KT3.3 new members of the two-pore K(+) channel (KT) family. Based on amino acid sequence and phylogenetic analysis, KT3.2, KT3.3, and TASK-1 constitute a subfamily within the KT channel mammalian family. When Xenopus oocytes were injected with KT3.2 cRNA, the resting membrane potential was brought close to the potassium equilibrium potential. At low extracellular K(+) concentrations, two-electrode voltage-clamp recordings revealed the expression of predominantly outward currents. With high extracellular K(+) (98 mM), the current-voltage relationship exhibited weak outward rectification. Measurement of reversal potentials at different [K(+)](o) revealed a slope of 48 mV per 10-fold change in K(+) concentration as expected for a K(+)-selective channel. Unlike TASK-1, which is highly sensitive to changes of pH in the physiological range, KT3.2 currents were relatively insensitive to changes in intracellular or extracellular pH within this range due to a shift in the pH dependency of KT3.2 of 1 pH unit in the acidic direction. On the other hand, the phorbol ester phorbol 12-myristate 13-acetate (PMA), which does not affect TASK-1, produces strong inhibition of KT3.2 currents. Human KT3.2 mRNA expression was most prevalent in the cerebellum. In rat, KT3.2 is exclusively expressed in the brain, but it has a wide distribution within this organ. High levels of expression were found in the cerebellum, medulla, and thalamic nuclei. The hippocampus has a nonhomogeneous distribution, expressing at highest levels in the lateral posterior and inferior portions. Medium expression levels were found in neocortex. The KT3.2 gene is located at chromosome 8q24 1-3, and the KT3.3 gene maps to chromosome 20q13.1

1.Voltage-gated K+ channels containing Kv3 subunits play specific roles in the repolarization of action potentials. Kv3 channels are expressed in selective populations of CNS neurons and are thought to be important in facilitating sustained and/or repetitive high frequency firing. Regulation of the activity of Kv3 channels by neurotransmitters could have profound effects on the repetitive firing characteristics of those neurons. 2.Kv3 channels are found in several neuronal populations in the CNS that express nitric oxide synthases (NOSs). We therefore investigated whether Kv3 channels are modulated by the signalling gas nitric oxide (NO). 3.We found that Kv3.1 and Kv3.2 currents are potentially suppressed by D-NONOate and other NO donors. The effects of NO on these currents are mediated by the activation of guanylyl cyclase (GC), since they are prevented by Methylene Blue, an inhibitor of GC, and by ODQ, a specific inhibitor of the soluble form of GC. Moreover, application of 8-Br-cGMP, a permeant analogue of cGMP, also blocked Kv3.1 and Kv3.2 currents. 4.KT5283, a cGMP-dependent protein kinase (PKG) blocker, prevented the inhibition of Kv3.1 and Kv3.2 currents by D-NONOate and 8-Br-cGMP. This indicates that activation of PKG as a result of the increase in intracellular cGMP levels produced by D-NONOate or 8-Br-cGMP is necessary for channel block. 5.Although the effects of NO on Kv3.1 and Kv3.2 channels require PKG activity, two observations suggest that they are not mediated by phosphorylation of channel proteins: (a) the reagents affect both Kv3.2 and Kv3.1 channels, although only Kv3.2 proteins have a putative PKA-PKG phosphorylation site, and (b) mutation of the PKA-PKG phosphorylation site in Kv3.2 does not interfere with the effects of NO or cGMP. 6.The inhibitory effects of NO and cGMP on Kv3.1 and Kv3.2 currents appear to be mediated by the activation of serine-threonine phosphatase, since they are blocked by low doses of okadaic acid. Furthermore, direct intracellular application of the catalytic subunit of protein phosphatase 2A inhibited Kv3.2 currents, indicating that activity of PKG-induced phosphatase is necessary and sufficient to inhibit these channels. 7.The results suggest that basal phosphorylation of Kv3 channel proteins is required for proper channel function. Activation of phosphatases via NO or other signals that increase cGMP might be a potent mechanism to regulate Kv3 channel activity in neurons