Faculty Bibliography

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 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

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

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

1. Subthreshold-operating transient (A-type) K(+) currents (I(SA)s) are important in regulating neuronal firing frequency and in the modulation of incoming signals in dendrites. It is now known that Kv4 proteins are the principal, or pore-forming, subunits of the channels mediating I(SA)s(.) In addition, accessory subunits of Kv4 channels have also been identified. These either have no effect or slow down the inactivation kinetics of Kv4 channels. However, in many neuronal populations the I(SA) is faster, not slower, than the current generated by channels containing only Kv4 proteins. 2. Evidence is presented for the presence in rat cerebellar mRNA of transcripts encoding a molecular factor, termed KAF, that accelerates the kinetics of Kv4 channels. Size-fractionation of cerebellar mRNA in sucrose gradients separated the high molecular weight mRNAs (4-7 kb) encoding KAF from the low molecular weight ones (1.5-3 kb) encoding factors that slow down the inactivation kinetics of Kv4 channels. The latter were identified as KChIPs using anti-KChIP antisense oligonucleotides. 3. Both anti-KChIP and anti-Kv4 antisense oligonucleotides failed to eliminate KAF's activity from the high molecular weight mRNA fraction, thus suggesting that KAF might be a novel subunit(s) that can contribute to generating native I(SA) channel diversity. 4. The time course of the currents expressed by KAF-modified Kv4 channels resembles more closely the time course of the native I(SA) in cerebellar granule cells

Frequenin, a Ca(2+)-binding protein, has previously been implicated in the regulation of neurotransmission, possibly by affecting ion channel function. Here, we provide direct evidence that frequenin is a potent and specific modulator of Kv4 channels, the principal molecular components of subthreshold activating A-type K(+) currents. Frequenin increases Kv4.2 current amplitudes (partly by enhancing surface expression of Kv4.2 proteins) and it slows the inactivation time course in a Ca(2+)-dependent manner. It also accelerates recovery from inactivation. Closely related Ca(2+)-binding proteins, such as neurocalcin and visinin-like protein (VILIP)-1 have no such effects. Specificity for Kv4 currents is suggested because frequenin does not modulate Kv1.4 or Kv3.4 currents. Frequenin has negligible effects on Kv4.1 current inactivation time course. By using chimeras made from Kv4.2 and Kv4.1 subunits, we determined that the differential effects of frequenin are mediated by means of the Kv4 N terminus. Immunohistochemical analysis demonstrates that frequenin and Kv4.2 channel proteins are coexpressed in similar neuronal populations and have overlapping subcellular localizations in brain. Coimmunoprecipitation experiments demonstrate that a physical interaction occurs between these two proteins in brain membranes. Together, our data provide strong support for the concept that frequenin may be an important Ca(2+)-sensitive regulatory component of native A-type K(+) currents

Analysis of the Kv3 subfamily of K(+) channel subunits has lead to the discovery of a new class of neuronal voltage-gated K(+) channels characterized by positively shifted voltage dependencies and very fast deactivation rates. These properties are adaptations that allow these channels to produce currents that can specifically enable fast repolarization of action potentials without compromising spike initiation or height. The short spike duration and the rapid deactivation of the Kv3 currents after spike repolarization maximize the quick recovery of resting conditions after an action potential. Several neurons in the mammalian CNS have incorporated into their repertoire of voltage-dependent conductances a relatively large number of Kv3 channels to enable repetitive firing at high frequencies - an ability that crucially depends on the special properties of Kv3 channels and their impact on excitability

The members of the three subfamilies (eag, erg, and elk) of the ether-a-go-go (EAG) family of potassium channel pore-forming subunits express currents that, like the M-current (I(M)), could have considerable influence on the subthreshold properties of neuronal membranes, and hence the control of excitability. A nonradioactive in situ hybridization (NR-ISH) study of the distribution of the transcripts encoding the eight known EAG family subunits in rat brain was performed to identify neuronal populations in which the physiological roles of EAG channels could be studied. These distributions were compared with those of the mRNAs encoding the components of the classical M-current (Kcnq2 and Kcnq3). NR-ISH was combined with immunohistochemistry to specific neuronal markers to help identify expressing neurons. The results show that each EAG subunit has a specific pattern of expression in rat brain. EAG and Kcnq transcripts are prominent in several types of excitatory neurons in the cortex and hippocampus; however, only one of these channel components (erg1) was consistently expressed in inhibitory interneurons in these areas. Some neuronal populations express more than one product of the same subfamily, suggesting that the subunits may form heteromeric channels in these neurons. Many neurons expressed multiple EAG family and Kcnq transcripts, such as CA1 pyramidal neurons, which contained Kcnq2, Kcnq3, eag1, erg1, erg3, elk2, and elk3. This indicates that the subthreshold current in many neurons may be complex, containing different components mediated by a number of channels with distinct properties and neuromodulatory responses

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