Faculty Bibliography

G-protein-gated inward rectifier potassium (GIRK) channels are coupled to numerous neurotransmitter receptors in the brain and can play important roles in modulating neuronal function, depending on their localization in a given neuron. Site-directed antibodies to the extreme C terminus of GIRK1 (or KGA1), a recently cloned component of GIRK channels, have been used to determine the relative expression levels and distribution of the protein in different regions of the rat brain by immunoblot and immunohistochemical techniques. We report that the GIRK1 protein is expressed prominently in the olfactory bulb, hippocampus, dentate gyrus, neocortex, thalamus, cerebellar cortex, and several brain stem nuclei. In addition to the expected localization in somas and dendrites, where GIRK channels may mediate postsynaptic inhibition, GIRK1 proteins were also found in axons and their terminal fields, suggesting that GIRK channels can also modulate presynaptic events. Furthermore, the distribution of the protein to either somatodendritic or axonal-terminal regions of neurons varied in different brain regions, which would imply distinct functions of these channels in different neuronal populations. Particularly prominent staining of the cortical barrels of layer IV of the neocortex, and the absence of this staining with unilateral kainate lesions of the thalamus, suggest that the GIRK1 protein is expressed in thalamocortical nerve terminals in which GIRK channels may mediate the actions of mu opiate receptors

The expression of the voltage-gated K(+)-channel subunit Kv3.1b in the developing hippocampus was determined by immunoblot and immunohistochemical techniques. Kv3.1b protein was detected first at postnatal day (P) 8. The Kv3.1b-immunopositive cell number per tissue section reached a maximum at P14 and was maintained through P40. In contrast, the Kv3.1b protein content of isolated membrane vesicles in immunoblots progressively increased through P40, suggesting an increase in Kv3.1b content per cell throughout this time period. Kv3.1b protein was expressed selectively in the somata, proximal dendrites, and axons of cells lying within or near the pyramidal cell layer, consistent with their being GABAergic inhibitory interneurons. Kv3.1b was present in approximately 80% of parvalbumin-positive interneurons. The developmental onset of Kv3.1b and parvalbumin immunoreactivity was identical. In contrast, Kv3.1b was mostly absent from the subset of somatostatin-positive inhibitory interneurons. Electrophysiological recordings were made from stratum pyramidale interneurons in which morphology and Kv3.1b-positive immunoreactivity were confirmed post hoc. Outward currents had voltage-dependent and biophysical properties resembling those of channels formed by Kv3.1b. The current blocked by low concentrations of 4-aminopyridine (4-AP) showed marked inactivation, suggesting that Kv3.1b may coassemble with other members of the Kv3 subfamily. In current-clamp recordings, concentrations of 4-AP that blocked the current through Kv3.1b channels allowed us tentatively to assign a role to Kv3.1b-containing channels in action-potential repolarization. These data demonstrate that Kv3.1b is regulated developmentally in a specific subpopulation of hippocampal interneurons and that channels containing this subunit may be a major determinant in imparting 'fast-spiking' characteristics to these and other cells throughout the central nervous system containing the Kv3.1b subunit

Endothelin 1 (ET1) desensitizes endothelin A receptor for 90-110 min while neurokinin A (NKA) desensitizes neurokinin A receptor for 25-35 min in Xenopus laevis oocytes. In the present study, endothelin A receptor and neurokinin A receptor were coexpressed in Xenopus laevis oocytes in an effort to characterize heterologous desensitization of the receptors that activate phospholipase C-beta. ET1 desensitizes both the endothelin A receptor and the neurokinin A receptor for 90-110 min, whereas stimulation with NKA desensitizes the same two receptors for only 25-35 min. Homologous and heterologous desensitization experiments were also carried out with endothelin 3 (ET3), a ligand that exhibits lower affinity to the endothelin A receptor and a quicker dissociation rate than ET1. ET3 was unable to desensitize endothelin A receptor and the neurokinin A receptor; this is in contrast to ET1 that desensitizes both receptors. These results suggests that the receptors that undergo homologous desensitization are able to heterologously desensitize other receptors that activate PLC-beta. Furthermore, the agonist-specific dissociation constant dictates the extent of desensitization and time of recovery of the receptor-mediated response

The protein tyrosine kinase PYK2, which is highly expressed in the central nervous system, is rapidly phosphorylated on tyrosine residues in response to various stimuli that elevate the intracellular calcium concentration, as well as by protein kinase C activation. Activation of PYK2 leads to modulation of ion channel function and activation of the MAP kinase signalling pathway. PYK2 activation may provide a mechanism for a variety of short- and long-term calcium-dependent signalling events in the nervous system

Mammalian voltage-activated Shaker K+ channels associate with at least three cytoplasmic proteins: Kv beta 1, Kv beta 2 and Kv beta 3. These beta subunits contain variable N-termini, which can modulate the inactivation of Shaker alpha subunits, but are homologous throughout an aldo-keto reductase core. Human and ferret beta 3 proteins are identical with rat beta 1 throughout the core while beta 2 proteins are not; beta 2 also contains a shorter N-terminus and has no reported physiological role. We report that human beta 1 and beta 3 are derived from the same gene and that beta 2 modulates the inactivation properties of Kv1.4 alpha subunits

The finding that some K+ channel mRNAs are restricted to certain populations of neurons in the CNS suggests that there are K+ channels tailored to certain neuronal circuits. One such example are the transcripts from the KV3.2 gene, the majority of which are expressed in thalamic relay neurons. To gain insights into the specific roles of KV3.2 subunits, site specific antibodies were raised to determine their localization in thalamic relay neurons. Immunohistochemical and focal lesioning studies demonstrate that KV3.2 proteins are localized to the terminal fields of thalamocortical projections. It is also shown that KV3.2 channels expressed in vitro are strongly inhibited through phosphorylation by cAMP-dependent protein kinase (PKA). Channels containing KV3.1 subunits, which otherwise exhibit nearly identical electrophysiological properties in heterologous expression systems but have a different and less restricted pattern of expression in the CNS, are not affected by PKA. Therefore, this modulation might be associated with the specific roles of KV3.2 subunits. Furthermore, we demonstrate that KV3.2 proteins can be phosphorylated in situ by intrinsic PKA. KV3.2 subunits display properties and have a localization consistent with a role in the regulation of the efficacy of the thalamocortical synapse, and could thereby participate in the neurotransmitter-mediated control of functional states of the thalamocortical system associated with global states of awareness