Brian A. McCool

Heterologous expression of metabotropic glutamate receptors in adult rat sympathetic neurons: Subtype-specific coupling to ion channels

A novel heterologous expression system was used to examine the coupling of metabotropic glutamate receptors (mGluRs) to neuronal voltage-gated ion channels. Cytoplasmic injection of mGluR2 cRNA into adult rat sympathetic neurons resulted in the expression of receptors that negatively coupled to N-type Ca2+ channels through a pertussis toxin-sensitive pathway. Injection of mGluR1 alpha cRNA resulted in the expression of receptors that inhibited M-type K+ channels via a pertussis toxin-insensitive pathway. Coupling was restricted to specific transduction elements and effectors, since mGluR2 did not inhibit M channels and mGluR1 alpha had minimal effects on Ca2+ channels. These findings demonstrate that heterologously expressed, and thus unambiguously identified, mGluR subtypes modulate specific neuronal ion channels through discrete signal transduction pathways.

Ifenprodil inhibition of the 5-Hydroxytryptamine3 receptor

The anti-hypertensive drug ifenprodil is known to interact potently with the alpha 1-adrenergic receptor as well as a number of other second messenger-linked receptors. In addition to these properties, ifenprodil has been shown to prevent glutamate-mediated excitotoxicity via non-competitive antagonism of NMDA receptors [Legendre and Westbrook (1991) Molec. Pharmac. 40: 289-298; Shalaby et al. (1992) J. Pharmac. Exp. Ther. 260: 925-932]. With these things in mind, we have begun to examine the specificity of ifenprodil for various ligand-gated ion channels using electrophysiological methods. While ifenprodil effectively inhibits NMDA-mediated currents in cortical neurons in culture, it does not interact with either kainate or GABA receptors. Surprisingly, ifenprodil also acts as a relatively potent antagonist of the 5-hydroxytryptamine3 (5-HT3) receptor in the NG108-15 neuroblastoma x glioma cell line. Furthermore, several aspects of ifenprodil action on the 5-HT3 receptor resemble its interaction with the NMDA receptor. Namely, inhibition of 5-HT3-mediated cation currents is readily reversible, has relatively slow onset, is non-competitive, and is not voltage dependent. Since most of the known 5-HT3 antagonists are competitive, it is possible that ifenprodil may define a unique modulatory site(s) on this neurotransmitter receptor.

Effects of Chronic Ethanol Consumption on Rat GABAA and Strychnine-sensitive Glycine Receptors Expressed by Lateral/Basolateral Amygdala Neurons

It is well known that the anxiolytic potential of ethanol is maintained during chronic exposure. We have confirmed this using a light-dark box paradigm following chronic ethanol ingestion via a liquid diet. However, cessation from chronic ethanol exposure is known to cause severe withdrawal anxiety. These opposing effects on anxiety likely result from neuro-adaptations of neurotransmitter systems within the brain regions regulating anxiety. Recent work highlights the importance of amygdala ligand-gated chloride channels in the expression of anxiety. We have therefore examined the effects of chronic ethanol exposure on GABA(A) and strychnine-sensitive glycine receptors expressed by acutely isolated adult rat lateral/basolateral amygdala neurons. Chronic ethanol exposure increased the functional expression of GABA(A) receptors in acutely isolated basolateral amygdala neurons without altering strychnine-sensitive glycine receptors. Neither the acute ethanol nor benzodiazepine sensitivity of either receptor system was affected. We explored the likelihood that subunit composition might influence each receptors response to chronic ethanol. Importantly, when expressed in a mammalian heterologous system, GABA(A) receptors composed of unique alpha subunits were differentially sensitive to acute ethanol. Likewise, the presence of the beta subunit appeared to influence the acute ethanol sensitivity of glycine receptors containing the alpha(2) subunit. Our results suggest that the facilitation of GABA(A) receptors during chronic ethanol exposure may help explain the maintenance of ethanols anti-anxiety effects during chronic ethanol exposure. Furthermore, the subunit composition of GABA(A) and strychnine-sensitive glycine receptors may ultimately influence the response of each system to chronic ethanol exposure.

Subunit composition of strychnine-sensitive glycine receptors expressed by adult rat basolateral amygdala neurons: Glycine α2/β-subunits in adult amygdala neurons

In neonatal rats, strychnine‐sensitive glycine receptors are widely expressed in the spinal cord, brainstem and forebrain. During development, these ‘neonatal’ receptors are replaced by an adult isoform, the expression of which becomes restricted primarily to brain stem and spinal cord. Unlike most forebrain regions, functional strychnine‐sensitive glycine receptors appear to persist within adult rat amygdala. However, the subunit composition of glycine receptors expressed by amygdala neurons and its relationship to the adult isoform in brain stem/spinal cord has not been defined precisely. In this report, we have utilized RT‐PCR and single‐cell RT‐PCR to demonstrate that the ‘neonatal’α2‐subunit mRNA persists in adult rat amygdala neurons and is the predominant α‐subunit. We further demonstrate that native amygdala glycine receptors are relatively insensitive to the receptor antagonist picrotoxin, suggesting that α2‐ and β‐subunits may be present together in the same multisubunit complex. We further demonstrate that α2‐ and β‐subunits cloned from adult rat amygdala can form functional channels when expressed in a heterologous system. Together, these studies highlight both the unique characteristics of strychnine‐sensitive glycine receptors in the adult rat amygdala as well as the possibility that α2/β channels may represent the adult forebrain isoform of the strychnine‐sensitive glycine receptor.

Molecular Genetics of Transketolase in the Pathogenesis of the Wernicke-Korsakoff Syndrome

Thiamine deficiency, a frequent complication of alcoholism, plays an important role in the pathogenesis of the Wernicke-Korsakoff syndrome [WKS]. Previous work by a number of investigators has implicated the thiamine-utilizing enzyme transketolase [Tk] as being involved mechanistically in the genetic predisposition to WKS. In particular, Tk derived from fibroblasts has been found to have an increased Km app for its cofactor thiamine pyrophosphate [TPP] and/or exist in different isoelectric forms in alcoholic patients with WKS as compared with unaffected individuals. We have demonstrated that these differences are not due to different Tk alleles, tissue-specific Tk isozymes, or differential mRNA splicing. These findings point to other mechanisms to explain the biochemical Tk variants, such as differences in assembly of the functional holoenzyme or differences in modification of the primary translation product. Tk assembly or modification, once biochemically characterized, may be found to be subject to genetic variation.

Relative contributions of G protein, channel, and receptor to voltage-dependent inhibition of neuronal N-type and P/Q-type calcium channels in HEK 293 cell lines

The voltage-dependent modulation of neuronal voltage-gated calcium channels by heterotrimeric G protein-coupled receptors potentially provides a means for activity-dependent modulation of synaptic efficacy. Recent attention has focused upon the molecular mechanisms by which such G proteins influence the biophysical properties of calcium channels. We have used an HEK 293-based heterologous system which stably expresses human neuronal calcium channels to address the relative contributions of receptor, G protein, and channel to voltage-dependent inhibition. We find that the receptor and channel subtype only insignificantly influence the time it takes to re-establish modulation following voltage-dependent relief of inhibition. In contrast, the G protein subtype mediating inhibition appears to play a significant part in this process. These results emphasize the importance of G protein subtype in the modulation of neuronal calcium channels.