Johannes J. Krupp

N-Terminal Domains in the NR2 Subunit Control Desensitization of NMDA Receptors

Recent molecular studies of glutamate channels have provided increasingly detailed models of the agonist-binding site and of the channel pore. However, little information is available on the domains involved in channel gating. We examined the molecular determinants for the NR2-subunit specificity of glycine-independent desensitization of NMDA channels using NR2C/NR2A chimeric subunits expressed in HEK 293 cells. We show that glycine-independent desensitization is controlled by N-terminal domains of the NR2 subunit that flank the putative agonist-binding domain: a four amino acid (aa) segment immediately preceding the first transmembrane domain (M1) and a region containing the leucine/isoleucine/valine-binding protein-like (LIVBP-like) domain. Our results provide evidence for a functional role of the region containing the LIVBP-like domain in glutamate receptor channels. We suggest that the pre-M1 segment, presumably situated near the entrance to the pore, serves as a dynamic link between ligand binding and channel gating.

Calcineurin acts via the C-terminus of NR2A to modulate desensitization of NMDA receptors.

Phosphatase IIb (calcineurin, CaN) can reduce N-methyl-D-aspartate (NMDA) synaptic responses by enhancing glycine-independent desensitization. We examined the action of CaN on desensitization in recombinant NMDA receptors comprised of NMDA receptor 1 (NR1) and NR2A subunits. The C-terminus of NR2A, but not NR1, was critical for modulation of desensitization by CaN. Alanine-scanning mutagenesis indicated that serines 900 and 929 in NR2A altered desensitization, as did inhibition of tyrosine phosphatases. Our data suggest that dephosphorylation-dependent regulation of the C-terminus of NR2A increases desensitization of NMDA receptors, providing an additional mechanism for modulation of synaptic signals.

Voltage-gated sodium channels in neurological disorders.

Voltage-gated sodium channels play an essential biophysical role in many excitable cells such as neurons. They transmit electrical signals through action potential (AP) generation and propagation in the peripheral (PNS) and central nervous systems (CNS). Each sodium channel is formed by one alpha-subunit and one or more beta-subunits. There is growing evidence indicating that mutations, changes in expression, or inappropriate modulation of these channels can lead to electrical instability of the cell membrane and inappropriate spontaneous activity observed during pathological states. This review describes the biochemical, biophysical and pharmacological properties of neuronal voltage-gated sodium channels (VGSC) and their implication in several neurological disorders.

Capsaicin augments synaptic transmission in the rat medial preoptic nucleus.

The medial preoptic nucleus (MPN) is the major nucleus of the preoptic area (POA), a hypothalamic area involved in the regulation of body-temperature. Injection of capsaicin into this area causes hypothermia in vivo. Capsaicin also causes glutamate release from hypothalamic slices. However, no data are available on the effect of capsaicin on synaptic transmission within the MPN. Here, we have studied the effect of exogenously applied capsaicin on spontaneous synaptic activity in hypothalamic slices of the rat. Whole-cell patch-clamp recordings were made from visually identified neurons located in the MPN. In a subset of the studied neurons, capsaicin enhanced the frequency of spontaneous glutamatergic EPSCs. Remarkably, capsaicin also increased the frequency of GABAergic IPSCs, an effect that was sensitive to removal of extracellular calcium, but insensitive to tetrodotoxin. This suggests an action of capsaicin at presynaptic GABAergic terminals. In contrast to capsaicin, the TRPV4 agonist 4alpha-PDD did not affect GABAergic IPSCs. Our results show that capsaicin directly affects synaptic transmission in the MPN, likely through actions at presynaptic terminals as well as on projecting neurons. Our data add to the growing evidence that capsaicin receptors are not only expressed in primary afferent neurons, but also contribute to synaptic processing in some CNS regions.

Distribution of the voltage‐gated sodium channel Nav1.7 in the rat: Expression in the autonomic and endocrine systems

It is generally accepted that the voltage‐gated, tetrodotoxin‐sensitive sodium channel, NaV1.7, is selectively expressed in peripheral ganglia. However, global deletion in mice of NaV1.7 leads to death shortly after birth (Nassar et al. [ 2004 ] Proc. Natl. Acad. Sci. U. S. A. 101:12706–12711), suggesting that this ion channel might be more widely expressed. To understand better the potential physiological function of this ion channel, we examined NaV1.7 expression in the rat by in situ hybridization and immunohistochemistry. As expected, highest mRNA expression levels are found in peripheral ganglia, and the protein is expressed within these ganglion cells and on the projections of these neurons in the central nervous system. Importantly, we found that NaV1.7 is present in discrete rat brain regions, and the unique distribution pattern implies a central involvement in endocrine and autonomic systems as well as analgesia. In addition, NaV1.7 expression was detected in the pituitary and adrenal glands. These results indicate that NaV1.7 is not only involved in the processing of sensory information but also participates in the regulation of autonomic and endocrine systems; more specifically, it could be implicated in such vital functions as fluid homeostasis and cardiovascular control. J. Comp. Neurol. 504:680–689, 2007.

Biophysical Properties of Human Nav1.7 Splice Variants and Their Regulation by Protein Kinase A

The sodium channel Na(v)1.7 is preferentially expressed in nociceptive neurons and is believed to play a crucial role in pain sensation. Four alternative splice variants are expressed in human dorsal root ganglion neurons, two of which differ in exon 5 by two amino acids in the S3 segment of domain I (exons 5A and 5N). Two others differ in exon 11 by the presence (11L) or absence (11S) of an 11 amino acid sequence in the loop between domains I and II, an important region for PKA regulation. In the present study, we used the whole cell configuration of the patch-clamp technique to investigate the biophysical properties and 8-bromo-cyclic adenosine monophosphate (8Br-cAMP) modulation of these splice variants expressed in tsA201 cells in the presence of the beta(1)-subunit. The alternative splicing of Na(v)1.7 had no effect on most of the biophysical properties of this channel, including activation, inactivation, and recovery from inactivation. However, development of inactivation experiments revealed that the isoform containing exon 5A had slower kinetics of inactivation for negative potentials than that of the variant containing exon 5N. This difference was associated with higher ramp current amplitudes for isoforms containing exon 5A. Moreover, 8Br-cAMP-mediated phosphorylation induced a negative shift of the activation curve of variants containing exon 11S, whereas inactivation properties were unchanged. Isoforms with exon 11L were not modulated by 8Br-cAMP-induced phosphorylation. We conclude that alternative splicing of human Na(v)1.7 can specifically modulate the biophysical properties and cAMP-mediated regulation of this channel. Changing the proportions of these variants may thus influence neuronal excitability and pain sensation.

Reduced effect of propofol at human α1β2(N289M)γ2 and α2β3(N290M)γ2 mutant GABAA receptors

BACKGROUND Propofol is an i.v. anaesthetic commonly used during general anaesthesia and intensive care. It is known that the second transmembrane segment of the beta subunit in the GABA(A) receptor is an important target for the effects of propofol; however, this has not been investigated in human receptors. The aim of this study was to investigate the effect of propofol on human beta2 and beta3 GABA(A) subunits with point mutations corresponding to the N265M mutation in the rat beta2 and beta3 subunits. METHODS Asparagine-to-methionine replacement at amino acid position 289 and 290 (N289M and N290M) in the beta2 and beta3 GABA(A) receptor subunits, respectively, was accomplished by site-directed mutagenesis. Thereafter, subunits for three human wild-type (alpha1beta2gamma2, alpha2beta2gamma2, and alpha2beta3gamma2) and two mutant GABA(A) receptor channels [alpha1beta2(N289M)gamma2 and alpha2beta3(N290M)gamma2] were introduced into Xenopus oocytes and studied with two-electrode voltage clamp. RESULTS The mutant receptors left-shifted the GABA concentration-response curve. In comparison with the wild-type receptors, both the positive modulatory and the agonistic effects of propofol were strongly reduced in potency and amplitude at both mutated GABA(A) channels. CONCLUSIONS We demonstrate that N289M or N290M mutation in human GABA(A) beta2 and beta3 subunits increases sensitivity to GABA, which is in contrast to the corresponding rat N265M mutation. Furthermore, the N289M and N289M mutations reduce both the potentiation of GABA-induced currents and the direct effect of propofol on channels incorporating either of the mutated subunits, which confirms earlier findings concerning the corresponding mutation in rat receptors and knock-in mice.

Ion channel screening technology.

Ion channels are at present the third biggest target class in drug discovery. Primary research is continually uncovering potential new ion channel targets in indications such as cancer, diabetes and respiratory diseases, as well as the more established fields of pain, cardiovascular disease, and neurological disorders. Despite the physiological significance and therapeutic relevance in a wide variety of biological systems, ion channels still remain under exploited as drug targets. This is to a large extent resulting from the historical lack of screening technologies to provide the throughput and quality of data required to support medicinal chemistry. Although technical challenges still lie ahead, this historic bottleneck in ion channel drug discovery is now being overcome by novel technologies that can be integrated into lead generation stages of ion channel drug discovery to allow the development of novel therapeutic agents. This review describes the variety of technologies available for ion channel screening and discusses the opportunities these technologies provide. The challenges that remain to be addressed are highlighted.

Propofol and AZD3043 Inhibit Adult Muscle and Neuronal Nicotinic Acetylcholine Receptors Expressed in Xenopus Oocytes.

Propofol is a widely used general anaesthetic with muscle relaxant properties. Similarly as propofol, the new general anaesthetic AZD3043 targets the GABAA receptor for its anaesthetic effects, but the interaction with nicotinic acetylcholine receptors (nAChRs) has not been investigated. Notably, there is a gap of knowledge about the interaction between propofol and the nAChRs found in the adult neuromuscular junction. The objective was to evaluate whether propofol or AZD3043 interact with the α1β1δε, α3β2, or α7 nAChR subtypes that can be found in the neuromuscular junction and if there are any differences in affinity for those subtypes between propofol and AZD3043. Human nAChR subtypes α1β1δε, α3β2, and α7 were expressed into Xenopus oocytes and studied with an automated voltage-clamp. Propofol and AZD3043 inhibited ACh-induced currents in all of the nAChRs studied with inhibitory concentrations higher than those needed for general anaesthesia. AZD3043 was a more potent inhibitor at the adult muscle nAChR subtype compared to propofol. Propofol and AZD3043 inhibit nAChR subtypes that can be found in the adult NMJ in concentrations higher than needed for general anaesthesia. This finding needs to be evaluated in an in vitro nerve-muscle preparation and suggests one possible explanation for the muscle relaxant effect of propofol seen during higher doses.

Reduced efficacy of the intravenous anesthetic agent AZD3043 at GABAA receptors with β2 (N289M) and β3 (N290M) point-mutations

AZD3043 (previously named THRX-918661) is a novel short-acting intravenous anesthetic agent in clinical trials. Although AZD3043 is a positive modulator at the γ-aminobutyric acid (GABA)(A)-receptor, its potency and efficacy have not been characterized in detail. Nor is it known whether the point-mutations in the β-subunit of the GABA(A)-receptor that dramatically reduce the anesthetic effect of propofol (i.e. β2 (N289M) and β3 (N290M)), also influence the effect of AZD3043. This study investigated the in vitro pharmacology of AZD3043 at the most common human GABA(A) receptor subtypes. Subunits of four human wild-type (α1β2, α1β2γ2, α2β2γ2 and α2β3γ2) and two mutant (α1β2(N289M)γ2 and α2β3(N290M)γ2) GABA(A) receptor channels were introduced into Xenopus oocytes and studied with two-electrode voltage-clamp. AZD3043 potentiated and directly activated the α1β2γ2, α2β2γ2 and α2β3γ2 GABA(A) receptor subtypes. Moreover, both potency and efficacy of AZD3043 were reduced at the mutant α1β2(N289M)γ2 and α2β3(N290M)γ2 subtypes. AZD3043 increased the GABA response also in GABA(A) receptors lacking the γ2-subunit, i.e. α1β2. In conclusion, AZD3043 is a positive modulator and a direct agonist at human GABA(A) receptors and is not dependent on the γ2-subunit for its effect. Similar to propofol, the effect of AZD3043 is dramatically reduced by point-mutations in the β2(N289M) and β3(N290M) subunits, indicating similar molecular mechanisms of action for propofol and AZD3043 at the human GABA(A) receptor.