Modulation of hippocampal synaptic transmission by the kynurenine pathway member xanthurenic acid and other VGLUT inhibitors.

Xanthurenic acid (XA), an endogenous kynurenine, is a known vesicular glutamate transport (VGLUT) inhibitor and has also been proposed as an mGlu2/3 receptor agonist. Changes in these systems have been implicated in the pathophysiology of schizophrenia and other psychiatric disorders; however, little is known of how XA affects synaptic transmission. We therefore investigated the effects of XA on synaptic transmission at two hippocampal glutamatergic pathways and evaluated the ability of XA to bind to mGlu2/3 receptors. Field excitatory postsynaptic potentials (fEPSPs) were recorded from either the dentate gyrus (DG) or CA1 region of mouse hippocampal slices in vitro. Addition of XA to the bathing medium (1-10 mM) resulted in a dose-related reduction of fEPSP amplitudes (up to 52% reduction) in both hippocampal regions. In the DG, the VGLUT inhibitors Congo Red and Rose Bengal, and the mGlu2/3 agonist LY354740, also reduced fEPSPs (up to 80% reduction). The mGlu2/3 antagonist LY341495 reversed the LY354740 effect, but not the XA effect. LY354740, but not XA, also reduced DG paired-pulse depression. XA had no effect on specific binding of 1 nM [(3)H]LY341495 to membranes with human mGlu2 receptors. We conclude that XA can modulate synaptic transmission via a mechanism that may involve VGLUT inhibition rather than activation of mGlu2/3 receptors. This could be important in the pathophysiology of nervous system disorders including schizophrenia and might represent a target for developing novel pharmacological therapies.

Painful nerve injury decreases sarco-endoplasmic reticulum Ca²âº-ATPase activity in axotomized sensory neurons.

The sarco-endoplasmic reticulum Ca(2+)-ATPase (SERCA) is a critical pathway by which sensory neurons sequester cytosolic Ca(2+) and thereby maintain intracellular Ca(2+) homeostasis. We have previously demonstrated decreased intraluminal endoplasmic reticulum Ca(2+) concentration in traumatized sensory neurons. Here we examine SERCA function in dissociated sensory neurons using Fura-2 fluorometry. Blocking SERCA with thapsigargin (1 µM) increased resting [Ca(2+)](c) and prolonged recovery (τ) from transients induced by neuronal activation (elevated bath K(+)), demonstrating SERCA contributes to control of resting [Ca(2+)](c) and recovery from transient [Ca(2+)](c) elevation. To evaluate SERCA in isolation, plasma membrane Ca(2+) ATPase was blocked with pH 8.8 bath solution and mitochondrial buffering was avoided by keeping transients small (≤ 400 nM). Neurons axotomized by spinal nerve ligation (SNL) showed a slowed rate of transient recovery compared to control neurons, representing diminished SERCA function, whereas neighboring non-axotomized neurons from SNL animals were unaffected. Injury did not affect SERCA function in large neurons. Repeated depolarization prolonged transient recovery, showing that neuronal activation inhibits SERCA function. These findings suggest that injury-induced loss of SERCA function in small sensory neurons may contribute to the generation of pain following peripheral nerve injury.

Minimal alterations in T-type calcium channel gating markedly modify physiological firing dynamics.

T-type calcium channel isoforms expressed in heterologous systems demonstrate marked differences in the biophysical properties of the resulting calcium currents. Such heterogeneity in gating behaviour not only reflects structural differences but is also observed following the regulation of channel activity by a number of ligands. However, the physiological impact of these differences in gating parameters of the T channels has never been evaluated in situ where the unique interplay between T-type calcium and other intrinsic currents is conserved, and T channel activation can be triggered by synaptic stimulation. Here, using the dynamic clamp technique, artificial T conductances were re-incorporated in thalamic neurons devoid of endogenous T currents to dissect the physiological role of the T current gating diversity on neuronal excitability. We demonstrate that the specific kinetics of the T currents in thalamocortical and nucleus reticularis thalami neurons determine the characteristic firing patterns of these neurons. We show that subtle modifications in T channel gating that are at the limit of the resolution achieved in classical biophysical studies in heterologous expression systems have profound consequences for synaptically evoked firing dynamics in native neurons. Moreover, we demonstrate that the biophysical properties of the T current in the voltage region corresponding to the foot of the activation and inactivation curves drastically condition physiologically evoked burst firing with a high degree of synaptic input specificity.

Cloning and functional expression of two families of beta-subunits of the large conductance calcium-activated K+ channel.

We report here a characterization of two families of calcium-activated K(+) channel beta-subunits, beta2 and beta3, which are encoded by distinct genes that map to 3q26.2-27. A single beta2 family member and four alternatively spliced variants of beta3 were investigated. These subunits have predicted molecular masses of 27. 1-31.6 kDa, share approximately 30-44% amino acid identity with beta1, and exhibit distinct but overlapping expression patterns. Coexpression of the beta2 or beta3a-c subunits with a BK alpha-subunit altered the functional properties of the current expressed by the alpha-subunit alone. The beta2 subunit rapidly and completely inactivated the current and shifted the voltage dependence for activation to more polarized membrane potentials. In contrast, coexpression of the beta3a-c subunits resulted in only partial inactivation of the current, and the beta3b subunit conferred an apparent inward rectification. Furthermore, unlike the beta1 and beta2 subunits, none of the beta3 subunits increased channel sensitivity to calcium or voltage. The tissue-specific expression of these beta-subunits may allow for the assembly of a large number of distinct BK channels in vivo, contributing to the functional diversity of native BK currents.

Distinct domains of the voltage-gated K+ channel Kv beta 1.3 beta-subunit affect voltage-dependent gating.

The Kvbeta1.3 subunit confers a voltage-dependent, partial inactivation (time constant = 5.76 +/- 0.14 ms at +50 mV), an enhanced slow inactivation, a hyperpolarizing shift in the activation midpoint, and an increase in the deactivation time constant of the Kv1.5 delayed rectifier. Removal of the first 10 amino acids from Kvbeta1.3 eliminated the effects on fast and slow inactivation but not the voltage shift in activation. Addition of the first 87 amino acids of Kvbeta1.3 to the amino terminus of Kv1.5 reconstituted fast and slow inactivation without altering the midpoint of activation. Although an internal pore mutation that alters quinidine block (V512A) did not affect Kvbeta1.3-mediated inactivation, a mutation of the external mouth of the pore (R485Y) increased the extent of fast inactivation while preventing the enhancement of slow inactivation. These data suggest that 1) Kvbeta1.3-mediated effects involve at least two distinct domains of this beta-subunit, 2) inactivation involves open channel block that is allosterically linked to the external pore, and 3) the Kvbeta1.3-induced shift in the activation midpoint is functionally distinct from inactivation.

Molecular analysis of a binding site for quinidine in a human cardiac delayed rectifier K+ channel. Role of S6 in antiarrhythmic drug binding.

The antiarrhythmic agent quinidine blocks the human cardiac hKv1.5 channel expressed in mammalian cells at therapeutically relevant concentrations (EC50, 6.2 mumol/L). Mechanistic analysis has suggested that quinidine acts as a cationic open-channel blocker at a site in the internal mouth of the ionic pore and that binding is stabilized by hydrophobic interactions. We tested these hypotheses using site-directed mutagenesis of residues proposed to line the internal mouth of the channel or of nearby residues. Amino acid substitutions in the midsection of S6 (T505I, T505V, T505S, and V512A) reduced the dissociation rate for quinidine, increased the affinity (0.7, 1.5, 3.4, and 1.4 mumol/L, respectively), and preserved both the voltage-dependent open channel-block mechanism and the electrical binding distance (0.19 to 0.22). In contrast, smaller or nonsignificant effects were observed for: deletion of the intracellular C-terminal domain, charge neutralizations in the region immediately C-terminal to S6, elimination of aromatic residues in S6, and mutations at the putative internal turn of the P loop, at the external entrance of the pore, and at sites in the S4S5 linker. The approximately 10-fold increase in affinity with T505I and the reduction of the dissociation rate constant with the mutations that increased affinity are consistent with a hydrophobic stabilization of binding. Moreover, the T505 and V512 residues align on the same side of the putative alpha-helical S6 segment. Taken together, these results localize the hydrophobic binding site for this antiarrhythmic drug in the internal mouth of this human K+ channel and provide molecular support for the open channel-block model and the role of S6 in contributing to the inner pore.

Functional differences in Kv1.5 currents expressed in mammalian cell lines are due to the presence of endogenous Kv beta 2.1 subunits.

The voltage-sensitive currents observed following hKv1.5 alpha subunit expression in HEK 293 and mouse L-cells differ in the kinetics and voltage dependence of activation and slow inactivation. Molecular cloning, immunopurification, and Western blot analysis demonstrated that an endogenous L-cell Kv beta 2.1 subunit assembled with transfected hKv 1.5 protein. In contrast, both mRNA and protein analysis failed to detect a beta subunit in the HEK 293 cells, suggesting that functional differences observed between these two systems are due to endogenous L-cell Kv beta 2.1 expression. In the absence of Kv beta 2.1, midpoints for activation and inactivation of hKv1.5 in HEK 293 cells were -0.2 +/- 2.0 and -9.6 +/- 1.8 mV, respectively. In the presence of Kv beta 2.1 these values were -14.1 +/- 1.8 and -22.1 +/- 3.7 mV, respectively. The beta subunit also caused a 1.5-fold increase in the extent of slow inactivation at 50 mV, thus completely reconstituting the L-cell current phenotype in the HEK 293 cells. These results indicate that 1) the Kv beta 2.1 subunit can alter Kv1.5 alpha subunit function, 2) beta subunits are not required for alpha subunit expression, and 3) endogenous beta subunits are expressed in heterologous expression systems used to study K+ channel function.

A novel K+ channel beta-subunit (hKv beta 1.3) is produced via alternative mRNA splicing.

Voltage-gated K+ channels can form multimeric complexes with accessory beta-subunits. We report here a novel K+ channel beta-subunit cloned from human heart, hKv beta 1.3, that has 74-83% overall identity with previously cloned beta-subunits. Comparison of hKv beta 1.3 with the previously cloned hKv beta 3 and rKv beta 1 proteins indicates that the carboxyl-terminal 328 amino acids are identical, while unique variable length amino termini exist. Analysis of human beta-subunit cDNA and genomic nucleotide sequences confirm that these three beta-subunits are alternatively spliced from a common beta-subunit gene. Co-expression of hKv beta 1.3 in Xenopus oocytes with the delayed rectifier hKv1.5 indicated that hKv beta 1.3 has unique functional effects. This novel beta-subunit induced a time-dependent inactivation during membrane voltage steps to positive potentials, induced a 13-mV hyperpolarizing shift in the activation curve, and slowed deactivation (tau = 13 +/- 0.5 ms versus 35 +/- 1.7 ms at -40 mV). Most notably, hKv beta 1.3 converted the Kv1.5 outwardly rectifying current voltage relationship to one showing strong inward rectification. These data suggest that Kv channel current diversity may arise from association with alternatively spliced Kv beta-subunits. A simplified nomenclature for the K+ channel beta-subunit subfamilies is suggested.

Characterization of a voltage-gated K+ channel beta subunit expressed in human heart.

Voltage-gated K+ channels are important modulators of the cardiac action potential. However, the correlation of endogenous myocyte currents with K+ channels cloned from human heart is complicated by the possibility that heterotetrameric alpha-subunit combinations and function-altering beta subunits exist in native tissue. Therefore, a variety of subunit interactions may generate cardiac K+ channel diversity. We report here the cloning of a voltage-gated K+ channel beta subunit, hKv beta 3, from adult human left ventricle that shows 84% and 74% amino acid sequence identity with the previously cloned rat Kv beta 1 and Kv beta 2 subunits, respectively. Together these three Kv beta subunits share > 82% identity in the carboxyl-terminal 329 aa and show low identity in the amino-terminal 79 aa. RNA analysis indicated that hKv beta 3 message is 2-fold more abundant in human ventricle than in atrium and is expressed in both healthy and diseased human hearts. Coinjection of hKv beta 3 with a human cardiac delayed rectifier, hKv1.5, in Xenopus oocytes increased inactivation, induced an 18-mV hyperpolarizing shift in the activation curve, and slowed deactivation (tau = 8.0 msec vs. 35.4 msec at -50 mV). hKv beta 3 was localized to human chromosome 3 by using a human/rodent cell hybrid mapping panel. These data confirm the presence of functionally important K+ channel beta subunits in human heart and indicate that beta-subunit composition must be accounted for when comparing cloned channels with endogenous cardiac currents.

Deletion of highly conserved C-terminal sequences in the Kv1 K+ channel sub-family does not prevent expression of currents with wild-type characteristics.

The C-terminal regions of Kv1 K+ channels show little conservation between isoforms except for the last four C-terminal residues, (E/L)TDV, which are well conserved from Drosophila to man. Deletions of the 4, 16, and 57 C-terminal residues of the human Kv1.5 channel did not affect whole cell current amplitude, midpoint of activation, degree of inactivation, or activation kinetics following expression in mouse L-cells. Similar results were obtained with the rat Kv1.1 channel. Therefore, the conserved (E/L)TDV motif, and most of the C-terminal amino acids, are not required for Kv1 channel expression.