Kv3 channels contribute to the delayed rectifier current in small cultured mouse dorsal root ganglion neurons.

Delayed rectifier voltage-gated K(+) (K(V)) channels are important determinants of neuronal excitability. However, the large number of K(V) subunits poses a major challenge to establish the molecular composition of the native neuronal K(+) currents. A large part (∼60%) of the delayed rectifier current (I(K)) in small mouse dorsal root ganglion (DRG) neurons has been shown to be carried by both homotetrameric K(V)2.1 and heterotetrameric channels of K(V)2 subunits with silent K(V) subunits (K(V)S), while a contribution of K(V)1 channels has also been demonstrated. Because K(V)3 subunits also generate delayed rectifier currents, we investigated the contribution of K(V)3 subunits to I(K) in small mouse DRG neurons. After stromatoxin (ScTx) pretreatment to block the K(V)2-containing component, application of 1 mM TEA caused significant additional block, indicating that the ScTx-insensitive part of I(K) could include K(V)1, K(V)3, and/or M-current channels (KCNQ2/3). Combining ScTx and dendrotoxin confirmed a relevant contribution of K(V)2 and K(V)2/K(V)S, and K(V)1 subunits to I(K) in small mouse DRG neurons. After application of these toxins, a significant TEA-sensitive current (∼19% of total I(K)) remained with biophysical properties that corresponded to those of K(V)3 currents obtained in expression systems. Using RT-PCR, we detected K(V)3.1-3 mRNA in DRG neurons. Furthermore, Western blot and immunocytochemistry using K(V)3.1-specific antibodies confirmed the presence of K(V)3.1 in cultured DRG neurons. These biophysical, pharmacological, and molecular results demonstrate a relevant contribution (∼19%) of K(V)3-containing channels to I(K) in small mouse DRG neurons, supporting a substantial role for K(V)3 subunits in these neurons.

Decrease in uric acid in acute ischemic stroke correlates with stroke severity, evolution and outcome.

BACKGROUND: Although uric acid (UA) is one of the most important antioxidants in plasma and appears to be neuroprotective in animal models, results from human studies are controversial. In this study, we investigated the kinetics of serum UA concentrations in the acute, subacute and chronic phase of ischemic stroke and its relation with initial stroke severity, stroke evolution in the subacute phase and long-term stroke outcome. METHODS: Serum concentrations of UA were measured in 199 stroke patients at admission (median, 2.8 h after stroke onset), at 24 h, 72 h, day 7, month 1 and month 3 after onset of stroke. We evaluated the relationship between changes in UA concentrations and (a) stroke severity [patients with transient ischemic attack (TIA) vs. stroke patients, National Institutes of Health Stroke Scale (NIHSS) score at admission], (b) stroke evolution (stroke progression, infarct volume at 72 h), and (c) stroke outcome [modified Rankin scale (mRS) score at month 3, mortality]. RESULTS: UA concentrations decreased significantly during the first 7 days after stroke onset before returning to baseline (p < 0.001). Mean plasma UA concentrations decreased from 336.66 +/- 113.01 micromol/L at admission to 300.37 +/- 110.04 micromol/L at day 7 (p < 0.001) in patients with stroke, but did not change significantly in patients with TIA. Changes in UA concentrations from admission to day 7 (DeltaUA(day 7)) correlated with the NIHSS score (rho = 0.32; p < 0.001), stroke progression (rho = 0.29; p = 0.001), infarct volume (rho = 0.37; p < 0.001), mRS score (rho = 0.28; p = 0.001) and mortality (p = 0.010). CONCLUSIONS: Decreases in UA during the first week after onset of stroke correlates with more severe stroke, unfavorable stroke evolution, and poor long-term stroke outcome.

The contribution of Kv2.2-mediated currents decreases during the postnatal development of mouse dorsal root ganglion neurons.

Delayed rectifier voltage-gated K(+)(Kv) channels play an important role in the regulation of the electrophysiological properties of neurons. In mouse dorsal root ganglion (DRG) neurons, a large fraction of the delayed rectifier current is carried by both homotetrameric Kv2 channels and heterotetrameric channels consisting of Kv2 and silent Kv (KvS) subunits (i.e., Kv5-Kv6 and Kv8-Kv9). However, little is known about the contribution of Kv2-mediated currents during the postnatal development ofDRGneurons. Here, we report that the Stromatoxin-1 (ScTx)-sensitive fraction of the total outward K(+)current (IK) from mouseDRGneurons gradually decreased (~13%,P < 0.05) during the first month of postnatal development. Because ScTx inhibits both Kv2.1- and Kv2.2-mediated currents, this gradual decrease may reflect a decrease in currents containing either subunit. However, the fraction of Kv2.1 antibody-sensitive current that only reflects the Kv2.1-mediated currents remained constant during that same period. These results suggested that the fractional contribution of Kv2.2-mediated currents relative toIKdecreased with postnatal age. SemiquantitativeRT-PCRanalysis indicated that this decrease can be attributed to developmental changes in Kv2.2 expression as themRNAlevels of the Kv2.2 subunit decreased gradually between 1 and 4 weeks of age. In addition, we observed age-dependent fluctuations in themRNAlevels of the Kv6.3, Kv8.1, Kv9.1, and Kv9.3 subunits. These results support an important role of both Kv2 and KvS subunits in the postnatal maturation ofDRGneurons.

Kv2.1 and silent Kv subunits underlie the delayed rectifier K+ current in cultured small mouse DRG neurons.

Silent voltage-gated K(+) (K(v)) subunits interact with K(v)2 subunits and primarily modulate the voltage dependence of inactivation of these heterotetrameric channels. Both K(v)2 and silent K(v) subunits are expressed in the mammalian nervous system, but little is known about their expression and function in sensory neurons. This study reports the presence of K(v)2.1, K(v)2.2, and silent subunit K(v)6.1, K(v)8.1, K(v)9.1, K(v)9.2, and K(v)9.3 mRNA in mouse dorsal root ganglia (DRG). Immunocytochemistry confirmed the protein expression of K(v)2.x and K(v)9.x subunits in cultured small DRG neurons. To investigate if K(v)2 and silent K(v) subunits are underlying the delayed rectifier K(+) current (I(K)) in these neurons, K(v)2-mediated currents were isolated by the extracellular application of rStromatoxin-1 (ScTx) or by the intracellular application of K(v)2 antibodies. Both ScTx- and anti-K(v)2.1-sensitive currents displayed two components in their voltage dependence of inactivation. Together, both components accounted for approximately two-thirds of I(K). A comparison with results obtained in heterologous expression systems suggests that one component reflects homotetrameric K(v)2.1 channels, whereas the other component represents heterotetrameric K(v)2.1/silent K(v) channels. These observations support a physiological role for silent K(v) subunits in small DRG neurons.

Involvement of voltage- and ligand-gated Ca2+ channels in the neuroexcitatory and synergistic effects of putative uremic neurotoxins.

BACKGROUND: Renal failure has been viewed as a state of cellular calcium toxicity due to the retention of small fast-acting molecules. We have tested this hypothesis and identified potentially neuroexcitatory compounds among a number of putative uremic neurotoxins by examining the acute in vitro effects of these compounds on cultured central neurons. The in vitro neuroexcitatory and synergistic effects of guanidinosuccinate and spermine were also examined in vivo. METHODS: The acute effects of 17 candidate uremic neurotoxins on murine spinal cord neurons in primary dissociated cell culture were investigated using the tight-seal whole-cell recording technique. The compounds studied comprised low-molecular-weight solutes like urea, indoles, guanidino compounds, polyamines, purines and phenoles, homocysteine, orotate, and myoinositol. Currents evoked by these compounds were further examined using various ligand- and voltage-gated ion channel blockers. The acute in vivo effects of guanidinosuccinate and spermine were behaviorally assessed following their injection in mice. RESULTS: It was shown that 3-indoxyl sulfate, guanidinosuccinate, spermine, and phenol evoked significant whole-cell currents. Inward whole-cell current evoked by 3-indoxyl sulfate was not blocked by any of the applied ligand- or voltage-gated ion channel blockers, and the compound appeared to influence miscellaneous membrane ionic conductances, probably involving voltage-gated Ca2+ channels as well. Phenol-evoked outward whole-cell currents were at least partly due to the activation of voltage-gated K+ channels, but may also involve a variety of other ionic conductances. On the other hand, inward whole-cell currents evoked by guanidinosuccinate and spermine were shown to be due to specific interaction with voltage- and ligand-gated Ca2+ channels. Guanidinosuccinate-evoked current was caused by activation of N-methyl-d-aspartate (NMDA) receptor-associated ion channels. Low (micromol/L) concentrations of spermine potentiated guanidinosuccinate-evoked current through the action of spermine on the polyamine binding site of the NMDA receptor complex, whereas current evoked by high (mmol/L) concentrations of spermine alone involved direct activation of voltage-gated Ca2+ channels. Finally, intracerebroventricular administration of 0.25 micromol/L spermine potentiated clonic convulsions induced by guanidinosuccinate. These neuroexcitatory and synergistic effects of guanidinosuccinate and spermine could take place at pathophysiologic concentrations. CONCLUSION: The observed in vitro and in vivo effects of uremic retention solutes suggest that the identified compounds could play a significant role in uremic pathophysiology. Some of the compounds tested displayed in vitro and in vivo neuroexcitatory effects that were mediated by ligand- and voltage-gated Ca2+ channels. The findings suggest a mechanism for the involvement of calcium toxicity in the central nervous system complications in renal failure with particular reference to guanidinosuccinate and spermine.