Dale Sawyer

Interactions of dextromethorphan with theN-methyl-d-aspartate receptor-channel complex: single channel recordings

The actions of dextromethorphan (DXM) on the 50 pS conductance state of the N-methyl-D-aspartate (NMDA) receptor-operated channel were studied using outside-out patches obtained from cultured rat hippocampal pyramidal neurons. DXM (5-50 microM) had no effect on the amplitudes of unitary currents but caused concentration-dependent reductions in channel mean open times and the frequency of channel openings. Channel open probability was reduced in a concentration-dependent manner by DXM and was one-half of the control value at a DXM concentration of 6 microM, with the patch potential held at -60 mV. An IC50 value of 4 microM was obtained for the reduction by DXM of NMDA-evoked rises in [Ca2+]i in cultured rat hippocampal pyramidal neurons loaded with Fura-2. The results were consistent with drug block of the open NMDA channel with an onward (blocking) rate constant of 7.7 x 10(6) M-1.s-1 (at -60 mV). The estimated unblocking rate constant was about 10 s-1, a value considerably higher compared to the off-rate constant found for dizocilpine block of the NMDA channel.

Cation and anion unitary ion channel currents in cultured bovine microglia

The use of excised patches has led to the identification and characterization of two channels not previously reported in microglia. A calcium-dependent K+ channel K(Ca) was activated in inside-out patches obtained from cultured bovine microglia and had a unitary conductance of 240 pS with symmetrical 140 mM K+ across patches. Mean open times of K(Ca) were exponentially dependent on patch potential and were increased with patch depolarization. Channel open probability (Popen) was increased with patch depolarization with either 5 mM or 140 mM internal K+ and was attributable to potential dependent enhancement of both opening frequency and mean open time. Whole cell currents showed the presence of a slowly activating K+ conductance, blocked by external TEA (at 2 mM) and likely the macroscopic correlate of the unitary K(Ca); half-maximal activation of the current occurred at +25 mV. An anion channel (unitary conductance of 325 pS with symmetrical Cl- across patches) was activated in inside-out patches with depolarizing and hyperpolarizing potential steps from 0 mV. Channel activation was not dependent on internal Ca2+. The anion currents inactivated during maintained potential steps with the time constant for inactivation faster with increased patch depolarization. The properties of the K(Ca) and anion channels in microglia membrane may have relevance to cell function in response to neuronal damage in the CNS.

Effects of divalent cations on the activation of a calcium-dependent potassium channel in hippocampal neurons

The activation of a calcium-dependent K+ channel K(Ca) in cultured hippocampal neurons has been studied after the addition, to the internal solution, of the divalent cations magnesium (Mg2+), strontium (Sr2+) or barium (Ba2+). With physiological K+ across inside-out patches and Ca2+ present at 0.2 mM in the bath solution, a 90-pS channel was activated with an open probability in excess of 75%. When the internal Ca2+ was reduced to levels near 5 μM, the channel-open probability was significantly diminished. However, if 0.2 mM concentrations of either Mg2+ or Sr2+ were added to the internal solution, the open probability was increased to a value close to original level. In the presence of internal Ca2+ at 0.1 μM, the K(Ca) channel was not active and was not activated with the addition of 0.2 mM Mg2+ or Sr2+ to the internal solution. Thus, Mg2+ or Sr2+ were not able to active K(Ca) in the absence of Ca2+; however, both of these divalent cations could potentiate the Ca2+-induced activation of K(Ca) if internal Ca2+ was near 5 μM. The results indicate that Mg2+ could have a role as an internal modulator of K(Ca) in the Ca2+-dependent regulation of excitability in nerve membrane. Replacement of Ca2+ with Ba2+, or addition of Ba2+ to Ca-containing solutions, caused significant decreases in the channel-open probability for K(Ca). The action of Ba2+ was primarily mediated by a decrease in the frequency of channel opening. At a concentration of 5 μM, Ba2+ diminished the channel-open probability by one-half.

Tedisamil blocks a calcium-dependent potassium channel in cultured motoneurons

A calcium-dependent potassium channel K(Ca) has been isolated in mouse motoneurons. With physiological concentrations of potassium across inside-out patches, a 100 pS K(Ca) channel was activated when the bath solution of Ca2+ was in excess of 1 microM. Introduction of the drug tedisamil, a blocker of repolarizing potassium channels in cardiac cells, at concentrations in the range 0.2-10 microM, caused a dose-dependent decrease in the mean open times for K(Ca). The drug action was consistent with open channel block of K(Ca) with an onward (blocking) rate constant of 6 x 10(7) M-1 s-1. Tedisamil, at concentrations of 1 microM and 5 microM, also blocked the K(Ca) channel when applied to outside-out patches with a similar potency as found with internal application. A large conductance K(Ca) channel in hippocampal neurons is also blocked by a number of putative Class III antiarrhythmic drugs, including tedisamil; thus, these agents may have utility in the characterization of the properties of K(Ca) channels in various cells.

The actions of L-687,384, a σ receptor ligand, on NMDA-induced currents in cultured rat hippocampal pyramidal neurons

The actions of the sigma receptor ligand L-687,384 were studied on N-methyl-D-aspartate (NMDA)-induced currents recorded from outside-out patches obtained from cultured rat hippocampal pyramidal neurons and on NMDA-evoked rises in [Ca2+]i in the same preparation. L-687,384 did not change the magnitudes of unitary NMDA currents or frequency of channel-openings but diminished channel-open probability by decreasing mean open time. This action was consistent with a voltage-dependent open-channel block of the NMDA channel by L-687,384 with a blocking rate constant of 5.9 x 10(6) M-1 s-1 at -80 mV. L-687,384 also reduced NMDA-evoked rises in [Ca2+]i in Fura-2-loaded neurons with an apparent IC50 value of 49 +/- 8 microM. The results demonstrate that L-687,384 acts as an antagonist at the NMDA receptor-channel complex.

Effects of volatile anaesthetics on a high conductance calcium dependent potassium channel in cultured hippocampal neurons.

(1) The effects of isoflurane and halothane (at nominal concentrations of 4%) were studied on a high conductance Ca2+-dependent K+ channel using inside-out patches from cultured rat hippocampal neurons. (2) With physiological-like concentrations of K+ across patches (140 mM internal and 5 mM external), unitary activity from a 4 pA channel (conductance near 100 pS) was evident in excised patches. (3) Isoflurane or halothane had no effects on unitary properties of K (Ca) including channel amplitudes and open and closed times. The channel open probability was also unchanged in the presence of the volatile agent. The results suggest that macroscopic high conductance K (Ca) currents in hippocampal neurons would be relatively insensitive to high concentrations of inhalational anaesthetics.

Dependence of single channel properties of the N-methyl-d-aspartate ion channel on stereoisomer agonists

The cell-attached patch-clamp configuration has been used to determine the single channel properties of the N-methyl-D-aspartate (NMDA) ion channel with activation of the NMDA receptor by stereoisomer agonists. All of the agonists studied, including the L and d forms of N-methyl-aspartate and the L and d forms of homocysteate, activated a 42-pS conductance channel in cultured hippocampal neurons. For all agonists, the mean open times of the channel were diminished with increased patch hyperpolarization and exhibited an exponential dependence on potential over the range -40 mV to -120 mV. The mean open times, for patch potentials close to resting potential, and the mean frequencies of channel openings, at all patch potentials, were significantly different between each member of the stereoisomer pairs. For both L-homocysteate and NMLA, a fourfold increase in the patch pipette concentration caused an approximate quadrupling in the frequency of unitary events, with no significant change in mean open time. The open channel probability was used as a measure of agonist potency, and, at a concentration of 30 μM, NMDA and L-homocysteate were significantly more potent (P open in excess of 1.5%) than the corresponding stereoisomer compounds NMLA and D-homocysteate (P open near 0.3%). The relative potencies of the stereoisomer pairs were in reasonable agreement with the potency ratios measured in binding studies.

(−)-β-Cyclazocine is an antagonist of NMDA receptor-mediated responses and a potent neuroprotectant in rat cortical neurons

Microspectrofluorimetry and excitotoxicity experiments were performed to study the NMDA receptor-blocking and neuroprotective actions of (-)- and (+)-beta-cyclazocine in cultured rat cortical neurons. (-)-beta-Cyclazocine potently antagonized NMDA-induced[Ca2+]i increases (IC50 = 220 nM) in neurons loaded with the Ca2+ fluorophore, fura-2. (-)-beta-Cyclazocine was specific for NMDA receptor-mediated responses versus those mediated through non-NMDA receptors or voltage-activated Ca2+ channels. The agent was active against NMDA-induced neurotoxicity, even at 1 microM. In all experiments, the (+)-enantiomer was found to be considerably less potent than the (-)-enantiomer. These results indicate that (-)-beta-cyclazocine is a specific NMDA receptor antagonist with potent neuroprotective properties in rat cortical neurons.

The effects of (-)- and (+)-β-cyclazocine on NMDA-evoked responses and NMDA-mediated cell damage in cultured rat hippocampal neurons

Abstract Microspectrofluorimetric measurements of excitatory amino acid-evoked rises in intracellular free calcium concentration ([Ca 2+ ] i ), electrophysiological measurements of currents through single NMDA receptor-operated ion channels and estimates of cellular viability following NMDA challenge were employed to examine the interactions of (−)- and (+)-β-cyclazocine with the NMDA receptor-channel complex in cultured rat hippocampal neurons. Rises in [Ca 2+ ] i evoked by NMDA, but not those evoked by kainate, AMPA or 50 mM K + , were reduced by (−)-β-cyclazocine in a concentration- and use-dependent manner with an estimated IC 50 value of 272 nM. In outside-out pathces, (−)-β-cyclazocine did not change the magnitudes of unitary NMDA-evoked currents but diminished both the frequency of channel openings and their mean open time. The IC 50 for (−)-β-cyclazocine against NMDA channel open state probability was estimated at 84 nM. The actions of (−)-β-cyclazocine were consistent with a voltage-dependent open channel block of the NMDA channel with a blocking rate constant of 7.03·10 7 M −1 ·s −1 at −40 mV. Neurons exposed to a high concentration of NMDA in vitro were protected from death by 1 and 10 μM (−)-β-cyclazocine. In all of the above assays, (+)-β-cyclazocine was considerably less potent an NMDA antagonist and neuroprotective agent than (−)-β-cyclazocine; the IC 50 for (+)-β-cyclazocine against channel open state probability was estimated at 14 μM. The results demonstrate that (−)-β-cyclazocine is a potent and selective inhibitor of NMDA-evoked responses in cultured rat hippocampal neurons and an effective neuroprotective agent in vitro.