Ca2+ channel antagonist U-92032 inhibits both T-type Ca2+ channels and Na+ channels in hippocampal CA1 pyramidal neurons.

The effects of 7-[[4-[bis(4-fluorophenyl)-methyl]-1-piperazinyl]methyl] -2-[(2-hydroxyethyl)amino] 4-(1-methylethyl)-2,4,6-cycloheptatrien-1-one (U-92032), a newly described Ca2+ channel blocker, on voltage-gated ionic currents were measured. Whole cell voltage-clamp records were obtained from acutely isolated CA1 hippocampal pyramidal neurons from 7- to 14-day-old rats. Dimethyl sulfoxide, at either 0.01% or 0.1%, partially inhibited T-type Ca2+ currents (approximately 20% inhibition) but not high-voltage-activated (HVA) Ca2+ currents. Ethanol (0.2%) did not affect Ca2+ currents. U-92032 selectively inhibited T-type Ca2+ currents (median inhibiting concentration approximately 500 nM). HVA Ca2+ currents were less sensitive, with approximately 75% of the current resistant at 10 microM. Inhibition of Ca2+ currents was reversible. U-92032 inhibited Na+ currents at concentrations similar to those required for T-type currents (> 33% block at 1 microM). Block of Na+ currents took several minutes to develop and was irreversible. Voltage-gated K+ currents were insensitive to U-92032 (1 or 10 microM). These results indicate that U-92032 inhibits both T-type Ca2+ channels and Na+ channels, constraining its utility in certain studies. Among Ca2+ channels, however, U-92032 should prove a useful tool for distinguishing physiological contributions of T-type channels.

Dihydropyridine-sensitive, voltage-gated Ca2+ channels contribute to the resting intracellular Ca2+ concentration of hippocampal CA1 pyramidal neurons.

1. Whole cell recordings and high-speed fluorescence imaging were used to investigate the contribution of voltage-gated Ca2+ channels to the resting Ca2+ concentration ([Ca2+]i) in hippocampal CA1 pyramidal neurons. 2. Prolonged membrane hyperpolarization produced, in a voltage-dependent manner, sustained decreases in [Ca2+]i in the somatic and apical dendritic regions of the neuron. This hyperpolarization-induced decrease in [Ca2+]i occurred with a time constant of approximately 1 s and was maintained for as long as the membrane potential was held at the new level. Ratiometric measures showed that [Ca2+]i is significantly elevated at holding potentials of -50 mV compared with -80 mV. 3. The hyperpolarization-induced decrease in [Ca2+]i was reduced significantly by 200 microM Cd2+ and 10 microM nimodipine, but was only slightly inhibited by 50 microM Ni2+. The largest amplitude decrease in [Ca2+]i was observed in the proximal apical dendrites with the amplitude of the Ca2+ change decreasing with further distance from the soma. 4. Whole cell recordings from acutely isolated hippocampal pyramidal neurons reveal a slowly inactivating Ca2+ current with similar voltage dependence and pharmacology to the hyperpolarization-induced decrease in [Ca2+]i. 5. The data suggest that a population of dihydropyridine-sensitive Ca2+ channels are active at resting membrane potentials and that this channel activation significantly contributes to the resting [Ca2+]i. These channels appear to be present throughout the neuron and may be located most densely in the proximal apical dendrites.

Multiple channel types contribute to the low-voltage-activated calcium current in hippocampal CA3 pyramidal neurons.

Hippocampal neurons exhibit low-voltage-activated (LVA) and high-voltage-activated (HVA) calcium currents. We characterized the LVA current by recording whole-cell Ca2+ currents from acutely isolated rat hippocampal CA3 pyramidal neurons in 2 mM Ca2+. Long depolarizing steps to -50 mV revealed two components to the LVA current: transient and sustained. The transient phase had a fast decay time constant of 59 msec. The sustained phase persisted throughout the depolarization, even for steps lasting several seconds. The transient current was inhibited by the classic T-type channel antagonists Ni2+ and amiloride. The anticonvulsant phenytoin preferentially blocked the sustained phase, but ethosuximide had no effect. Steady-state inactivation of the transient component was half-maximal at -80 mV. Nimodipine, an L-type channel antagonist, partly inhibited the sustained current. BayK-8644, an L-type channel agonist, potentiated the sustained current. Calciseptine, another L-type channel antagonist, inhibited the sustained component. omega-Conotoxin-MVIIC, a nonselective toxin for HVA channels, had no effect on either of the LVA current components. omega-Grammotoxin-SIA, another nonselective toxin, partially inhibited the sustained component. The voltage dependence of activation of the nimodipine-sensitive current could be fit with a single Boltzmann, consistent with a homogenous population of L-type channels in CA3 neurons. Half-maximal activation of the nimodipine-sensitive current occurred at -30 mV, considerably more negative than the remaining HVA current. These results suggest that in physiologic Ca2+ more than one type of Ca2+ channel contributes to the LVA current in CA3 neurons. The transient current is carried by T-type channels. The sustained current is carried, at least in part, by dihydropyridine-sensitive channels. Thus, the designation "low-voltage-activated" should not be limited to T-type channels. These findings challenge the traditional designation of L-type channels as exclusively HVA and reveal a possible role in subthreshold Ca2+ signaling.

Partition of parinaroylphosphatidylethanolamines and parinaroylphosphatidylglycerols in immiscible phospholipid mixtures.

Partitioning of two parinaroyl phosphatidylethanolamines and two parinaroyl phosphatidylglycerols between solid and fluid phase phospholipids was examined. Fluorescence quantum yields and fluorescence polarization measurements were used to calculate Ks/fp, the solid to fluid partition coefficient of each probe (Sklar, L.A., Miljanich, G.P. and Dratz, E.A. (1979) Biochemistry 18, 1707-1716). In the immiscible mixture dipalmitoylphosphatidylcholine and dilinoleylphosphatidylcholine, both 1-palmitoyl-2-trans-parinaroylphosphatidylethanolamine and 1-palmitoyl-2-transparinaroylphosphatidylglycerol partitioned preferentially into solid phase lipid with mean Ks/fp values (calculated from quantum yields) of 3.4 +/- 1.5 and 2.1 +/- 0.7, respectively. In contrast, 1-oleoyl-2-cis-parinaroylphosphatidylethanolamine and 1-oleoyl-2-cis-parinaroylphosphatidylglycerol partitioned preferentially into fluid phase lipid in the same model system with mean Ks/fp values (calculated from quantum yields) of 0.44 +/- 0.26 and 0.16 +/- 0.07, respectively. Fluorescence polarization data on the same four parinaroyl phospholipids in mixtures of solid-phase dimyristoylphosphatidyl ethanolamine and fluid-phase dilinoleoylphosphatidylglycerol were similar to those obtained in the immiscible phosphatidylcholine system, demonstrating that the partitioning of these probes is not strongly dependent on head group. Knowledge of the partition properties of these fluorescent probes is relevant to use of these probes in investigation of the phase behavior of Escherichia coli inner membrane lipids, since phosphatidylethanolamine and phosphatidylglycerol species account for approximately 95% of these lipids.

Cross-linking of phosphatidylethanolamine neighbors with dimethylsuberimidate is sensitive to the lipid phase.

Dimethylsuberimidate was reacted with aqueous dispersions of dipalmitoylphosphatidylethanolamine, dimyristoylphosphatidylethanolamine, dilauroylphosphatidylethanolamine, and dielaidoylphosphatidylethanolamine at pH 10 and at pH 8. The amount of amidine dimer formation was about four times greater above the gel-to-fluid phase transition of each lipid than below the transition. The transition temperature of each phosphatidylethanolamine, measured by steady-state fluorescence anisotropy of cis-parinaric acid, was lower at pH 10 than at pH 8 or in water. The ability of dimethylsuberimidate to discriminate between phosphatidylethanolamines in the fluid and gel phases should allow use of this reagent to identify phosphatidylethanolamine species within the gel or fluid lipid phase.