Conductance changes produced by acetylcholine in lipidic membranes containing a proteolipid from Electrophorus.

Ultrathin lipidic membranes containing one ten-thousandth of a special proteolipid from electric organ of Electrophorus reacted to the addition of acetylcholine by a rapid and transient increase in conductance. Such a change was not induced by choline and is greatly reduced by a previous application of d-tubocurarine. These properties, resembling those from chemically excitable membranes, were not observed with another proteolipid from the same tissue.

Enhancement of the glutamate response by cAMP-dependent protein kinase in hippocampal neurons.

Receptor channels activated by glutamate, an excitatory neurotransmitter in the mammalian brain, are involved in processes such as long-term potentiation and excitotoxicity. Studies of glutamate receptor channels expressed in cultured hippocampal pyramidal neurons reveal that these channels are subject to neuromodulatory regulation through the adenylate cyclase cascade. The whole-cell current response to glutamate and kainate [a non-NMDA (N-methyl-D-aspartate) receptor agonist] was enhanced by forskolin, an activator of adenylate cyclase. Single-channel analysis revealed that an adenosine 3',5'-monophosphate-dependent protein kinase (PKA) increases the opening frequency and the mean open time of the non-NMDA-type glutamate receptor channels. Analysis of synaptic events indicated that forskolin, acting through PKA, increased the amplitude and decay time of spontaneous excitatory postsynaptic currents.

Prolonged Ca2+-dependent afterhyperpolarizations in hippocampal neurons of aged rats.

The possibility that calcium is elevated in brain neurons during aging was examined by quantifying afterhyperpolarizations induced by spike bursts in CAl neurons of hippocampal slices from young and aged rats. The afterhyperpolarizations result from Ca2+-dependent K+ conductance increases and are blocked in medium low in Ca2+ and prolonged in medium high in Ca2+. The afterhyperpolarization and associated conductance increases were considerably prolonged in cells from aged rats, although inhibitory postsynaptic potentials did not differ with age. Since elevated intracellular Ca2+ can exert deleterious effects on neurons, the data suggest that altered Ca2+ homeostasis may play a significant role in normal brain aging.

Subconductance behavior in a maxi Ca2(+)-activated K+ channel induced by dendrotoxin-I.

Toxin I (DTX-I), a 60-residue peptide belonging to the dendrotoxin family of Mamba snake neurotoxins, is a potent inhibitor of various types of voltage-gated K+ currents. To investigate the sensitivity of another major class of K+ channels to DTX-I, the effect of this toxin was studied on single Ca2(+)-activated K+ channels from rat skeletal muscle incorporated into planar bilayers. Internal (intracellular) DTX-I was found to induce reversibly a long-lived (tau = 40 s), inwardly rectifying subconductance state with 66% of the normal open-state current at +20 mV. Analysis of the kinetics of substate formation and the current-voltage behavior of the substate suggest that binding of DTX-I modifies conduction of K+ ions through the pore without affecting the Ca2+ dependence or voltage dependence of gating. These results identify a unique internal binding site for DTX-I (Kd = 90 nM in 50 mM KCl) on a ubiquitous class of high-conductance, Ca2(+)-activated K+ channels.

Glycine-insensitive desensitization of NMDA responses in cultured mouse embryonic neurons.

The influence of glycine on the desensitization of NMDA-induced currents was studied using cultured embryonic mouse neurons. Although glycine often appeared to reduce desensitization in the whole-cell mode, it had no effect on desensitization in outside-out patches. Various interpretations can be proposed for this discrepancy, such as the presence in intact cells of an intracellular factor regulating desensitization, or the masking of desensitization in intact cells by restricted diffusion of the agonist in the extracellular space. The fact that glycine potentiates the NMDA responses under conditions where it does not regulate desensitization indicates that the potentiation cannot be explained by a reduction of desensitization.

Muscarinic suppression of the M-current is mediated by a rise in internal Ca2+ concentration.

The role of intracellular Ca2+ in the muscarinic suppression of M-current was examined. Intracellular injection of Ca2+ buffer into cells in the intact ganglion reduced the response to muscarinic agonist. In similar experiments on isolated cells, Ca2+ buffer was introduced into the cytoplasm using a perfused recording pipette. Ca2+ buffer (20 mM) with the free Ca2+ concentration set to normal resting levels produced a reversible reduction of the muscarinic response. In a second line of investigation, it was found that pharmacological procedures designed to deplete internal stores of Ca2+ produced a decrease in the muscarinic response. These results, taken together with previous work, support the hypothesis that the muscarinic suppression of M-current is mediated by the release of Ca2+ from intracellular stores.

Ca(2+)-activated K+ currents underlying the afterhyperpolarization in guinea pig vagal neurons: a role for Ca(2+)-activated Ca2+ release.

We examined the possibility that Ca2+ released from intracellular stores could activate K+ currents underlying the afterhyperpolarization (AHP) in neurons. In neurons of the dorsal motor nucleus of the vagus, the current underlying the AHP had two components: a rapidly decaying component that was maximal following the action potential (GkCa,1) and a slower component that had a distinct rising phase (GkCa,2). Both components required influx of extracellular Ca2+ for their activation, and neither was blocked by extracellular TEA (10 mM). GkCa,1 was selectively blocked by apamin, whereas GkCa,2 was selectively reduced by noradrenaline. The time course of GkCa,2 was markedly temperature sensitive. GkCa,2 was selectively blocked by application of ryanodine or sodium dantrolene, or by loading cells with ruthenium red. These results suggest that influx of Ca2+ directly gates one class of K+ channels and leads to release of Ca2+ from intracellular stores, which activates a different class of K+ channel.

Seizure-like activity and cellular damage in rat hippocampal neurons in cell culture.

Neurons dissociated from the hippocampal formations of neonatal rats were grown in medium containing kynurenic acid (a glutamate receptor antagonist) and elevated Mg2+. Such chronically blocked neurons, when first exposed to medium without blockers (after 0.5-5.0 months), generated intense seizure-like activity. This consisted of bursts of synchronous electrical responses that resembled paroxysmal depolarization shifts and sustained depolarizations that, in some neurons, nearly abolished the resting potential. Sustained depolarizations were usually reversed by timely application of kynurenate or 2-amino-5-phosphonovalerate, indicating that continuous activation of glutamate receptors was required for their maintenance. Prolonged periods of intense seizure-like activity usually killed most neurons in the culture. This system allows seizure-related cellular mechanisms to be studied in long-term cell culture.

L-glutamate conditionally modulates the K+ current of Müller glial cells.

L-Glutamate inhibits the K+ conductance that dominates the electrical behavior of a Müller glial cell. The effect of glutamate is enhanced by simultaneous exposure to dopamine. L-Glutamate acts at a metabotropic receptor that controls the K+ conductance through two pathways. A rapid pathway produces a partial inhibition in less than 2 s. Thereafter, a slow pathway progressively inhibits the conductance with a half-time of minutes. Pathways initiated by L-glutamate and dopamine appear to converge on and stimulate adenylyl cyclase. A subsequent step is the activation of a cAMP-dependent protein kinase, PKA. The local overflow of L-glutamate from active synapses may functionally remove K+ channels from nearby glial membranes. A uniform rise in extracellular L-glutamate concentration, as might occur during pathological conditions, should suppress a glial cell's K+ conductance and allow other voltage-dependent processes to be influenced by depolarization.

Competition for block of a Ca2(+)-activated K+ channel by charybdotoxin and tetraethylammonium.

Single high-conductance Ca2(+)-activated K+ channels were incorporated into planar lipid bilayers, and the discrete block by charybdotoxin (CTX), a protein inhibitor of this channel, was studied. In particular, the effect of externally added tetraethylammonium (TEA) on CTX blocking kinetics was investigated. TEA decreases the on-rate of CTX in exact proportion to its blocking of the single-channel current. The CTX off-rate is unaffected by TEA. The results demonstrate that TEA and CTX are mutually exclusive in their binding to the channel. Since the site of TEA binding is known to be located on the external side of the conduction pore, this result further strengthens the proposal that the CTX binding site is located in the external mouth of the channel.

A diffusible second messenger mediates one of the pathways coupling receptors to calcium channels in rat sympathetic neurons.

Muscarinic and alpha-adrenergic suppression of current through Ca2+ channels was studied in adult rat superior cervical ganglion neurons using whole-cell and cell-attached configurations of the patch-clamp technique. Oxotremorine methiodide suppressed ICa by both a rapid (much less than 1 s) and a slow (greater than 4 s) process, whereas norepinephrine suppressed ICa only by a rapid process. The slow muscarinic suppression could be prevented by adding 20 mM BAPTA, a Ca2+ chelator, to the recording pipette, whereas the adrenergic suppression was not affected. Muscarinic, but not alpha-adrenergic, receptors can couple to Ca2+ channels by a second messenger capable of diffusing into an on-cell patch. This signal seems not to be carried by intracellular Ca2+, cGMP, cAMP, or protein kinase C.

Serotonin receptor activation reduces calcium current in an acutely dissociated adult central neuron.

The release of serotonin (5-HT) from the terminals of serotonergic (raphe) neurons is under inhibitory feed-back control. 5-HT, acting on raphe cell body autoreceptors, also mediates inhibitory postsynaptic potentials as a result of release from collaterals from neighboring raphe neurons. This may involve a ligand (5-HT)-gated increase in the membrane potassium conductance, leading to a decrease in action potential frequency, which could indirectly reduce calcium influx into nerve terminals. In this report we demonstrate that 5-HT can also directly reduce calcium influx at potentials including and bracketing the peak of calcium current activation. Using acutely isolated, patch-clamped dorsal raphe neurons, we found that low concentrations of 5-HT and the 5-HT1A-selective agonist 8-OH-DPAT reversibly decrease whole-cell calcium current. This effect is antagonized by the putative 5-HT1A-selective antagonist NAN 190. Hence, the inhibition of calcium current may serve a physiological role in these cells and elsewhere in the brain.

Zinc has opposite effects on NMDA and non-NMDA receptors expressed in Xenopus oocytes.

Pharmacological characterization of Zn2+ effects on glutamate ionotropic receptors was investigated in Xenopus oocytes injected with rat brain mRNA, using a double microelectrode, voltage-clamp technique. At low concentration, Zn2+ inhibited NMDA currents (IC50 = 42.9 +/- 1.3 microM) and potentiated both AMPA (EC50 = 30.0 +/- 1.2 microM) and desensitized kainate responses (EC50 = 13.0 +/- 0.1 microM). At higher concentrations, Zn2+ inhibited non-NMDA responses with IC50 values of 1.3 +/- 0.1 mM and 1.2 +/- 0.3 mM for AMPA and kainate, respectively. The potentiation of AMPA or quisqualate currents by Zn2+ was more than 2-fold, whereas that of the kainate current was only close to 30%. This potentiating effect of Zn2+ on AMPA current modified neither the affinity of the agonist for its site nor the current-voltage relationship. In addition, 500 microM Zn2+ differentially affected NMDA and non-NMDA components of the glutamate-induced response. The possible physiological relevance of Zn2+ modulation is discussed.

Specific inhibitors of protein kinase C block transmitter-induced modulation of sensory neuron calcium current.

Modulation of neuronal, voltage-dependent calcium current has been described for a number of transmitters and peptides, but the biochemical basis for this phenomenon has not been completely identified. In several cases protein kinase C (PKC) is thought to mediate transmitter inhibition of calcium current; however, a lack of specific PKC inhibitors has hampered a direct physiological test of this idea. We have used the whole-cell, tight-seal configuration of the patch-clamp technique to apply intracellularly two specific PKC inhibitors to the cell bodies of embryonic chick sensory neurons. Both inhibitors, a 17 kd protein purified from bovine brain and a synthetic 13 amino acid "pseudosubstrate" peptide, blocked inhibition of calcium current by either norepinephrine or an exogenously applied PKC activator. These results provide strong evidence that activation of PKC is a prerequisite for the modulation of sensory neuron calcium current by norepinephrine.

Antibodies to the GTP binding protein, Go, antagonize noradrenaline-induced calcium current inhibition in NG108-15 hybrid cells.

The voltage-dependent calcium current in chemically differentiated NG108-15 cells is depressed by noradrenaline acting on alpha-adrenoreceptors. The response is absent in cells pretreated with pertussis toxin, implicating the involvement of a G-protein. To identify this G-protein, we have studied the response to noradrenaline in cells preinjected with antibodies specific for two G-proteins, Gi and Go. Cells injected with the Gi antibody responded normally to noradrenaline. In contrast, the response to noradrenaline in cells injected with the Go antibody was markedly attenuated. We conclude that Go is employed in coupling alpha-adrenoreceptors to the calcium channels in NG108-15 cells.

Estrogen induction of a small, putative K+ channel mRNA in rat uterus.

Estrogen causes dramatic long-term changes in the activity of the uterus. Here we report the molecular cloning of a small (700 base) uterine mRNA species capable of inducing a slow K+ current in Xenopus oocytes. The 130 amino acid protein encoded by this mRNA species has a predicted structure that does not resemble that of previously described voltage-dependent channels from mammalian sources. It is, however, similar to structural motifs found in certain prokaryotic ion channels. The induction of this mRNA by estrogen is rapid; this uterine mRNA species is not detectable in uteri from estrogen-deprived rats, but is substantially induced after 3 hr of estrogen treatment. These results support a critical role for regulation of ion channel expression by estrogen in the uterus.

Neurosteroids act on recombinant human GABAA receptors.

The endogenous steroid metabolites 3 alpha,21dihydroxy-5 alpha-pregnan-20-one and 3 alpha-hydroxy-5 alpha-pregnan-20-one potentiate GABA-activated Cl- currents recorded from a human cell line transfected with the beta 1, alpha 1 beta 1, and alpha 1 beta 1 gamma 2 combinations of human GABAA receptor subunits. These steroids are active at nanomolar concentrations in potentiating GABA-activated Cl- currents and directly elicit bicuculline-sensitive Cl- currents when applied at micromolar concentrations. The potentiating and direct actions of both steroids were expressed with every combination of subunits tested. However, an examination of single-channel currents recorded from outside-out patches excised from these transfected cells suggests that despite the common minimal structural requirements for expressing steroid and barbiturate actions, the mechanism of GABAA receptor modulation by these pregnane steroids may differ from that of barbiturates.

Prostaglandin E2 inhibits sodium transport in rabbit cortical collecting duct by increasing intracellular calcium.

The mechanism by which prostaglandin E2 (PGE2) inhibits sodium absorption (JNa) in the rabbit cortical collecting duct (CCD) was explored. PGE2 activates at least three signaling mechanisms in the CCD: (a) by itself PGE2 increases cAMP generation (b) PGE2 also inhibits vasopressin-stimulated cAMP accumulation, and (c) PGE2 raises intracellular calcium([Ca++]i). We tested the contribution of these signaling pathways to PGE2's effect on Na+ absorption, measuring 22Na flux (JNa) and [Ca++]i (using fura-2) in microperfused rabbit CCDs. In control studies PGE2 reduced JNa from 28.2 +/- 3.4 to 15.6 +/- 2.6 pmol.mm-1.min-1. Lowering bath calcium from 2.4 to 45 nM did not by itself alter JNa but in this setting PGE2 failed to inhibit JNa (28.6 +/- 5.4 to 38.5 +/- 4.0). In separate tubules, PGE2 raised [Ca++]i in a spike-like fashion followed by a sustained elevation. However, in 45 nM bath Ca++, PGE2 failed to produce a sustained [Ca++]i elevation. While pretreatment of CCDs with pertussis toxin blocked PGE2 inhibition of vasopressin-stimulated water permeability, it did not block the effect of PGE2 on JNa. To see if cAMP generation contributes to the effect of PGE2 on JNa, we tested the effect of exogenous cAMP, (8-chlorophenylthio(CPT)cAMP) on JNa. 0.1 mM 8-CPTcAMP reduced JNa from 35.75 +/- 2.3 to 21.6 +/- 2.2. However, the addition of PGE2 further blunted JNa to 15.9 +/- 1.3. In CCDs pretreated with indomethacin, 8-CPTcAMP did not significantly decrease JNa 33.6 +/- 2.8 vs. 28.4 +/- 2. However, superimposed PGE2 reduced JNa to 19.0 +/- 3.0. We conclude that PGE2 inhibits sodium transport predominantly by increasing intracellular calcium. This action is not mediated by a pertussis toxin-sensitive G protein. Finally, cAMP, through a cyclooxygenase-dependent mechanism, also inhibits CCD JNa and may contribute to the effects of PGE2 on JNa in the rabbit CCD.

Short-term effects of uninephrectomy on electrical properties of the cortical collecting duct from rabbit remnant kidneys.

Microelectrode techniques were used to assess the electrical properties of the collecting duct cell in the isolated perfused cortical collecting duct from remnant kidneys 3, 6, and 24 h after uninephrectomy (UNX); results were compared with those from sham-operated kidneys. Plasma aldosterone levels did not change during the time course after UNX. The lumen-negative transepithelial voltage was elevated significantly 3 h after UNX, and was increased further 24 h after UNX. The basolateral membrane voltage (VB) was elevated 6 h after UNX, and then was increased further at 24 h. Although the tight junction conductance and the fractional apical membrane resistance (fRA) were not altered at any time points after UNX, the apical membrane conductance as well as the transepithelial (GT) and basolateral membrane conductances increased 6 and 24 h after UNX. The changes in apical membrane voltage, GT, and fRA upon addition of luminal amiloride increased just 3 h after UNX, and then remained elevated at 6 and 24 h. The changes in apical membrane voltage and GT upon addition of luminal Ba2+, the changes in VB upon addition of bath ouabain, and the changes in VB, GT, and fRA upon raising bath K+ were not influenced 3 h after UNX, but increased at 6 and 24 h. At these latter periods after UNX, the transference number of Cl- of the basolateral membrane decreased significantly, whereas the transference number of K+ of the basolateral membrane increased significantly. Simultaneously, addition of Ba2+ to the bath caused the VB to hyperpolarize in parallel with decreases in GT and fRA. We conclude: (a) the initial effect of UNX (3 h) in the collecting duct cell is an increase in apical membrane Na+ conductance; (b) the delayed effects of UNX (6 and 24 h) are increases in apical membrane K+ conductance as well as basolateral membrane Na(+)-K+ pump activity and K+ conductance; (c) the hyperpolarization of VB at 6 and 24 h after UNX may result in the decrease of the ratio of the relative Cl- conductance to the relative K+ conductance of the basolateral membrane and also may increase passive K+ entry into the cell across the basolateral membrane; (d) these time-dependent electrical changes occur independently of plasma aldosterone levels.

Ca-mediated stimulation of Cl secretion by reactive oxygen metabolites in human colonic T84 cells.

Monochloramine (NH2Cl), a granulocyte-derived reactive oxygen metabolite (ROM), increases short-circuit current (Isc) in cultured T84 monolayers in a concentration-dependent manner up to nonlethal concentrations of 75 microM. Isc increases slowly after NH2Cl, reaching a peak value of 18 +/- 2 microA/cm2 20 min after addition. The Isc changes are persistent (lasting over 20-30 min), depend on medium Cl, and are inhibitable with bumetanide. 36Cl flux studies demonstrated that NH2Cl increases serosa-to-mucosa flux of Cl without changing mucosa-to-serosa flux, consistent with stimulation of electrogenic Cl secretion. Isc responses to NH2Cl, but not PGE2, are dependent on medium calcium. As demonstrated in fura-2-loaded T84 cells, NH2Cl increases free cytosolic calcium by influx of extracellular Ca2+ and by release of Ca2+ from endogenous stores. However, NH2Cl had no effect on phosphatidylinositol metabolism or cyclic nucleotide levels. We conclude that ROM directly stimulate electrolyte secretion, an effect in part mediated by increases in cytosolic Ca2+, possibly through increasing Ca2+ permeability of cellular membranes.