Interaction of the M4 segment with other transmembrane segments is required for surface expression of mammalian α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptors.

Ionotropic glutamate receptors (GluRs) are ligand-gated ion channels with a modular structure. The ion channel itself shares structural similarity, albeit an inverted membrane topology, with P-loop channels. Like P-loop channels, prokaryotic GluR subunits (e.g. GluR0) have two transmembrane segments. In contrast, eukaryotic GluRs have an additional transmembrane segment (M4), located C-terminal to the ion channel core. However, the structural/functional significance of this additional transmembrane segment is poorly defined. Although topologically similar to GluR0, mammalian AMPA receptor (GluA1) subunits lacking the M4 segment do not display surface expression. This lack of expression is not due to the M4 segment serving as an anchor to the ligand-binding domain because insertion of an artificial polyleucine transmembrane segment does not rescue surface expression. Specific interactions between M4 and the ligand-binding domain are also unlikely because insertion of polyglycines into the linker connecting them has no deleterious effects on function or surface expression. However, tryptophan and cysteine scanning mutagenesis of the M4 segment, as well as recovery of function in the polyleucine background, defined a unique face of the M4 helix that is required for GluR surface expression. In the AMPA receptor structure, this face forms intersubunit contacts with the transmembrane helices of the ion channel core (M1 and M3) from another subunit within the homotetramer. Thus, our experiments show that a highly specific interaction of the M4 segment with an adjacent subunit is required for surface expression of AMPA receptors. This interaction may represent a mechanism for regulating AMPA receptor biogenesis.

Pulses of extracellular K+ produce two cytosolic Ca2+ transients that display different temperature dependence and carbonyl cyanide m-chlorophenyl sensitivity in SH-SY5Y cells.

In SH-SY5Y cells we have shown that stimulation with high extracellular K+ ([K+]e) evokes a transient increase in cytoplasmic Ca2+ ([Ca2+]cyt) (K+on) that is triggered by the opening of voltage-dependent Ca2+ channels and followed by Ca2+ -induced Ca2+ release from the endoplasmic reticulum (Xu, F., Zhang, J., Recio-Pinto, E. and Blanck, T.J., Halothane and isoflurane augment depolarization-induced cytosolic CA2+ transients and attenuate carbachol-stimulated CA2+ transients, Anesthesiology, 92 (2000) 1746-56). The removal of high-[K+]e results in a second transient increase in [Ca2+]cyt (K+off) that is independent of extracellular Ca2+ (Corrales, A., Montoya, G.J., Sutachan, J.J., Cornillez-Ty, G., Garavito-Aguilar, Z., Xu, F., Blanck, T.J. and Recio-Pinto, E., Transient increases in extracellular K+ produce two pharmacological distinct cytosolic Ca2+ transients, Brain Res., 1031 (2005) 174-184). In this study we further characterize the properties of K+off. We found that K+off was detectable at near physiological temperatures (34-36 degrees C) but, depending on the level of [K+]e, it was undetectable or highly diminished at room temperature. In contrast, K+on was increased by lowering the temperature. Extracellular Na+ -replacement with K+ did not affect K+off, indicating that K+off was not generated by osmolarity changes. Replacement of extracellular Na+ with choline+ did not affect K+off, indicating that K+off did not result from activity changes of the plasma membrane Na+/Ca2+ exchanger. Caffeine decreased K+on but not K+off. CCCP (carbonyl cyanide m-chlorophenyl), a protonophore uncoupler that decreases mitochondrial Ca2+ uptake, decreased K+on but not K+off. CGP37157, an inhibitor of the mitochondria Na+/Ca2+ exchanger, decreased K+off when added alone but not when added simultaneously with CCCP. Clonazepam had similar effects as CGP37157. These findings indicate that the generation of K+off is strongly temperature-dependent and its pharmacology is distinct from the [Ca2+]cyt changes observed previously at room temperature.

Transient increases in extracellular K+ produce two pharmacological distinct cytosolic Ca2+ transients.

Transient increases in extracellular K+ are observed under various conditions, including repetitive neuronal firing, anoxia, ischemia and hypoglycemic coma. We studied changes in cytoplasmic Ca2+ ([Ca2+]cyt) evoked by pulses of KCl in human neuroblastoma SH-SY5Y cells and rat dorsal root ganglia (DRG) neurons at 37 degrees C. A "pulse" of KCl evoked two transient increases in [Ca2+]cyt, one upon addition of KCl (K+on) and the other upon removal of KCl (K+off). The K+on transient has been described in many cell types and is initiated by the activation of voltage-dependent Ca2+ channels followed by Ca2+-evoked Ca2+ release from intracellular Ca2+ stores. The level of KCl necessary to evoke the K+off transient depends on the type of neuron, in SH-SY5Y cells it required 100 mM KCl, in most (but not all) of dorsal root ganglia neurons it could be detected with 100-200 mM KCl and in a very few dorsal root ganglia neurons it was detectable at 20-50 mM KCl. In SH-SY5Y cells, reduction of extracellular Ca2+ inhibited the K+on more strongly than the K+off and slowed the decay of K+off. Isoflurane (1 mM) reduced the K+on)- but not the K+off-peak. However, isoflurane slowed the decay of K+off. The nonspecific cationic channel blocker La3+ (100 microM) had an effect similar to that of isoflurane. Treatment with thapsigargin (TG) at a concentration known to only deplete IP3-sensitive Ca2+ stores did not affect K+on or K+off, suggesting that Ca2+ release from the IP3-sensitive Ca2+ stores does not contribute to K+on and K+off transients and that the thapsigargin-sensitive Ca2+ ATPases do not contribute significantly to the rise or decay rates of these transients. These findings indicate that a pulse of extracellular K+ produces two distinct transient increases in [Ca2+]cyt.

Isoflurane reduces the carbachol-evoked Ca2+ influx in neuronal cells.

BACKGROUND: The authors previously reported that the isoflurane-caused reduction of the carbachol-evoked cytoplasmic Ca transient increase ([Ca]cyt) was eliminated by K or caffeine-pretreatment. In this study the authors investigated whether the isoflurane-sensitive component of the carbachol-evoked [Ca]cyt transient involved Ca influx through the plasma membrane. METHODS: Perfused attached human neuroblastoma SH-SY5Y cells were exposed to carbachol (1 mm, 2 min) in the absence and presence of isoflurane (1 mm) and in the absence and presence of extracellular Ca (1.5 mm). The authors studied the effect of the nonspecific cationic channel blocker La (100 microm), of the L-type Ca channel blocker nitrendipine (10 microm), and of the N-type Ca channel blocker omega-conotoxin GVIA (0.1 microm) on isoflurane modulation of the carbachol-evoked [Ca]cyt transient. [Ca]cyt was detected with fura-2 and experiments were carried out at 37 degrees C. RESULTS: Isoflurane reduced the peak and area of the carbachol-evoked [Ca]cyt transient in the presence but not in the absence of extracellular Ca. La had a similar effect as the removal of extracellular Ca. Omega-conotoxin GVIA and nitrendipine did not affect the isoflurane sensitivity of the carbachol response although nitrendipine reduced the magnitude of the carbachol response. CONCLUSIONS: The current data are consistent with previous observations in that the carbachol-evoked [Ca]cyt transient involves both Ca release from intracellular Ca stores and Ca entry through the plasma membrane. It was found that isoflurane attenuates the carbachol-evoked Ca entry. The isoflurane sensitive Ca entry involves a cationic channel different from the L- or N- type voltage-dependent Ca channels. These results indicate that isoflurane attenuates the carbachol-evoked [Ca]cyt transient at a site at the plasma membrane that is distal to the muscarinic receptor.

Differential thapsigargin-sensitivities and interaction of Ca2+ stores in human SH-SY5Y neuroblastoma cells.

In human SH-SY5Y neuroblastoma cells, two distinct intracellular Ca2+ stores, a KCl-/caffeine-sensitive and a carbachol-/IP3-sensitive store, were demonstrated previously. In this study, responses of these two intracellular Ca2+ stores to thapsigargin were characterized. Ca2+-release from these stores was evoked either by high K+ (100 mM KCl) or by 1 mM carbachol, and changes in the intracellular Ca2+ level were monitored using Fura-2 fluorimetry. A sequential stimulation protocol (KCl-->carbachol or vice versa) allowed evaluation of the individual contribution of different Ca2+ stores to the evoked intracellular Ca2+ ([Ca2+]i)-transients and the dynamic interaction between them. Thapsigargin (0.05 nM - 20 microM) alone induced a [Ca2+]i-transient. Both the carbachol- and the KCl-evoked [Ca2+]i-transients were inhibited by thapsigargin, but with very different sensitivities. Thapsigargin inhibited the carbachol-evoked [Ca2+]i-transients with (IC50 = 0.353 nM) or without (IC50 = 0.448 nM) a KCl-prestimulation, but an additional small component, with a much lower sensitivity (IC50=4814 nM), was observed in the absence of a KCl-prestimulation. In contrast, the KCl-evoked [Ca2+]i-transients displayed only one component with a very low sensitivity to thapsigargin in both absence (IC50=3343 nM) and presence (IC50=6858 nM) of a carbachol-prestimulation. These findings suggest that the sarco-/endoplasmic reticular Ca2+ ATPases associated with the KCl-/caffeine- and carbachol-/IP3-sensitive intracellular Ca2+ stores differ from each other, either in types or in their post-translational modification. Such difference might play important role in the regulation of neuronal Ca2+ homeostasis.

Isoflurane reduction of carbachol-evoked cytoplasmic calcium transients is dependent on caffeine-sensitive calcium stores.

BACKGROUND: Many muscarinic functions are relevant to anesthesia, and alterations in muscarinic activity affect the anesthetic/analgesic potency of various drugs. Volatile anesthetics have been shown to depress muscarinic receptor function, and inhibition of the muscarinic signaling pathway alters the minimal alveolar anesthetic concentration of inhaled anesthetics. The purpose of this investigation was to determine in a neuronal cell which source of Ca2+ underlying the carbachol-evoked transient increase in cytoplasmic Ca2+ was reduced by isoflurane. METHODS: Experiments were performed at 37 degrees C on continuously perfused monolayers of human neuroblastoma SH-SY5Y cells using Fura-2 as the cytoplasmic Ca2+ indicator. Carbachol (1 mm) was applied to evoke a transient increase in cytoplasmic Ca2+. RESULTS: Isoflurane (1 mm) reduces the carbachol-evoked transient increase in cytoplasmic Ca2+, and this isoflurane action is eliminated when the cells are continuously stimulated with 200 mm KCl or pretreated with 10 mm caffeine or 200 microm ryanodine. CONCLUSIONS: Isoflurane reduction of the carbachol-evoked transient increase in cytoplasmic Ca2+ requires full caffeine-sensitive Ca2+ stores and Ca2+ release from the caffeine-sensitive stores through the ryanodine-sensitive Ca2+ release channels. The results indicate that isoflurane interferes with a muscarinic Ca2+ signaling through a mechanism downstream from the muscarinic receptors.