Development of an inducible NMDA receptor stable cell line with an intracellular Ca2+ reporter.

Cytotoxicity associated with NMDA receptor activation has impeded the establishment of cell lines expressing recombinant subtypes of this ligand-gated ion channel class. To circumvent this toxicity, we describe in this report the use of a potent inducible promoter in the construction of a cell line stably expressing the NR1a/NR2A subtype of the NMDA receptor. Western blot analysis using subunit selective antibodies revealed that NR2A subunits were constitutively expressed in this cell line, whereas expression of NR1a subunits was tightly regulated by tetracycline. Upon tetracycline removal, electrophysiological recordings using the patch clamp technique indicated the expression of functional receptors with biophysical and pharmacological properties corresponding to those expected of the NR1a/NR2A subtype. In addition, we utilized this cell line with the recombinant membrane targeted Ca2+ reporter, aequorin, in a functional assay of NMDA receptor activation. An evaluation of the coupling efficiency of NMDA receptor activation and aequorin response, as well as the pharmacological profile of this assay, illustrates the suitability of this cell line and the Ca2+ reporter assay to functionally identify novel NMDA receptor antagonists.

Cloning and characterization of a novel human inwardly rectifying potassium channel predominantly expressed in small intestine.

A new member of the two transmembrane domain potassium (K+) channel family was identified and isolated from a human brain cDNA library. The cDNA clone contains an open reading frame which encodes a 360 amino acid sequence with a characteristic P domain flanked by two hydrophobic regions representing the membrane spanning segments. The closest homologue of this gene product is the inwardly rectifying potassium channel subunit, Kir1.2 (identity approximately 42%). Northern blot analysis of human tissues with a selective cDNA probe for this new K+ subunit showed a single major transcript of 3.4 kb predominantly expressed at high levels in small intestine, with lower levels in stomach, kidney and brain. The main regions of expression in the central nervous system were medulla, hippocampus and corpus callosum. cRNA-injected oocytes and transiently transfected HEK293 cells expressed a K+ conductance which displays an inward rectification. This conductance is blocked by cesium and barium but is insensitive to tolbutamide and diazoxide even upon co-transfection of this novel subunit with the plasmid encoding the sulfonylurea receptor SUR1. Taken together, these results demonstrate that we have isolated and characterized a novel K+ channel subunit belonging to the inwardly rectifying K+ (Kir) channel family to which, upon homology classification, we have given the nomenclature Kir7.1.

Development of stable cell lines expressing different subtypes of GABAA receptors.

The experiments reported here were motivated by our interest to express in stably-transfected cells large amounts of recombinant rat GABAA receptors. For this, we developed an original two step selection strategy, in which the first step consisted of transfecting HEK 293 cells with rat GABAA receptor alpha and beta subunits. G 418 resistant colonies isolated at this step were screened for [3H] muscimol binding to select for those that coexpressed alpha- and beta-subunits. The best alpha and beta subunit expressing colony was then supertransfected with a plasmid coding for the gamma rat GABAA receptor subunit and a mutant DHFR gene. After a second round of selection, this time in presence of methotrexate, those colonies that coexpressed ternary alpha beta gamma GABAA receptor combinations were distinguished using [3H] flumazenil as a probe. This strategy was applied to the isolation of 3 GABAA receptor clones, alpha 1 beta 2 gamma 2s, alpha 3 beta 2 gamma 2s and alpha 5 beta 3 gamma 2s, that expressed relatively high levels of these proteins. These 3 cell lines exhibited pharmacological and functional properties similar to cells transiently-transfected with equivalent subunit combinations. These cell lines therefore provide attractive models with which to evaluate the intrinsic activity and potency of compounds at recombinant GABAA receptor subtypes.

Chemical synthesis, structural and functional characterisation of noxiustoxin, a powerful blocker of lymphocyte voltage-dependent K+ channels.

Two forms of the Centrudoides noxius scorpion noxiustoxin, containing an amidated and an acid C-terminus, were synthesized on a solid support by using Fmoc-chemistry and 2-(1H-benzotriazol-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate (HBTU) coupling. Comparison of the two synthetic forms with the native toxin by tryptic mapping and CD spectroscopy shows that noxiustoxin possesses an amidated C-terminus and the same fold as all short scorpion toxins. Patch-clamp assays on B lymphocytes demonstrate that noxiustoxin inhibits the voltage-dependent K+ channels with 2 nM affinity, but does not affect the Ca(2+)-activated K+ channels. This toxin, because of its high affinity and specificity for voltage-gated K+ channel, may provide a powerful tool in the investigation of the role(s) of these channels in the T and B lymphocyte activation and proliferation.

A primary T-cell immunodeficiency associated with defective transmembrane calcium influx.

We investigated a T-cell activation deficiency in a 3-month-old boy with protracted diarrhea, serious cytomegalovirus pneumonia, and a family history (in a brother) of cytomegalovirus infection and toxoplasmosis. In spite of detection of normal number of peripheral lymphocytes, T cells did not proliferate after activation by anti-CD3 and anti-CD2 antibodies, although proliferation induced by antigens was detectable. We sought to determine the origin of this defect as it potentially represented a valuable tool to analyze T-cell physiology. T-cell activation by anti-CD3 antibody or phytohemagglutinin (PHA) led to reduced interleukin-2 (IL-2) production and abnormal nuclear factor-activated T cell (NF-AT; a complex regulating the IL-2 gene transcription) binding activity to a specific oligonucleotide. T-cell proliferation was restored by IL-2. Early events of T-cell activation, such as anti-CD3 antibody-induced cellular protein tyrosine phosphorylation, p59fyn and p56lck kinase activities, and phosphoinositide turnover, were found to be normal. In contrast, anti-CD3 antibody-induced Ca2+ flux was grossly abnormal. Release from endoplasmic reticulum stores was detectable as tested in the presence of anti-CD3 antibody or thapsigargin after cell membrane depolarization in a K+ rich medium, whereas extracellular entry of Ca2+ was defective. The latter abnormality was not secondary to defective K+ channel function, which was found to be normal. A similar defect was found in other hematopoietic cell lineages and in fibroblasts as evaluated by both cytometry and digital video imaging experiments at a single-cell level. This primary T-cell immunodeficiency appears, thus, to be due to defective Ca2+ entry through the plasma membrane. The same abnormality did not alter B-cell proliferation, platelet function, and polymorphonuclear neutrophil (PMN) function. Elucidation of the mechanism underlying this defect would help to understand the physiology of Ca2+ mobilization in T cells.

The calcium current activated by T cell receptor and store depletion in human lymphocytes is absent in a primary immunodeficiency.

Stimulation of antigen receptors of lymphocytes triggers a transitory release of Ca2+ from internal stores and the opening of a transmembrane Ca2+ conductive pathway. The latter underlies the sustained increase of intracellular free calcium concentration, and it seems to be a key event in the Ca(2+)-dependent biochemical cascade leading to T cell proliferation. Alternatively, pharmacological depletion of internal stores by itself activates Ca2+ influx. This has led to the hypothesis that antigen-triggered Ca2+ influx is secondary to Ca2+ release from internal stores. However, the precise relationship between antigen and Ca2+ release-activated Ca2+ currents remains unclear, particularly since neither of them has been electrophysiologically recorded in normal lymphocytes. Using the whole-cell and the perforated configurations of the patch clamp technique on peripheral blood lymphocytes, we found that a low amplitude Ca(2+)-selective current was triggered when intracellular stores were depleted by stimuli such as the intracellular perfusion of inositol triphosphate or thapsigargin and the extracellular perfusion of ionomycin. A similar current was elicited by the cross-linking of the T cell receptor-CD3 complex. This current displayed an inward rectification below 0 mV and was completely blocked by the divalent cation Cd2+. It was very selective for Ca2+ over Na+ and insensitive to changes in chloride concentration. The physiological relevance of this conductance was investigated with the analysis of abnormal Ca2+ signaling in lymphocytes from a patient suffering from a primary immunodeficiency associated with a defective T cell proliferation. Using fura-2 video imaging, an absence of Ca2+ influx was established in the patient's lymphocytes, whereas the Ca2+ release from internal stores was normal. This was the case whether cells were stimulated physiologically through their antigen receptors or with store depleting pharmacological agents. Most importantly, no Ca(2+)-selective current was elicited in these cells. Our data strongly suggest that the Ca2+ release-activated current underlies the sustained Ca2+ influx during antigenic stimulation and that it plays a key role in the immune function.

Defective transmembrane calcium influx demonstrated in a primary immunodeficiency by video-imaging.

A rise of intracellular calcium concentration triggered by the engagement of various membrane receptors is a key event in the control of cell growth. This increase involves both a release of calcium from intracellular stores and the opening of a transmembrane calcium conductive pathway. Using video imaging to measure intracellular calcium concentration in individual fura-2-loaded cells, we detected a defect in calcium influx in lymphocytes and fibroblasts collected from patients affected by a rare and new form of primary immunodeficiency. In these cells, pharmacological agents such as thapsigargin or ionomycin, and the physiological activator bradykinin, only induced transient increases in cytoplasmic calcium level, due to the emptying of internal stores, while in control cells, this initial step is followed by an additional and sustained transmembrane calcium influx. The fact that calcium influx is absent in patient's fibroblasts indicates that the related deficiency, which is clinically associated with a lack of proliferation of T lymphocytes, also affects cells of the non-hematopoietic lineages. This study emphasizes the adequacy of single cell imaging for determining whether some forms of pathologies are associated with a disregulation of ionic fluxes, and for identifying them accurately.

Pattern of potassium channel expression in proliferating B lymphocytes depends upon the mode of activation.

Ionic channel expression is highly regulated during mitogenesis. But it is not clear whether these regulations only follow intrinsic programs during the course of the cell cycle or if they also depend upon the external factors used to promote cell activation. B lymphocytes express two classes of potassium channels and can be stimulated to enter the cell cycle by distinct pathways. Thus, we have analyzed, with the patch-clamp technique, if the expression of channels varies when the cells are activated by different signals that lead to cell proliferation. We found that stimulation through Ag receptors increases the expression of calcium- and voltage-activated potassium channels, whereas a bacterial mitogen, LPS, only enhances the expression of the latter. Moreover, channel expression can still be modified in proliferating cells because stimulation of LPS-activated cells through Ag receptors induces rapid expression of calcium-activated channels. The use of inhibitors of mRNA synthesis revealed that this process depends upon gene transcription. Thus, differential induction of the expression of potassium channels is not only linked to the entry into the cell cycle but depends also on pathways of stimulation.

Cross-linking of IgG receptors inhibits membrane immunoglobulin-stimulated calcium influx in B lymphocytes.

By cross-linking membrane immunoglobulins (mIg), the antigenic stimulation of B lymphocytes induces an increase in intracellular free calcium levels ([Ca2+]i) because of a combination of release from intracellular stores and transmembrane influx. It has been suggested that both events are linked, as in a number of other cases of receptor-induced increase in [Ca2+]i. Conversely, in B lymphocytes, type II receptors for the Fc fragment of IgG (Fc gamma RII) inhibit mIg-mediated signaling. Thus, we have investigated at the level of single cells if these receptors could act on specific phases of mIg Ca2+ signaling. Lipopolysaccharide-activated murine B splenocytes and B lymphoma cells transfected with intact or truncated Fc gamma RII-cDNA were used to determine the domains of Fc gamma RII implicated in the inhibition of the Ca2+ signal. [Ca2+]i was measured in single fura-2-loaded cells by microfluorometry. The phases of release from intracellular stores and of transmembrane influx were discriminated by using manganese, which quenches fura-2, in the external medium as a tracer for bivalent cation entry. The role of membrane potential was studied by recording [Ca2+]i in cells voltage-clamped using the perforated patch-clamp method. Cross-linking of mIgM or mIgG with F(ab')2 fragments of anti-Ig antibodies induced a sustained rise in [Ca2+]i due to an extremely fast and transitory release of Ca2+ from intracellular stores and a long lasting transmembrane Ca2+ influx. The phase of influx, but not that of release, was inhibited by membrane depolarization. The increase in [Ca2+]i occurred after a delay inversely related to the dose of ligand. Co-cross-linking mIgs and Fc gamma RII with intact anti-Ig antibodies only triggered transitory release of Ca2+ from intracellular stores but no Ca2+ influx, even when the cell was voltage-clamped at negative membrane potentials. These transitory Ca2+ rises had similar amplitudes and delays to those induced by cross-linking mIgs alone. Thus, our data show that Fc gamma RII does not mediate an overall inhibition of mIg signaling but specifically affects transmembrane Ca2+ influx without affecting the release of Ca2+ from intracellular stores. Furthermore, this inhibition is not mediated by cell depolarization. Thus, Fc gamma RII represents a tool to dissociate physiologically the phases of release and transmembrane influx of Ca2+ triggered through antigen receptors.

Differential regulation of voltage- and calcium-activated potassium channels in human B lymphocytes.

The expression and characteristics of K+ channels of human B lymphocytes were studied by using single and whole-cell patch-clamp recordings. They were gated by depolarization (voltage-gated potassium current, IKv, 11-20 pS) and by an increase in intracellular Ca2+ concentration (calcium-activated potassium current, IKCa, 26 pS), respectively. The level of expression of these channels was correlated with the activational status of the cell. Both conductances are blocked by tetraethylammonium, verapamil, and charybdotoxin, and are insensitive to apamin; 4-aminopyridine blocks IK, preferentially. We used a protein kinase C activator (PMA) or antibodies to membrane Ig (anti-mu) to activate resting splenocytes in culture. Although IKv was recorded in the majority of the resting lymphocytic population, less than 20% of the activated cells expressed this conductance. However, in this subset the magnitude of IKv was 20-fold larger than in resting cells. On the other hand, IKCa was detected in nearly one half of the resting cells, whereas all activated cells expressed this current. The magnitude of IKCa was, on average, 30 times larger in activated than in nonactivated cells. These results probably reflect that during the course of activation 1) the number of voltage-dependent K+ channels per cell decreases and increases in a small subset and 2) the number of Ca(2+)-dependent K+ channels per cell increases in all cells. We suggest that the expression of functional Ca(2+)- and voltage-activated K+ channels are under the control of different regulatory signals.