Monospecific antibodies as probes for the stoichiometry of recombinant GABA(A) receptors

GABA(A) receptors composed of alpha1beta3 gamma2 and alpha1beta3 subunits were expressed in insect Sf9 cells and solubilized in 1% Triton X100. In sucrose density gradients, [3H]-Ro15-1788 binding activity, in the case of alpha1beta3 gamma2, and [3H]-muscimol binding activity, in the case of alpha1beta3 containing receptors sedimented as a single sharp peak suggesting the formation of receptors containing a defined number of subunits. When alpha1beta3gamma2 -containing receptors were incubated with an alpha-subunit specific antibody (bd24), a single class of antibody receptor complex was formed irrespective of the receptor-antibody ratio. This is consistent with two alpha subunits cross-linked within the receptor by the antibody. Similar results were obtained using a beta-subunit specific antibody (bd17). Several classes of antibody-receptor complex were formed when receptors were pre-incubated with a gamma specific antibody (anti gamma(2) 1-15 Cys). This profile is consistent with the presence of a single gamma subunit in each complex. Experiments with alpha1beta3 subunit containing receptors and antibody bd24 produced a profile similar to that seen with alpha1beta3 gamma2 receptors, consistent with two alpha subunits per receptor complex. In this case, the anti-beta subunit antibody, bd17, produced a unique and complex profile consistent with three beta subunits per receptor. This method permits the rapid determination of subunit stoichiometries of homogeneous receptor populations     

Voltage-dependent Ca2+ channel alpha2delta auxiliary subunit: structure, function and regulation.

Voltage-dependent Ca2+ channels are heteromultimeric proteins consisting minimally of a alpha1 main subunit and auxiliary alpha2delta, and beta subunits. The alpha1 subunit forms the ion-conducting pore and contains receptor sites for ligands that modify channel activity. The auxiliary subunits appear to be necessary for the expression of the native kinetic properties of the channel. In particular, the alpha2delta complex, with the transmembrane domain-containing delta subunit arising as a result of proteolysis from the C-terminal end of a alpha2delta precursor, has shown to modify the biophysical and pharmacological properties of the alpha1 subunit in different model systems. Structure-function studies have provided insight into the molecular mechanisms through which alpha2delta exerts its actions and revealed that both allosteric modulation and cellular localization of the Ca2+ channel complex are important mechanisms for alpha2delta regulation of ionic current. Recent studies have shown that the alpha2delta subunit can also support pharmacological interactions with therapeutic agents for the treatment of neurological disorders.     

Beta3 receptors mediate relaxation in stomach fundus whereas a fourth beta receptor mediates tachycardia in atria from transgenic beta3 receptor knockout mice

Pharmacological studies have revealed a non-beta1, beta2 or beta3 adrenergic receptor that mediates tachycardia in rat and human atria. The present studies utilized transgenic mice that lack the rodent beta3 receptor to explore, in a more definitive fashion, whether a non-beta1, beta2 or beta3 receptor can mediate atrial tachycardia. Insofar as the rat stomach fundus possesses a beta3 receptor mediating relaxation, we examined the stomach fundus from beta3 receptor knockout mice for the presence or absence of the beta3 relaxant receptor. Contractile responses to carbamylcholine were similar in potency and magnitude between mouse stomach fundus from wild type and beta3 receptor knockout animals. However, the classical beta3 receptor agonist CL316243, (10(-8)-10(-6)M) relaxed stomach fundus from wild type mice, but not from the beta3 receptor knockout animals. These data provide functional evidence for the absence of the beta3 receptor in beta3 receptor knockout animals and support the role of beta3 receptors mediating relaxation in mouse stomach fundus. Atria from mice lacking the beta3 receptor responded similarly (in potency and maximal increase in heart rate) to isoproterenol (10(-9)-10(-6)M) as atria from wild type mice. Furthermore, propranolol (3 x 10(-7) M) produced a dextral shift in the concentration response to isoproterenol in atria from both the beta3 receptor knockout and wild type mice with negative log K(B) values of 8.03 and 8.09, respectively. Thus, beta receptors mediating tachycardia to isoproterenol are intact and respond similarly in atria from both knockout and wild type mice. Furthermore, CGP12177, a prototypic 'atypical' beta receptor agonist produced tachycardia with a similar EC50 and maximal response in atria from both the wild type and beta3 receptor knockout mice. Cyanopindolol was a partial agonist relative to CGP12177 in both wild type and beta3 receptor knockout mice. Tachycardia to CGP12177 and cyanopindolol was not blocked by propranolol (3 x 10(-7) M) in atria from either group. These data provide definitive evidence that the receptor mediating tachycardia to CGP12177 and to cyanopindolol in atria from the transgenic beta3 receptor knockout mice is neither the beta1, beta2, nor beta3 adrenergic receptor.     

Cloning and expression of a jellyfish calcium channel beta subunit reveal functional conservation of the alpha1-beta interaction.

In high voltage-activated calcium channels, the binding between the pore-forming alpha1 subunit and the modulatory beta subunit is mediated by interaction domains in each molecule that are highly conserved among most known subunits. However, the interaction domain within CyCaalpha1, an alpha1 subunit cloned from the jellyfish Cyanea capillata, matches the canonical sequence of the alpha1 interaction domain at only four of nine sites. We have now cloned a cDNA from Cyanea neuromuscular tissue that encodes a Ca2+ channel beta subunit. The subunit, named CyCabeta, shares 47-54% identity with vertebrate beta subunit isoforms, but is most highly conserved within its interaction domain. Coexpression of CyCabeta with CyCaalpha1 in Xenopus oocytes increases the amplitude of the CyCaalpha1 current and shifts its activation to more hyperpolarized potentials. These responses are mimicked by coexpression of the rat beta2a subunit, demonstrating that the alpha1 beta interaction is functionally conserved between cnidarians and mammals. CyCabeta also markedly accelerates the rate of recovery of CyCaalpha1 from inactivation, an action that is modestly duplicated by beta2a and may represent an additional mechanism by which beta subunit isoforms differentially modulate alpha1 subunits. These findings establish that limited conservation within the alpha1 interaction domain is sufficient to allow full modulation by a beta subunit, as well as altered regulation by different beta isoforms.     

The bradykinin B2 receptor couples less efficiently than the angiotensin AT1 receptor to the G protein Gq in transiently transfected COS-7 cells

The purpose of this study was to compare the efficiency of two different Gq protein-coupled receptors (AT1 receptor for angiotensin II and B2 receptor for bradykinin) to activate phospholipase C (PLC). When the receptors were expressed at a similar level of 0.5 pmol/mg of protein, inositol trisphosphate (IP) accumulation elicited by AT1 receptor was four times higher than that elicited by B2 receptor. Genistein and pertussis toxin did not modify AT1 receptor- or B2 receptor-induced IP accumulation. These results indicate that in COS-7 cells, the two receptors activate PLC beta through G proteins of the Gq family. AT1 or B2 receptors were co-expressed with the alpha subunit of either Gq or G11. Both alpha subunits potentiated to the same extent AT1 receptor-induced IP accumulation. alpha 11 was also as efficient as alpha q to potentiate B2 receptor-induced response. Interestingly, however, the potentiating effect of alpha q and alpha 11 was more important (by 5-fold) on AT1 receptor-mediated response than on B2 receptor-mediated response. These results demonstrate that the extent of activation of PLC beta by different Gq-coupled receptors depends on the level of expression of these receptors and on their coupling efficiency. These are important parameters that determine the relative contribution of specific hormones to different biological processes.     

Evidence for voltage-gated potassium channel beta-subunits with oxidoreductase motifs in human and rodent pancreatic beta cells

Voltage-gated K(+) channel alpha subunits (K(V) alpha) have been previously identified in pancreatic islet beta-cells where it has been suggested they have a role in membrane repolarization and insulin secretion. Here we report the cloning of the three mammalian K(V) beta subunits, including splice variants of these subunits, from both human and rat pancreatic islets and from the rat insulinoma cell line INS-1. Two of the splice variants, K(V) beta1a and K(V) beta3, previously reported to be neuronal tissue specific, are expressed in islets and INS-1 cells. In addition, a splice variant of K(V) beta2 that lacks two potential protein kinase C phosphorylation sites at the amino terminus is present. Immunoblot analysis suggests a high level of K(V) beta2 subunit protein in rat pancreatic islets and immunoprecipitation with anti-K(V) beta2 antibody pulls down a protein from INS-1 cells that reacts with anti-aldose reductase antibody. The K(V) beta subunits, which are attached to the cytoplasmic face of the alpha subunits and are members of the aldose reductase superfamily of NADPH oxidoreductases, may have an as yet undetermined role in the regulation of insulin secretion by the intracellular redox potential. Finally, we suggest that a systematic nomenclature for K(V) beta subunits first proposed by McCormack et al. be adopted for this family of potassium channel subunits as it corresponds with the nomenclature used for their cognate K(V) alpha subunits.     

Immunocytochemical study of alpha 1 and beta 2/3 subunits of GABAA receptors in freehand isolated vestibular Deiters' neurons

Vestibular Deiters' neurons have been isolated from bovine brain by the Hydén's freehand dissection technique and challenged with monoclonal antibodies directed toward the alpha 1 and beta 2/3 subunits of the GABAA receptors. Subsequent challenge with fluorescent secondary antibodies and confocal microscopy allowed the study of the cellular distribution of such subunits. In Deiters' neurons the beta 2/3 subunit displayed a clear presence all along the cell body profile and the initial parts of the dendrites. The alpha 1 subunit was found highly present all over the cell interior except the nuclear profiles. The strong presence inside the cells possibly masked its presence on the plasma membrane. However, in part of the cells studied a distinct presence on the plasma membrane was evident. This subunit was visualized also all along the long dendrites of these neurons. The approach we describe here, involving freehand isolated mature neurons from adult animals, may allow a better characterization of the tridimensional distribution of different types of neuronal GABAA receptors in the respect of the approach with brain slices.     

Unmasking different constitutive activity of four chemoattractant receptors using Na+ as universal stabilizer of the inactive (R) state.

Neutrophils express receptors for the chemoattractants N-formyl-L-methionyl-L-leucyl-L-phenylalanine (FMLP) complement C5a, leukotriene B4 (LTB4) and platelet-activating factor (PAF). The aim of this study was to analyze the constitutive activity of chemoattractant receptors by studying binding of guanosine 5'-O-(3-thiotriphosphate) (GTP gamma S) to the G-protein Gi alpha 2 beta 1 gamma 2 expressed in Sf9 cells. We used Na+ as modulator of constitutive activity because there are no known inverse agonists for the C5a receptor (C5aR), LTB4 receptor (BLTR) and PAF receptor (PAFR). In the absence of NaCl, PAF and LTB4 exhibited larger relative stimulatory effects on GTP gamma S binding than fMLP and C5a. NaCl showed larger inhibitory effects on basal GTP gamma S binding in membranes expressing the formyl peptide receptor (FPR) and C5aR than in membranes expressing BLTR and PAFR. The order of potency of NaCl at inhibiting basal GTP gamma S binding was FPR > C5aR approximately BLTR > PAFR. As a result of the inhibitory effect of NaCl on basal GTP gamma S binding, the relative stimulatory effects of agonists were increased. By quantitatively analyzing the expression levels of chemoattractant receptors and Gi alpha 2 and the stoichiometry of receptor/G-protein coupling we obtained no evidence for structural instability of constitutively active receptors and catalytical G-protein activation. Taken together, the FPR and C5aR exhibit higher constitutive activity than the BLTR and PAFR. Na+ acts as a universal stabilizer of the inactive (R) state in chemoattractant receptors. The different potencies of NaCl at suppressing basal G-protein activity with different receptors indicate that chemoattractant receptors differ from each other in their Na(+)-affinity.     

Unaltered agonist potency upon inducible 5-HT7(a) but not 5-HT4(b) receptor expression indicates agonist-independent association of 5-HT7(a) receptor and Gs

We compared adenylyl cyclase (AC) activation by the G protein-coupled human serotonin (5-HT) receptors 5-HT4(b) and 5-HT7(a) using an ecdysone-inducible expression system, which allowed for reproducible expression of increasing receptor densities in clonal HEK293 (EcR293) cell lines. Low constitutive expression of receptors (2-70 fmol/mg protein) was observed and could be titrated up to 50-200-fold (approximately 400-7000 fmol/mg protein) by the ecdysone analogue ponasterone A. Although 5-HT-stimulated AC activity increased with receptor density, interclonal variation precluded comparisons of coupling efficiency. Interestingly, the potency of 5-HT to stimulate AC increased with increasing receptor density only in clones expressing 5-HT4(b) receptors. The potency for 5-HT did not change in clones expressing 5-HT7(a) receptors, even though 5-HT-stimulated AC activity approached asymptotic levels. This indicates that potency of 5-HT for stimulation of AC through the 5-HT7(a) receptor is independent of receptor-Gs stoichiometry and is consistent with a model where the 5-HT7(a) receptors are tightly associated with G protein, independent of agonist binding. This supports the existence of a complex between inactive receptor and G protein, as predicted by the cubic ternary complex model. In such a system, spare receptors do not lead to increased potency of an agonist with increased receptor density.     

Functional domains of delta- and mu-opioid receptors responsible for adenylyl cyclase inhibition

Opioid receptors (delta, mu) belong to the large superfamily of G protein coupled receptors that inhibit adenylyl cyclase. We have studied the effects of seven synthetic peptides representing selected cytoplasmic regions of the murine delta-opioid receptor on forskolin-mediated adenylyl cyclase activity in membranes of D2 and Neuro(2A) cells stably expressing the delta- and mu-opioid receptors respectively. The entire third intracellular loop (i3), its amino-terminal portion (i3.1) and the carboxyl-terminal region of the second cytoplasmic loop (i2.2) enhanced dose-dependently the agonist-mediated inhibition of cAMP accumulation. The peptide-mediated effects are blocked by pertussis toxin treatment and are not observed in parental cells that lack these receptors. The inhibitory effects of the peptides on adenylyl cyclase were markedly attenuated when membranes from D2 and Neuro(2A) cells were preincubated with antisera against Gi(2) alpha and G beta subunits of G proteins. Our results provide evidence on domains of the delta- and mu-opioid receptors responsible for adenylyl cyclase inhibition.     

Rat brain cholinergic, dopaminergic, noradrenergic and serotonergic neurons express GABAA receptors derived from the alpha3 subunit

In order to study the most abundant GABAA receptor subtypes expressed in cholinergic, dopaminergic, noradrenergic and serotonergic neurons (i.e., in neurons of the so-called "global" projection systems), we employed double-immunocytochemical techniques combining the labeling of GABAA receptor alpha1, alpha2 and alpha3 subunit with markers for these cells. Cholinergic neurons in the striatum, habenula, and pedunculo-pontine nucleus were immunonegative for the alpha1 subunit, and most were also alpha2-immunonegative. However, cholinergic neurons in the striatum, septum and pedunculo-pontine nucleus were alpha3 immunopositive. Dopaminergic neurons in the substantia nigra pars compacta were highly immunopositive for the alpha3, and noradrenergic neurons in the locus coeruleus were immunoreactive for the alpha3 and the alpha2-subunit; although neurons of these areas were negative for alpha1. Similarly, serotonergic neurons in raphe also showed a high level of labeling of alpha3, while there was a lack of immunoreactivity for the alpha1-subunit, and only some individual neurons were positive for the alpha2 subunit. As the presence of different alpha-subunits confers specific physiological and pharmacological properties to GABAA receptors, the abundance of receptors containing the alpha3 subunit (and the scarcity of receptor subtypes including the other alpha-subunits studied) may have important implications for the GABAergic regulation of brain "global" or "diffuse" projection systems.     

Cellular signaling mechanisms for muscarinic acetylcholine receptors

Signaling pathways for muscarinic acetylcholine receptors (mAChRs) include several enzymes and ion channels. Recent studies have revealed the importance of various isoforms of both alpha and betagamma subunits of G proteins in initiation of signaling as well as the role of the small monomeric G protein, Rho, in the activation of phospholipase D. Modulation of adenylyl cyclase activity by mAChRs appears more diverse as the interaction of various receptor subtypes with the many isoforms of the enzyme are studied. Both alpha and beta subunits of G(i/o) may be involved. Some mAChR responses arise through release of nitric oxide from nitrergic nerves, including salivary gland secretion and hippocampal slow wave activity. mAChRs utilize a variety of intracellular pathways to activate various mitogen-activated protein kinases. The kinases are involved in cholinergic regulation of kidney epithelial function, catabolism of amyloid precursor protein, hippocampal long-term potentiation, activation of phospholipase A(2), and gene induction. mAChR activation can also stimulate or inhibit cellular growth and apoptosis, dependent on prior levels of cellular activity. Modulation of ion channels by mAChR agonists appears increasingly complex, based on recent studies. K(+) channels may be activated by M(2) and M(4) mAChR stimulation, although in the rat superior cervical ganglion topographical constraints appear to limit the effect to the M(2) mAChR. Another ganglionic K(+) current, the M current, is inhibited by M(1) mAChR activation, but in murine hippocampus inhibition involves another receptor subtype. R-type Ca(2+) channels are both facilitated and inhibited by M(1) and M(2) mAChRs; facilitation being more pronounced with activation of M(1) mAChRs and inhibition with M(2) mAChRs.     

Stable expression of human homomeric and heteromeric AMPA receptor subunits in HEK293 cells

Human homomeric and heteromeric alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionate (AMPA)-type glutamate receptors (GluRs) were stably expressed in HEK293 cells with cDNAs encoding the flip splice variant of GluR1, GluR2, GluR3, GluR4 subunit, and the GluR1/GluR2, GluR3/GluR2, and GluR4/GluR2 combination. The lethal combination of GluR2 and GluR4 subunits was found in high expression levels of both receptors. The AMPA-evoked current voltage relationships demonstrated the functional channel properties, such as a double rectification in GluR1, GluR3, and GluR4 receptors, and a linear relation in receptors assembled from GluR2 alone and coexpression of GluR2 with the other subunits. All the transfectants exhibited higher selectivity for AMPA than glutamate in dose-dependent current responses. [3H]AMPA binding revealed that the homomeric and heteromeric receptors displayed a single binding site in Scatchard analysis, with dissociation constant (Kd) values in the range of 14.5-49.3 nM. The Bmax values were in the range of 0.57-7.66 pmol/mg protein. The ligand displacement potency for [3H]AMPA binding was CNQX > glutamate > NS257 in all of the transfectants. These results suggest that stable transformants expressing human homomeric and heteromeric AMPA receptors will be useful tools to define selectivity and potential site of action for AMPA receptor modulators.     

Coupling of the expressed cannabinoid CB1 and CB2 receptors to phospholipase C and G protein-coupled inwardly rectifying K+ channels.

Signaling of the cannabinoid CB1 and CB2 receptors through phospholipase C (PLC) and G protein-coupled inwardly rectifying K+ channels (GIRK) was studied after their expression in COS7 cells and Xenopus oocytes. The CB1 or CB2 receptor was co-expressed with alpha subunits of the Galphaq family (Galphaq, Galpha11, Galpha14, Galpha15 and Galpha16) in COS7 cells. Receptor-dependent activation of PLC was observed after co-expressing the CB1 receptor with Galpha14, Galpha15 or Galpha16 but not with Galphaq or Galpha11. Co-expression of Gbeta1 and Ggamma2 abolished the activation, indicating that the activation was mediated by Galpha. PLC activation was not observed when the CB2 receptor was expressed alone or co-expressed with any of the above Galpha subunits. Coupling to GIRK was observed with both CB1 and CB2 receptors after expression in Xenopus oocytes. Significantly larger currents were induced when the receptor was co-expressed with both GIRK1 and GIRK4 than with either GIRK alone. Co-expression of Galpha transducin with the receptor significantly reduced the K+ currents, indicating that GIRK activation was mediated by Gbetagamma but not by Galpha. These findings suggest two new signaling pathways for the cannabinoid receptors.     

Structural and functional characterization of Kv6.2 a new gamma-subunit of voltage-gated potassium channel.

We have cloned and functionally expressed Kv6.2, a new member of the Kv6 subfamily of voltage-gated potassium channel subunits. The human Kv6.2 (KCNF2) gene was mapped at 18q22-18q23. Kv6.2 mRNA is preferentially expressed in rat and human myocard. Rat and human Kv6.2 subunits appear to be unable to form functional Kv channels in a heterologous expression system, but, when coexpressed with Kv2.1 alpha subunits, heteromultimeric Kv channels were formed mediating voltage-activated delayed-rectifier type outward currents. Their kinetics and conductance-voltage relationship were different from those mediated by homomultimeric Kv2.1 channels. Yeast two-hybrid reporter assays indicated that Kv6.2 amino-termini are able to interact specifically with the Kv2.1 amino-terminus. It is proposed that this protein protein interaction underlies Kv2.1/Kv6.2 subunit assembly and the expression of functional heteromultimeric Kv2.1/Kv6.2 channels. The most resiliant feature of the Kv2.1/Kv6.2 channels was their submicromolar sensitivity to the antiarrhythmic drug propafenone. The data suggest that delayed-rectifier type channels containing Kv6.2 subunits may contribute to cardiac action potential repolarization.     

Lack of sumatriptan-induced aortic contraction or relaxation: 5-HT1B receptor protein detected in endothelium and smooth muscle of vasa vasorum but not aorta

Since 5-HT1B receptor mRNA was reported in rat aorta, and 5-HT1B receptor activation has been linked to vascular contraction, we explored sumatriptan-induced contractility and immunohistochemical density of 5-HT1B receptor protein in rat aorta. Sumatriptan (up to 10(-4) M), a 5-HT1B/1D receptor agonist, did not contract the endothelial intact or denuded rat aorta, even in the presence of L-NAME or after induction of modest tone with PGF2 alpha (10(-6) M). Sumatriptan also did not relax aortic preparations precontract with PGF2 alpha (3 x 10(-6) M) or phenylephrine (3 x 10(-7) M). Using a polyclonal antibody directed against the 5-HT1B receptor, minimal 5-HT1B receptor protein was detected in either the endothelium or smooth muscle of the rat aorta. However, dense 5-HT1B receptor protein was found in the vascular smooth muscle of the vasa vasorum supplying the aorta. Thus, the 5-HT1B receptor mRNA detected in tissue extracts of the rat aorta most likely reflects 5-HT1B receptor expression in the arterioles of the vasa vasorum. These studies support the link between the 5-HT1B receptor and vascular contraction in that the aorta with low density of 5-HT1B receptors lacked responses to sumatriptan, an agonist thought to contract blood vessels via 5-HT1B/1D receptors. Furthermore, caution must be observed when using 5-HT1B receptor mRNA to suggest receptor protein in tissues since this RT-PCR product may be derived predominantly from small blood vessels.     

Extracellular Ca2+ sensitivity of mGluR1alpha associated with persistent glutamate response in transfected CHO cells

We previously reported that the metabotropic glutamate receptor 1alpha (mGluR1alpha) can be activated not only by glutamate but also by extracellular Ca2+ (Ca2+o), and that Ser 166 in the extracellular domain determines the sensitivity to Ca2+o. In the present study, we investigated by intracellular Ca2+ (Ca2+i) imaging, the effect of Ca2+o on the glutamate responses of Chinese Hamster Ovary (CHO) cells stably expressing mGluR1alpha wild-type (CHO-wt). As a negative control, we carried out similar experiments using CHO cells expressing Ser166Asp mutant of mGluR1alpha (CHO-S166D) or the substance P receptor (CHO-SPR), which were not activated by Ca2+o application. We observed a remarkable prolongation of the duration of the glutamate response in CHO-wt cells in a Ca2+o concentration dependent manner. In CHO-S166D cells and CHO-SPR cells, only a small sustained component of the glutamate response was observed in the presence of Ca2+o. These sustained components were blocked by SKF-96365, a blocker of receptor-operated Ca2+-influx. Thus, it was concluded that the Ca2+o-sensing function of mGluR1alpha-wt induced the persistent opening of the receptor-operated Ca2+-permeable channels, probably by persistent activation of the receptor by glutamate. We additionally observed that the dose-response relationship of CHO-S166D and CHO-SPR shifted significantly by changing Ca2+o concentration, i.e. Ca2+o was required to maintain the normal ligand responses of these receptors.     

Phosphorylation of the 5-hydroxytryptamine3 (5-HT3) receptor expressed in HEK293 cells

The 5-hydroxytrptamine3 (5-HT3) receptor is a pentameric complex belonging to the family of ligand-gated ion channels. A variety of studies have suggested that phosphorylation regulates the rate of desensitisation and the size of amplitude of the receptor current. In this study we have examined the phosphorylation of the myc-tagged wild-type 5-HT3A receptor subunits from guinea-pig expressed in HEK293 cells (human embryonic kidney). Stably transfected cells were metabolically labelled with 32P-phosphoric acid. The results of immunoprecipitation and autoradiography demonstrate that both splicc variants of the 5-HT3A receptor subunit are phosphorylated in HEK293 cells. Site-specific mutagenesis revealed that phosphorylation occurs at serine 409, a potential target of protein kinase A. Thus the 5-HT3 receptor might be modulated by intracellular pathways, that allow variable 5-hydroxytryptamine action as responses to different extracellular stimuli.     

Isradipine interacts with the open state of the L-type calcium channel at high concentrations

Triple mutation of Tyr1485, Met1486 and Ile1493 in the IVS6 segment of alpha 1C-b subunit of the L-type calcium channel results in a loss of the high affinity inhibition by isradipine. The mutant channel (Ch30) yet exhibits a concentration-dependent inhibition by isradipine with a 110-fold lower affinity. The mechanisms underlying the remaining low affinity block were investigated. Isradipine accelerated the current decay in Ch30 but not in wild type channel in a concentration dependent manner. Dependence of the current amplitude inhibition on holding potential was parallel in Ch30 and in wild type channels, while the acceleration of current decay in Ch30 was independent of the membrane potential. The recovery from voltage-dependent inactivation was biphasic in both channels and was slowed down by isradipine in the wild type but not in the Ch30 channel. The change of the charge carrier (Ba2+ or Ca2+) and calcium chelator (EGTA or BAPTA) did not affect the acceleration of current decay indicating that isradipine did not interact with the Ca(2+)-inactivated state of the channel. These results demonstrate that the mutations of Ch30 affect selectively the high affinity inhibition of an inactivated channel and unmask a low affinity interaction of isradipine with an open state of the channel.