Interleukin 10 (IL-10) upregulates functional high affinity IL-2 receptors on normal and leukemic B lymphocytes.

Interleukin 10 (IL-10) has recently been shown to induce normal human B lymphocytes to proliferate and differentiate into immunoglobulin (Ig)-secreting cells. Herein, we show that IL-10 also promotes DNA synthesis and IgM production by anti-CD40 activated B cell chronic lymphocytic leukemia (B-CLL). Most strikingly, IL-2 and IL-10 were found to synergize to induce the proliferation and differentiation of B-CLL cells. This synergy between IL-2 and IL-10 was also observed with normal B cells which proliferated strongly and secreted large amounts of IgM, IgG, and IgA. The observed synergy is likely to be due to the IL-10-induced increase of high affinity IL-2 receptors on both normal and leukemic B cells. This increase of high affinity receptor is associated to an increase of Tac/CD25 expression that can be detected by flow cytometric analysis. Taken together, these results indicate that IL-10 permits anti-CD40 activated B cells to respond to IL-2 through an induction of high affinity IL-2 receptors. This effect of IL-10 may partly explain how T cells, which activate B cells in a CD40-dependent fashion, induce B cell proliferation and differentiation mostly through IL-2.

Localization of the human CD40 gene to chromosome 20, bands q12-q13.2.

CD40 is a surface glycoprotein, member of the nerve growth factor receptor family, which is expressed on B cells and plays an important role in their development, growth, and differentiation. Using chromosomal in situ hybridization, we localized the CD40 gene to the long arm of chromosome 20, bands q12-q13.2. This localization correlates well with the mapping of the murine CD40 gene to the distal region of chromosome 2, syntenic to the human 20q11-q13 region.

Up-regulation of [3H]-des-Arg10-kallidin binding to the bradykinin B1 receptor by interleukin-1 beta in isolated smooth muscle cells: correlation with B1 agonist-induced PGI2 production.

1. Binding of the specific bradykinin B1 receptor agonist, [3H]-des-Arg10-kallidin (-KD) was investigated in smooth muscle cells (SMC) isolated from rabbit mesenteric arteries (RMA). 2. [3H]-des-Arg10-KD specifically bound to interleukin-1 (IL-1)-treated RMA-SMC in a saturable fashion with an equilibrium dissociation constant (KD) of 0.3-0.5 nM. The number of binding sites per cell was 20,000-35,000. Kinins inhibited [3H]-des-Arg10-KD binding to RMA-SMC with an order of potency very similar to that observed in typical B1 specific bioassays: des-Arg9-bradykinin (BK) approximately KD >> BK. Furthermore, the B1 receptor antagonist [Leu8]des-Arg9-BK inhibited [3H]-des-Arg10-KD binding with an IC50 of 43 nM as expected for its effect at B1 receptors. The B2 receptor antagonists, NPC 567 and Hoe 140 only affected [3H]-des-Arg10-KD binding at very high concentrations (IC50 = 0.8 microM and IC50 > 10 microM, respectively). 3. Des-Arg9-BK (B1 agonist) and [Hyp3]Tyr(Me)8-BK (B2 agonist) did not induce prostacyclin (PGI2) production by RMA-SMC. Lipopolysaccharide (LPS) treatment of the cells did not affect the B1 agonist response whereas IL-1 beta treatment produced a 7 fold increase in des-Arg9-BK-stimulated PGI2 production. IL-1 beta also stimulated the response to B2 agonists. 4. Des-Arg9-BK-induced PGI2 secretion in IL-1-primed RMA-SMC was mediated by B1 receptors since it was inhibited by [Leu8]des-Arg9-BK (IC50 = 56-73 nM) but not by Hoe 140. High concentrations of NPC 567 (IC5o = 2.4 micro M) were required to inhibit PGI2 production induced by B1 agonists.5. IL- 1-treated RMA-SMC displayed a 5 fold increase in the number of B1 receptors without modification of the affinity constant, thus establishing a possible relationship between the receptor density and the IL-i-primed B1 response.6. LPS treatment of the cells induced a 4 fold increase in B1 receptor number without modifying PGI2 secretion. This observation suggests that IL-1 but not LPS, in addition to increase in the number of receptors, signals the cell to permit the coupling of B1 receptors to the PLA2/cyclo-oxygenase pathway.

[125I]-S36057: a new and highly potent radioligand for the melanin-concentrating hormone receptor.

Shortened, more stable and weakly hydrophobic analogues of melanin-concentrating hormone (MCH) were searched as candidates for radioiodination. Starting from the dodecapeptide MCH(6 - 17), we found that: (1) substitution of Tyr(13) by a Phe residue; (2) addition of a 3-iodo-Tyr residue at the N-terminus; and (3) addition of a hydrophilic spacer 8-amino-3,6-dioxyoctanoyl between the 3-iodo-Tyr and MCH(6 - 17) (compound S36057), led to an agonist more potent than MCH itself in stimulating [35S]-GTPgammaS binding at membranes from HEK293 cells stably expressing the human MCH receptor. Specific binding of [125I]-S36057 was found in HEK293 and CHO cell lines stably expressing the human MCH receptor. This radioligand recognized a similar number of binding sites (ca. 800 fmol mg(-1)) than [125I]-[3-iodo Tyr(13)]-MCH. However, the K(D) for [125I]-S36057 obtained from saturation studies (0.037 nM) or from binding kinetics (0.046 nM) was at least 10 fold higher to that of [125I]-[3-iodo Tyr(13)]-MCH (0.46 nM). Affinities determined for a series of MCH analogues were similar with both radioligands, S36057 being the most potent compound tested (K(i)=0.053 nM). Finally, [125I]-S36057 also potently labelled the MCH receptor in membranes from whole rat brain (K(D) 0.044 nM, B(max)=11 fmol mg(-1)). In conclusion, [125I]-S36057 is a more potent and more stable radioligand than [125I]-[3-iodo Tyr(13)]-MCH that will represent a reliable tool for binding assays in the search of novel MCH ligands. It should also provide great help for autoradiographic studies of the MCH receptor distribution in the central nervous system.

NPY receptor subtype in the rabbit isolated ileum.

1. The purpose of this work was to verify the hypothesis that the rabbit ileum is a selective preparation for the NPY Y5 receptor by using new selective antagonists recently synthesized. Spontaneous contractions of the rabbit isolated ileum were recorded and binding experiments were performed in cells expressing the human NPY Y1, Y2, Y4 or Y5 receptor subtype. 2. NPY analogues produced a concentration-dependent transient inhibition of the spontaneous contractions of the rabbit ileum with the following order of potency hPP > rPP > PYY > or = [Leu31,-Pro34]-NPY > NPY >> NPY13-36. Pre-exposure to rPP, PYY, [Leu31,Pro34]-NPY or NPY (but not NPY13-36) inhibited the effect of subsequent administration of hPP suggesting cross-desensitization of the preparation. The apparent affinity of the various agonists studied was correlated to the affinity reported for the human Y4 receptor subtype (and to a lesser extent for the rat Y4 subtype) but not to the affinity for the Y5 receptor subtype. 3. BIBO 3304, a selective NPY Y1 receptor antagonist, and CGP 71683A, a selective NPY Y5 receptor antagonist, did not affect the response to hPP. JCF 109, another NPY Y5 receptor antagonist, produced an inhibition of the response to hPP but only at the highest dose tested (10 microM) which also, by itself, produced intrinsic inhibitory effects. 4. 1229U91, a non-selective ligand for Y1, Y2, Y4 and Y5 receptors with high affinity toward the Y1 and Y4 receptor subtypes, produced a concentration-dependent transient inhibition of the spontaneous contractions of the rabbit ileum and a dose-dependent inhibition of the response to hPP (apparent pKB: 7.2). 5. These results suggest that in the rabbit ileum, the NPY receptor involved in the inhibition of the spontaneous contractile activity is a NPY Y4 receptor subtype.

NPY receptor subtypes involved in the contraction of the proximal colon of the rat.

Experiments were designed to determine the receptor subtype(s) involved in the contraction of the rat proximal colon to NPY. In this tissue, mRNA of Y2 and Y4 NPY receptor subtypes were highly expressed, whereas Y5 mRNA levels were very low and Y1 mRNA levels were intermediate. NPY analogues induced contractions with the following order of potency: rPP > hPP = PYY = NPY = [Leu31,Pro34]NPY > NPY(2-36) = [D-Trp32]NPY > NPY(33-36). Responses to NPY, PYY and NPY(13-36) were not or partially affected by tetrodotoxin, in contrast to the responses to [Leu31,Pro34]NPY, rPP, hPP and [D-Trp32]NPY which were fully blocked. Atropine did not inhibit the contractions to NPY, PYY and [Leu31,Pro34]NPY but significantly affected those to NPY(13-36), [D-Trp32]NPY, rPP and hPP. The specific Y1 receptor antagonist BIBP 3226 was ineffective but JCF 104 and JCF 105 (two compounds with preferential affinity toward the hY5 receptor versus the hY1 or hY2 receptor) abolished the contractions provoked by the NPY analogues. These results suggest that NPY activates three receptor subtypes, a Y2 subtype possibly by a direct action on the smooth muscle cells, as well as a Y4 and a Y5 (or 'Y5-like') subtype which, respectively, release acetylcholine and an unknown neurotransmitter.

Receptors for diphenylbutylpiperidine neuroleptics in brain, cardiac, and smooth muscle membranes. Relationship with receptors for 1,4-dihydropyridines and phenylalkylamines and with Ca2+ channel blockade.

Neuroleptic molecules of the diphenylbutylpiperidine series (DPBP), such as fluspirilene, penfluridol, pimozide and clopimozide, antagonize binding of (-)[3H]desmethoxyverapamil ((-)[3H]D888) and (+)[3H]PN 200-110 to rabbit brain, heart and smooth muscle membranes. The diphenylbutylpiperidine binding site in all these tissues is distinct but is allosterically related to the 1,4-dihydropyridine binding site and to the phenylalkylamine binding site. High and low affinity binding sites for (-)D888 were identified. (-)[3H]D888 binding at both types of sites was inhibited following the saturation of a single type of diphenylbutylpiperidine binding site. Half-maximal inhibition (K0.5) of brain, heart and smooth muscle membranes binding by different diphenylbutylpiperidines was in the range of 10-100 nM. These K0.5 values were one to two orders of magnitude higher than those found for the high affinity diphenylbutylpiperidine receptor in skeletal muscle membranes. The K0.5 values found in binding experiments in smooth muscle were similar to the (IC50) values for half-maximal inhibition by diphenylbutylpiperidine of voltage-dependent 45Ca2+ influx through the slow Ca2+ channel.

Binding of prostaglandins to human PPARgamma: tool assessment and new natural ligands.

The peroxisome proliferator-activated receptors (PPAR) form a family of nuclear receptors with a wide variety of biological roles from adipogenesis to carcinogenesis. More ligands (agonist and antagonist) are needed to explore the multiple functions of PPAR, particularly PPARgamma. In order to complete such ligand screening, a binding test should be assessed versus the classical transactivation reporter gene assay. In the present work, the full-length human PPARgamma protein as well as its ligand binding domain portion were expressed in Escherichia coli. Bacterial membrane preparations expressing those constructs were characterized using a classical binding competition assay [3H]rosiglitazone as the radioligand. When the receptor preparations were soluble, binding had to be measured with a new alternative method. The systems were assessed using a series of reference PPAR (alpha, beta and gamma) ligands. The full-length human PPARgamma fused to glutathione-S-transferase, expressed in E. coli and tested as a bacterial membrane-bound protein led to the most accurate results when compared to the literature. Furthermore, in an attempt to complete the panel of natural PPARgamma ligands, 29 commercially available prostaglandins were screened in the binding assay. Prostaglandins H(1) and H(2) were found to be modest ligands, however as potent as 15Delta(12-14 )prostaglandin J(2). These results were confirmed in the classical transactivation assay. The fact that these three prostaglandins were equally potent, suggests new pathways of PPARgamma-linked gene activation.

Human interleukin 4.

Human IL-4 is a mature 129 AA glycoprotein, mostly secreted by activated T cells. It is a pleiotropic cytokine which acts on T and B lymphocytes, monocytes, polymorphonuclear cells, fibroblasts and endothelial cells. In addition it acts at various stages of cell differentiation and its effects are also dependent on the cytokine environment. In particular, IL-4 blocks some of the effects of IL-2 whereas interferon gamma blocks some of the effects of IL-4. In vitro and in vivo experiments in mouse and in vitro experiments in man have shown that IL-4 plays a crucial role in the induction of IgE production, whereas interferons counteract this effect. Human IL-4 binds to a high affinity receptor which is composed at least of one 130-kDa glycoprotein of 800 AA, a member of the newly described hematopoietin receptor superfamily. IL-4 may prove useful as an antitumoral and antiinflammatory agent.

Receptors for interleukin 4, interleukin 5 and interleukin 6.

cDNA encoding IL-4, IL-5 and IL-6 have been isolated and the availability of recombinant proteins allowed the demonstration of the pleiotropic effects of these cytokines. IL-4, IL-5 and IL-6 bind to high affinity receptors (Kd = 1-6 x 10(-10) M) expressed in low numbers on cells. The IL-4R is expressed on virtually every cell type whereas the IL-6R and IL-5R are less widely distributed. Low affinity IL-6 and IL-5 binding sites have also been detected but the biological activity of these different cytokines appears to correlate with the presence of high affinity binding sites. Biochemical characterization of the IL-4R, IL-5R and IL-6R by crosslinking of the labelled ligands, immunoprecipitation or purification experiments suggests that these receptors are composed of several proteins. IL-4 binds to three species (130, 75 and 65 kDa) and cDNAs encoding the 130 kDa protein have been isolated. IL-6 binds to a 80 kDa protein for which a cDNA has been isolated. Furthermore the IL-6 80 kDa protein complex binds to a 130 kDa transducer for which a cDNA was recently isolated. The IL-5R consists of three proteins of 105, 75 and 60 kDa but corresponding cDNAs have not yet been isolated. The IL-4, IL-6R 80 kDA protein and the IL-6 transducer protein display significant homology in their extracellular domains. These proteins belong to the new cytokine receptor family (hematopoietin receptors superfamily) which also includes the G-CSFR, GM-CSFR, IL-3, IL-7R and IL-2R 70 kDa chain.

Aging and obesity induce distinct gene expression adaptation in the liver of C57BL/6J mice.

BACKGROUND: Aging and obesity induce complex transcriptomic changes in the liver, promoting the development of insulin resistance and type 2 diabetes. In spite of an increasing amount of studies on the role of aging and nutrient excess in metabolic disorders, the specific molecular events leading to insulin resistance are still poorly understood. METHODS: This study presents a comparative analysis of hepatic gene expression profiles between young adult C57BL/6J mice fed with a low- or a high-fat diet for 1 and 12 months. We evaluated the expression of a defined set of genes implicated in glucose and lipid metabolism as well as key nuclear receptors and their target genes, IGF1 signaling and clock genes. RESULTS: Aging and short-term high-fat consumption induced insulin resistance, albeit through two distinct processes. Hepatic gene expression changes were more pronounced in the context of aging. We further analyzed expression profiles together with plasma parameters by principal component analysis with regard to diet condition. CONCLUSIONS: Our results suggest that in the liver of C57BL/6J mice, the molecular mechanisms underlying high-fat feeding or aging which mediated insulin resistance were not identical.

Comment on "Obestatin, a peptide encoded by the ghrelin gene, opposes ghrelin's effects on food intake".

Zhang et al. (Research Articles, 11 November 2005, p. 996) reported that obestatin, a peptide derived from the ghrelin precursor, activated the orphan G protein-coupled receptor GPR39. However, we found that I125-obestatin does not bind GPR39 and observed no effects of obestatin on GPR39-transfected cells in various functional assays (cyclic adenosine monophosphate production, calcium mobilization, and GPR39 internalization). Our results indicate that obestatin is not the cognate ligand for GPR39.

Monoclonal antibodies that coimmunoprecipitate the 1,4-dihydropyridine and phenylalkylamine receptors and reveal the Ca2+ channel structure.

Monoclonal hybridoma cell lines secreting antibodies against the (+)-PN 200-110 and the (-)-demethoxyverapamil binding components of the voltage-dependent calcium channel from rabbit transverse-tubule membranes have been isolated. The specificity of these monoclonal antibodies was established by their ability to coimmunoprecipitate (+)-[3H]PN 200-110 and (-)-[3H]demethoxyverapamil receptors. Monoclonal antibodies described in this work cross-reacted with rat, mouse, chicken, and frog skeletal muscle Ca2+ channels but not with crayfish muscle Ca2+ channels. Cross-reactivity was also detected with membranes prepared from rabbit heart, brain, and intestinal smooth muscle. These antibodies were used in immunoprecipitation experiments with 125I-labeled detergent [3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate (CHAPS) and digitonin] solubilized membranes. They revealed a single immunoprecipitating component of molecular weight (Mr) 170,000 in nonreducing conditions. After disulfide bridge reduction the CHAPS-solubilized (+)-PN 200-110-(-)-demethoxyverapamil binding component gave rise to a large peptide of Mr 140,000 and to smaller polypeptides of Mr 30,000 and 26,000 whereas the digitonin-solubilized receptor appeared with subunits at Mr 170,000, 140,000, 30,000, and 26,000. All these results taken together are interpreted as showing that both the 1,4-dihydropyridine and the phenylalkylamine receptors are part of a single polypeptide chain of Mr 170,000.

A recombinant extracellular domain of the human interleukin 4 receptor inhibits the biological effects of interleukin 4 on T and B lymphocytes.

Human interleukin 4 (IL4) acts on various hematopoietic cell types through interaction with a specific cell surface receptor (IL4R), whose cDNA has been cloned. We have produced a cDNA encoding a soluble form of the extracellular domain of the human IL 4R (sIL4R) and describe here the capacity of sIL4R to antagonize the in vitro activities of IL4 on normal B and T lymphocytes. sIL4R inhibited IL4-induced proliferation of both phytohemagglutinin-preactivated peripheral blood mononuclear cells (PBMC) and anti-IgM co-stimulated tonsil B cells with similar efficiency. This inhibitory activity was specific since sIL4R did not affect IL2-dependent proliferation of these cells. sIL4R also blocked IL4-dependent induction of the low-affinity receptor for IgE on B cells and inhibited IgE production by IL4-activated PBMC. Thus, in contrast to the IL6R extracellular domain which stimulates IL6 biological activity, the IL4R extracellular domain is a powerful antagonist of its specific ligand.

Characterization of the Ca2+ coordination site regulating binding of Ca2+ channel inhibitors d-cis-diltiazem, (+/-)bepridil and (-)desmethoxyverapamil to their receptor site in skeletal muscle transverse tubule membranes.

Ca2+ inhibits (-)[3H]desmethoxyverapamil, d-cis-[3H]diltiazem and (+/-)[3H]bepridil binding to skeletal muscle transverse-tubule membranes with a half-maximum inhibition constant, K0.5 = 5 +/- 1 microM. This value is close to that of the high affinity Ca2+ binding site which controls the ionic selectivity of the Ca2+ channel found in electrophysiological experiments suggesting that the Ca2+ coordination site which regulates the ionic selectivity is also the one which alters binding of the Ca2+ channel inhibitors investigated here. Ca2+ and (-)D888 bind to distinct sites. Occupation of the Ca2+ coordination site decreases the affinity of (-)D888 for its receptor by a factor of 5. Other divalent cations have the same type of inhibition behavior with the rank order of potency Ca2+ (K0.5 = 5 microM) greater than Sr2+ (K0.5 = 25 microM) greater than Ba2+ (K0.5 = 50 microM) greater than Mg2+ (K0.5 = 170 microM).

Properties of receptors for the Ca2+-channel blocker verapamil in transverse-tubule membranes of skeletal muscle. Stereospecificity, effect of Ca2+ and other inorganic cations, evidence for two categories of sites and effect of nucleoside triphosphates.

The verapamil receptor associated with the voltage-dependent calcium channel of rabbit skeletal muscle transverse tubule membranes has the following properties. (i) This receptor is stereospecific and discriminates between the different stereoisomers of verapamil, gallopamil and diltiazem. (ii) Inorganic divalent cations inhibit the binding of [3H]verapamil to its receptor in an apparently non-competitive fashion. The rank order of potency is: Ca2+ = Mn2+ greater than Mg2+ greater than Sr2+ greater than Ba2+ much greater than Co2+ much greater than Ni2+. Ca2+ and Mn2+ have inhibition constants of 0.3 mM. Binding of [3H]verapamil is also sensitive to monovalent cations such as Cs+, K+, Li+ and Na+. The most active of these cations (Cs+ and K+) have inhibition constants in the range of 30 mM. (iii) Binding of [3H]verapamil is pH-dependent and reveals the presence on the verapamil receptor of an essential ionizable group with a pKa of 6.5. (iv) A low-affinity binding site for verapamil and for some other Ca2+ channel blockers is detected by studies of dissociation kinetics of the [3H]verapamil receptor in the presence of high concentrations of verapamil, gallopamil, bepridil and diltiazem. (v) GTP and nucleoside analogs change the properties of [3H]verapamil binding to verapamil binding sites. High-affinity binding sites seem to be transferred into low-affinity sites. Dissociation constants obtained from inhibition studies of [3H]verapamil binding are in the range of 0.1-0.3 mM for GTP, ATP and Gpp(NH)p.

[3H] verapamil binding sites in skeletal muscle transverse tubule membranes.

[3H]verapamil binding to muscle tubule membrane has the following properties. KD = 27 +/- 5 nM and maximum binding capacity Bmax = 50 +/- 5 pmol/mg of protein. A 1 = 1 stoichiometry of binding was found for the ratio of [3H]verapamil versus [3H] nitrendipine binding sites. The dissociation constant found at equilibrium is near that determined from the ratio of the rate constants for association (k1) and dissociation (k-1). Antiarrhythmic drugs like D600, diltiazem and bepridil are competitive inhibitors of [3H] verapamil binding with KD values between 40 and 200 nM. Dihydropyridine analogs are apparent non competitive inhibitors of [3H]verapamil binding with half-maximum inhibition values (K0.5) between 1 and 5 nM.

Neuroleptics of the diphenylbutylpiperidine series are potent calcium channel inhibitors.

[3H]Fluspirilene, a neuroleptic molecule of the diphenylbutylpiperidine series, binds to skeletal muscle transverse tubule membranes with a high affinity corresponding to a Kd of 0.11 +/- 0.04 nM, A 1:1 stoichiometry was found between [3H]fluspirilene binding and the binding of (-)-[3H]desmethoxyverapamil [(-)[3H]D888], one of the most potent Ca2+ channel inhibitors. Ca2+ channel inhibitors such as D888, verapamil, gallopamil, bepridil, or diltiazem antagonize [3H]fluspirilene binding besides antagonizing (-)[3H]-D888 binding. Neuroleptics, especially those of the diphenylbutylpiperidine family, also antagonize both (-)[3H]D888 binding and [3H]fluspirilene binding. There is an excellent correlation between affinities found from [3H]fluspirilene binding experiments and those found from (-)[3H]D888 binding experiments. Analysis of the properties of these cross-inhibitions indicates that [3H]fluspirilene binds to a site that is not identical to that for phenylalkylamine derivatives (gallopamil, verapamil, diltiazem, and bepridil). Voltage-clamp experiments have shown that fluspirilene is an efficient inhibitor of the voltage dependent Ca2+ channel, achieving a half-maximal effect near 0.1-0.2 nM and nearly complete blockade at 1 nM. Fluspirilene blockade has little voltage dependence.

Dihydropyridine-sensitive Ca2+ channels in mammalian skeletal muscle cells in culture: electrophysiological properties and interactions with Ca2+ channel activator (Bay K8644) and inhibitor (PN 200-110).

The whole-cell patch-clamp technique has been used to analyze the properties of the dihydropyridine-sensitive Ca2+ channel in rat skeletal muscle cells (myoballs) in culture. The potential dependence of Ca2+-channel activation is similar to that observed in cardiac cells. However, the skeletal muscle Ca2+ channel is activated more slowly (by a factor of about 10). The voltage dependence of Ca2+-channel inactivation indicates a half-maximal inactivation (Vh0.5) at -72 mV as compared to Vh0.5 = -35 mV for cardiac cells. Blockade of the skeletal muscle Ca2+ channel by the dihydropyridine (+)-PN 200-110 is voltage dependent, with a half-maximal effect (K0.5) of 13 nM for an application of the drug to the myoball membrane held at -90 mV and of 0.15 nM for an application at a potential of -65 mV. The 100-fold difference in apparent affinity is interpreted as a preferential association of PN 200-110 with the inactivated form of the Ca2+ channel. The K0.5 value found from electrophysiological experiments for the binding to the inactivated state (K0.5 = 0.15 nM) is nearly identical to the equilibrium dissociation constant found from binding experiments with (+)-[3H]PN 200-110 using transverse-tubular membranes (Kd = 0.22 nM). The dihydropyridine activator Bay K8644 acts by increasing Ca2+ current amplitude and by slowing down deactivation.