CD19 and CD22 expression reciprocally regulates tyrosine phosphorylation of Vav protein during B lymphocyte���signaling

B cell development and humoral immune responses are controlled by signaling thresholds established through the B lymphocyte antigen receptor (BCR) complex. BCR signaling thresholds are differentially regulated by the CD22 and CD19 cell surface receptors in vivo. B cells from CD22-deficient mice exhibit characteristics of chronic stimulation and are hyper-responsive to BCR crosslinking with augmented intracellular Ca2+ responses. By contrast, B cells from CD19-deficient mice are hypo-responsive to transmembrane signals. To identify signaling molecules involved in the positive and negative regulation of signaling thresholds, the signal transduction pathways activated after BCR crosslinking were examined in CD22- and CD19-deficient B cells. These comparisons revealed that tyrosine phosphorylation of Vav protein was uniquely augmented after BCR or CD19 crosslinking in CD22-deficient B cells, yet was modest and transient after BCR crosslinking in CD19-deficient B cells. Ligation of CD19 and CD22 in vivo is likely to positively and negatively regulate BCR signaling, respectively, because CD19 crosslinking was more efficient than BCR crosslinking at inducing Vav phosphorylation. However, simultaneous crosslinking of CD19 with the BCR resulted in a substantial decrease in Vav phosphorylation when CD22 was expressed. Thus, the differential regulation of Vav tyrosine phosphorylation by CD19 and CD22 may provide a molecular mechanism for adjusting BCR signaling thresholds.

A transient expansion of the native state precedes aggregation of recombinant human interferon-��

Aggregation of proteins, even under conditions favoring the native state, is a ubiquitous problem in biotechnology and biomedical engineering. Providing a mechanistic basis for the pathways that lead to aggregation should allow development of rational approaches for its prevention. We have chosen recombinant human interferon-�� (rhIFN-��) as a model protein for a mechanistic study of aggregation. In the presence of 0.9 M guanidinium hydrochloride, rhIFN-�� aggregates with first order kinetics, a process that is inhibited by addition of sucrose. We describe a pathway that accounts for both the observed first-order aggregation of rhIFN-�� and the effect of sucrose. In this pathway, aggregation proceeds through a transient expansion of the native state. Sucrose shifts the equilibrium within the ensemble of rhIFN-�� native conformations to favor the most compact native species over more expanded ones, thus stabilizing rhIFN-�� against aggregation. This phenomenon is attributed to the preferential exclusion of sucrose from the protein surface. In addition, kinetic analysis combined with solution thermodynamics shows that only a small (9%) expansion surface area is needed to form the transient native state that precedes aggregation. The approaches used here link thermodynamics and aggregation kinetics to provide a powerful tool for understanding both the pathway of protein aggregation and the rational use of excipients to inhibit the process.

Evidence for a voltage-dependent enhancement of neurotransmitter release mediated via the synaptic protein interaction site of N-type Ca2+ channels

Secretion of neurotransmitters is initiated by voltage-gated calcium influx through presynaptic, voltage-gated N-type calcium channels. These channels interact with the SNARE proteins, which are core components of the exocytosis process, via the synaptic protein interaction (synprint) site in the intracellular loop connecting domains II and III of their ��1B subunit. Interruption of this interaction by competing synprint peptides inhibits fast, synchronous transmitter release. Here we identify a voltage-dependent, but calcium-independent, enhancement of transmitter release that is elicited by trains of action potentials in the presence of a hyperosmotic extracellular concentration of sucrose. This enhancement of transmitter release requires interaction of SNARE proteins with the synprint site. Our results provide evidence for a voltage-dependent signal that is transmitted by protein���protein interactions from the N-type calcium channel to the SNARE proteins and enhances neurotransmitter release by altering SNARE protein function.

Soluble complexes of regulated upon activation, normal T cells expressed and secreted (RANTES) and glycosaminoglycans suppress HIV-1 infection but do not induce Ca2+ signaling

Chemokines comprise a family of low-molecular-weight proteins that elicit a variety of biological responses including chemotaxis, intracellular Ca2+ mobilization, and activation of tyrosine kinase signaling cascades. A subset of chemokines, including regulated upon activation, normal T cell expressed and secreted (RANTES), macrophage inflammatory protein-1�� (MIP-1��), and MIP-1��, also suppress infection by HIV-1. All of these activities are contingent on interactions between chemokines and cognate seven-transmembrane spanning, G protein-coupled receptors. However, these activities are strongly inhibited by glycanase treatment of receptor-expressing cells, indicating an additional dependence on surface glycosaminoglycans (GAG). To further investigate this dependence, we examined whether soluble GAG could reconstitute the biological activities of RANTES on glycanase-treated cells. Complexes formed between RANTES and a number of soluble GAG failed to induce intracellular Ca2+ mobilization on either glycanase-treated or untreated peripheral blood mononuclear cells and were unable to stimulate chemotaxis. In contrast, the same complexes demonstrated suppressive activity against macrophage tropic HIV-1. Complexes composed of 125I-labeled RANTES demonstrated saturable binding to glycanase-treated peripheral blood mononuclear cells, and such binding could be reversed partially by an anti-CCR5 antibody. These results suggest that soluble chemokine���GAG complexes represent seven-transmembrane ligands that do not activate receptors yet suppress HIV infection. Such complexes may be considered as therapeutic formulations for the treatment of HIV-1 infection.

Termination of Ca2+ release by a local inactivation of ryanodine receptors in cardiac myocytes

In heart, a robust regulatory mechanism is required to counteract the regenerative Ca2+-induced Ca2+ release from the sarcoplasmic reticulum. Several mechanisms, including inactivation, adaptation, and stochastic closing of ryanodine receptors (RyRs) have been proposed, but no conclusive evidence has yet been provided. We probed the termination process of Ca2+ release by using a technique of imaging local Ca2+ release, or ���Ca2+ spikes���, at subcellular sites; and we tracked the kinetics of Ca2+ release triggered by L-type Ca2+ channels. At 0 mV, Ca2+ release occurred and terminated within 40 ms after the onset of clamp pulses (0 mV). Increasing the open-duration and promoting the reopenings of Ca2+ channels with the Ca2+ channel agonist, FPL64176, did not prolong or trigger secondary Ca2+ spikes, even though two-thirds of the sarcoplasmic reticulum Ca2+ remained available for release. Latency of Ca2+ spikes coincided with the first openings but not with the reopenings of L-type Ca2+ channels. After an initial maximal release, even a multi-fold increase in unitary Ca2+ current induced by a hyperpolarization to ���120 mV failed to trigger additional release, indicating absolute refractoriness of RyRs. When the release was submaximal (e.g., at +30 mV), tail currents did activate additional Ca2+ spikes; confocal images revealed that they originated from RyRs unfired during depolarization. These results indicate that Ca2+ release is terminated primarily by a highly localized, use-dependent inactivation of RyRs but not by the stochastic closing or adaptation of RyRs in intact ventricular myocytes.

Interaction of the synprint site of N-type Ca2+ channels with the C2B domain of synaptotagmin���I

N-type Ca2+ channels mediate Ca2+ influx, which initiates fast exocytosis of neurotransmitters at synapses, and they interact directly with the SNARE proteins syntaxin and SNAP-25 (synaptosome-associated protein of 25 kDa) through a synaptic protein interaction (synprint) site in the intracellular loop connecting domains II and III of their ��1B subunits. Introduction of peptides containing the synprint site into presynaptic neurons reversibly inhibits synaptic transmission, confirming the importance of interactions with this site in synaptic transmission. Here we report a direct interaction of the synprint peptide from N-type Ca2+ channels with synaptotagmin I, an important Ca2+ sensor for exocytosis, as measured by an affinity-chromatography binding assay and a solid-phase immunoassay. This interaction is mediated by the second C2 domain (C2B) of synaptotagmin I, but is not regulated by Ca2+. Using both immobilized recombinant proteins and native presynaptic membrane proteins, we found that the synprint peptide and synaptotagmin competitively interact with syntaxin. This interaction is Ca2+-dependent because of the Ca2+ dependence of the interactions between syntaxin and these two proteins. These results provide a molecular basis for a physical link between Ca2+ channels and synaptotagmin, and suggest that N-type Ca2+ channels may undergo a complex series of Ca2+-dependent interactions with multiple presynaptic proteins during neurotransmission.

Voltage-controlled gating in a large conductance Ca2+-sensitive K+channel���(hslo)

Large conductance calcium- and voltage-sensitive K+ (MaxiK) channels share properties of voltage- and ligand-gated ion channels. In voltage-gated channels, membrane depolarization promotes the displacement of charged residues contained in the voltage sensor (S4 region) inducing gating currents and pore opening. In MaxiK channels, both voltage and micromolar internal Ca2+ favor pore opening. We demonstrate the presence of voltage sensor rearrangements with voltage (gating currents) whose movement and associated pore opening is triggered by voltage and facilitated by micromolar internal Ca2+ concentration. In contrast to other voltage-gated channels, in MaxiK channels there is charge movement at potentials where the pore is open and the total charge per channel is 4���5 elementary charges.

Ablation of P/Q-type Ca2+ channel currents, altered synaptic transmission, and progressive ataxia in mice lacking the ��1A-subunit

The Ca2+ channel ��1A-subunit is a voltage-gated, pore-forming membrane protein positioned at the intersection of two important lines of research: one exploring the diversity of Ca2+ channels and their physiological roles, and the other pursuing mechanisms of ataxia, dystonia, epilepsy, and migraine. ��1A-Subunits are thought to support both P- and Q-type Ca2+ channel currents, but the most direct test, a null mutant, has not been described, nor is it known which changes in neurotransmission might arise from elimination of the predominant Ca2+ delivery system at excitatory nerve terminals. We generated ��1A-deficient mice (��1A���/���) and found that they developed a rapidly progressive neurological deficit with specific characteristics of ataxia and dystonia before dying ���3���4 weeks after birth. P-type currents in Purkinje neurons and P- and Q-type currents in cerebellar granule cells were eliminated completely whereas other Ca2+ channel types, including those involved in triggering transmitter release, also underwent concomitant changes in density. Synaptic transmission in ��1A���/��� hippocampal slices persisted despite the lack of P/Q-type channels but showed enhanced reliance on N-type and R-type Ca2+ entry. The ��1A���/��� mice provide a starting point for unraveling neuropathological mechanisms of human diseases generated by mutations in ��1A.

Transient cardiac expression of constitutively active G��q leads to hypertrophy and dilated cardiomyopathy by calcineurin-dependent and independent pathways

Cardiac hypertrophy and dilatation can result from stimulation of signal transduction pathways mediated by heterotrimeric G proteins, especially Gq, whose �� subunit activates phospholipase C�� (PLC��). We now report that transient, modest expression of a hemagglutinin (HA) epitope-tagged, constitutively active mutant of the Gq �� subunit (HA��*q) in hearts of transgenic mice is sufficient to induce cardiac hypertrophy and dilatation that continue to progress after the initiating stimulus becomes undetectable. At 2 weeks, HA��*q protein is expressed at less than 50% of endogenous ��q/11, and the transgenic hearts are essentially normal morphologically. Although HA��*q protein declines at 4 weeks and is undetectable by 10 weeks, the animals develop cardiac hypertrophy and dilatation and die between 8 and 30 weeks in heart failure. As the pathology develops, endogenous ��q/11 rises (2.9-fold in atria; 1.8-fold in ventricles). At 2 weeks, basal PLC activity is increased 9- to 10-fold in atria but not ventricles. By 10 weeks, it is elevated in both, presumably because of the rise in endogenous ��q/11. We conclude that the pathological changes initiated by early, transient HA��*q expression are maintained in part by compensatory changes in signal transduction and other pathways. Cyclosporin A (CsA) prevents hypertrophy caused by activation of calcineurin [Molkentin, J. D., Lu, J.-R., Antos, C. L., Markham, B., Richardson, J., Robbins, J., Grant, S. R. & Olson, E. N. (1998) Cell 93, 215���228]. Because HA��*q acts upstream of calcineurin, we hypothesized that HA��*q might initiate additional pathways leading to hypertrophy and dilatation. Treating HA��*q mice with CsA diminished some, but not all, aspects of the hypertrophic phenotype, suggesting that multiple pathways are involved.

Ca2+-dependent and -independent interactions of the isoforms of the ��1A subunit of brain Ca2+ channels with presynaptic SNARE���proteins

Fast neurotransmission requires that docked synaptic vesicles be located near the presynaptic N-type or P/Q-type calcium channels. Specific protein���protein interactions between a synaptic protein interaction (synprint) site on N-type and P/Q-type channels and the presynaptic SNARE proteins syntaxin, SNAP-25, and synaptotagmin are required for efficient, synchronous neurotransmitter release. Interaction of the synprint site of N-type calcium channels with syntaxin and SNAP-25 has a biphasic calcium dependence with maximal binding at 10���20 ��M. We report here that the synprint sites of the BI and rbA isoforms of the ��1A subunit of P/Q-type Ca2+ channels have different patterns of interactions with synaptic proteins. The BI isoform of ��1A specifically interacts with syntaxin, SNAP-25, and synaptotagmin independent of Ca2+ concentration and binds with high affinity to the C2B domain of synaptotagmin but not the C2A domain. The rbA isoform of ��1A interacts specifically with synaptotagmin and SNAP-25 but not with syntaxin. Binding of synaptotagmin to the rbA isoform of ��1A is Ca2+-dependent, with maximum affinity at 10���20 ��M Ca2+. Although the rbA isoform of ��1A binds well to both the C2A and C2B domains of synaptotagmin, only the interaction with the C2A domain is Ca2+-dependent. These differential, Ca2+-dependent interactions of Ca2+ channel synprint sites with SNARE proteins may modulate the efficiency of transmitter release triggered by Ca2+ influx through these channels.

Transient ���deafness��� accompanies auditory development during metamorphosis from tadpole to���frog

During metamorphosis, ranid frogs shift from a purely aquatic to a partly terrestrial lifestyle. The central auditory system undergoes functional and neuroanatomical reorganization in parallel with the development of new sound conduction pathways adapted for the detection of airborne sounds. Neural responses to sounds can be recorded from the auditory midbrain of tadpoles shortly after hatching, with higher rates of synchronous neural activity and lower sharpness of tuning than observed in postmetamorphic animals. Shortly before the onset of metamorphic climax, there is a brief ���deaf��� period during which no auditory activity can be evoked from the midbrain, and a loss of connectivity is observed between medullary and midbrain auditory nuclei. During the final stages of metamorphic development, auditory function and neural connectivity are restored. The acoustic communication system of the adult frog emerges from these periods of anatomical and physiological plasticity during metamorphosis.

Predominance of the ��1D subunit in L-type voltage-gated Ca2+ channels of hair cells in the chicken���s���cochlea

The voltage-gated Ca2+ channels that effect tonic release of neurotransmitter from hair cells have unusual pharmacological properties: unlike most presynaptic Ca2+ channels, they are sensitive to dihydropyridines and therefore are L-type. To characterize these Ca2+ channels, we investigated the expression of L-type ��1 subunits in hair cells of the chicken���s cochlea. In PCRs with five different pairs of degenerate primers, we always obtained ��1D products, but only once an ��1C product and never an ��1S product. A full-length ��1D mRNA sequence was assembled from overlapping PCR products; the predicted amino acid sequence of the ��1D subunit was about 90% identical to those of the mammalian ��1D subunits. In situ hybridization confirmed that the ��1D mRNA is present in hair cells. By using a quantitative PCR assay, we determined that the ��1D mRNA is 100���500 times more abundant than the ��1C mRNA. We conclude that most, if not all, voltage-gated Ca2+ channels in hair cells contain an ��1D subunit. Furthermore, we propose that the ��1D subunit plays a hitherto undocumented role at tonic synapses.

Hair cell-specific splicing of mRNA for the ��1D subunit of voltage-gated Ca2+ channels in the chicken���s���cochlea

The L-type voltage-gated Ca2+ channels that control tonic release of neurotransmitter from hair cells exhibit unusual electrophysiological properties: a low activation threshold, rapid activation and deactivation, and a lack of Ca2+-dependent inactivation. We have inquired whether these characteristics result from cell-specific splicing of the mRNA for the L-type ��1D subunit that predominates in hair cells of the chicken���s cochlea. The ��1D subunit in hair cells contains three uncommon exons: one encoding a 26-aa insert in the cytoplasmic loop between repeats I and II, an alternative exon for transmembrane segment IIIS2, and a heretofore undescribed exon specifying a 10-aa insert in the cytoplasmic loop between segments IVS2 and IVS3. We propose that the alternative splicing of the ��1D mRNA contributes to the unusual behavior of the hair cell���s voltage-gated Ca2+ channels.

Construction of a high-affinity receptor site for dihydropyridine agonists and antagonists by single amino acid substitutions in a non-l-type Ca2+���channel

The activity of l-type Ca2+ channels is increased by dihydropyridine (DHP) agonists and inhibited by DHP antagonists, which are widely used in the therapy of cardiovascular disease. These drugs bind to the pore-forming ��1 subunits of l-type Ca2+ channels. To define the minimal requirements for DHP binding and action, we constructed a high-affinity DHP receptor site by substituting a total of nine amino acid residues from DHP-sensitive l-type ��1 subunits into the S5 and S6 transmembrane segments of domain III and the S6 transmembrane segment of domain IV of the DHP-insensitive P/Q-type ��1A subunit. The resulting chimeric ��1A/DHPS subunit bound DHP antagonists with high affinity in radioligand binding assays and was inhibited by DHP antagonists with high affinity in voltage clamp experiments. Substitution of these nine amino acid residues yielded 86% of the binding energy of the l-type ��1C subunit and 92% of the binding energy of the l-type ��1S subunit for the high-affinity DHP antagonist PN200���110. The activity of chimeric Ca2+ channels containing ��1A/DHPS was increased 3.5 �� 0.7-fold by the DHP agonist (���)Bay K8644. The effect of this agonist was stereoselective as in l-type Ca2+ channels since (+) Bay K8644 inhibited the activity of ��1A/DHPS. The results show conclusively that DHP agonists and antagonists bind to a single receptor site at which they have opposite effects on Ca2+ channel activity. This site contains essential components from both domains III and IV, consistent with a domain interface model for binding and allosteric modulation of Ca2+ channel activity by DHPs.

Inactivation of calcium-activated chloride channels in smooth muscle by calcium/calmodulin-dependent protein���kinase

To determine the mechanisms responsible for the termination of Ca2+-activated Cl��� currents (ICl(Ca)), simultaneous measurements of whole cell currents and intracellular Ca2+ concentration ([Ca2+]i) were made in equine tracheal myocytes. In nondialyzed cells, or cells dialyzed with 1 mM ATP, ICl(Ca) decayed before the [Ca2+]i decline, whereas the calcium-activated potassium current decayed at the same rate as [Ca2+]i. Substitution of AMP-PNP or ADP for ATP markedly prolonged the decay of ICl(Ca), resulting in a rate of current decay similar to that of the fall in [Ca2+]i. In the presence of ATP, dialysis of the calmodulin antagonist W7, the Ca2+/calmodulin-dependent kinase II (CaMKII) inhibitor KN93, or a CaMKII-specific peptide inhibitor the rate of ICl(Ca) decay was slowed and matched the [Ca2+]i decline, whereas H7, a nonspecific kinase inhibitor with low affinity for CaMKII, was without effect. When a sustained increase in [Ca2+]i was produced in ATP dialyzed cells, the current decayed completely, whereas in cells loaded with 5���-adenylylimidodiphosphate (AMP-PNP), KN93, or the CaMKII inhibitory peptide, ICl(Ca) did not decay. Slowly decaying currents were repeatedly evoked in ADP- or AMP-PNP-loaded cells, but dialysis of adenosine 5���-O-(3-thiotriphosphate) or okadaic acid resulted in a smaller initial ICl(Ca), and little or no current (despite a normal [Ca2+]i transient) with a second stimulation. These data indicate that CaMKII phosphorylation results in the inactivation of calcium-activated chloride channels, and that transition from the inactivated state to the closed state requires protein dephosphorylation.