Biomedical subjects

M De Waard

Publications and source records attributed to M De Waard.

At least 37 records · Page 2Linked to original sources

PubMed1999

A new beta subtype-specific interaction in alpha1A subunit controls P/Q-type Ca2+ channel activation.

The cytoplasmic beta subunit of voltage-dependent calcium channels modulates channel properties in a subtype-specific manner and is important in channel targeting. A high affinity interaction site between the alpha1 interaction domain (AID) in the I-II cytoplasmic loop of alpha1 and the beta interaction domain (BID) of the beta subunit is highly conserved among subunit subtypes. We describe a new subtype-specific interaction (Ss1) between the amino-terminal cytoplasmic domain of alpha1A (BI-2) and the carboxyl terminus of beta4. Like the interaction identified previously () between the carboxyl termini of alpha1A and beta4 (Ss2), the affinity of this interaction is lower than AID-BID, suggesting that these are secondary interactions. Ss1 and Ss2 involve overlapping sites on beta4 and are competitive, but neither inhibits the interaction with AID. The interaction with the amino terminus of alpha1 is isoform-dependent, suggesting a role in the specificity of alpha1-beta pairing. Coexpression of beta4 in Xenopus oocytes produces a reduced hyperpolarizing shift in the I-V curve of the alpha1A channel compared with beta3 (not exhibiting this interaction). Replacing the amino terminus of alpha1A with that of alpha1C abolishes this difference. Our data contribute to our understanding of the molecular organization of calcium channels, providing a functional basis for variation in subunit composition of native P/Q-type channels.

Amino Acid Sequence↗

PubMed1999

Antibodies against the beta subunit of voltage-dependent calcium channels in Lambert-Eaton myasthenic syndrome.

Lambert-Eaton myasthenic syndrome is an autoimmune disease that impairs neuromuscular transmission. Several studies suggest that neurotransmitter release is reduced by an immune response directed against the calcium channel complex of nerve terminals. The immunoglobulin G fractions from Lambert-Eaton myasthenic syndrome patients immunoprecipitate solubilized neuronal N- and P/Q-type channels and in certain cases brain, skeletal and cardiac muscle L-type channels [El Far O. et al. (1995) J. Neurochem. 64, 1696-1702; Lennon V. A. and Lambert E. H. (1989) Mayo Clin. Proc. 64, 1498-1504; Sher E. et al. (1989) Lancet ii, 640-643; Suenaga A. et al. (1996) Muscle Nerve 19, 1166-1168]. These channel immunoprecipitation assays are considered as useful for the diagnosis of this syndrome. In this study, we demonstrate that two predominant neuronal voltage-dependent calcium channel beta subunits (beta3 and beta4, of mol. wt 58,000) are general targets of Lambert-Eaton myasthenic syndrome autoantibodies. Of 20 disease sera tested, 55% were able to immunoprecipitate 35S-labeled beta subunits. All five patients affected with small-cell lung carcinoma were positive for the beta-subunit immunoprecipitation assay. Interestingly, only a fraction of the beta-subunit-positive sera was also able to immunoprecipitate N- and P/Q-type channels, suggesting that several of the beta-subunit epitopes are masked in native channels. In accordance with this observation, we found that several beta-positive sera were able to prevent the interaction between calcium channel alpha1 and beta subunits in vitro. In cases where sera were able to immunoprecipitate beta subunits, N- and P/Q-type channels, the immunoprecipitation of both channel types was either partially or entirely mediated by beta-subunit antibodies. Our results suggest that assays based on the immunoprecipitation of beta subunits can be used as an additional test to assist in the diagnosis of Lambert-Eaton myasthenic syndrome.

Antibodies↗

PubMed1999

Scorpion alpha-like toxins, toxic to both mammals and insects, differentially interact with receptor site 3 on voltage-gated sodium channels in mammals and insects.

alpha-Like toxins, a unique group designated among the scorpion alpha-toxin class that inhibit sodium channel inactivation, are highly toxic to mice but do not compete for alpha-toxin binding to receptor site 3 on rat brain sodium channels. We analysed the sequence of a new alpha-like toxin, which was also highly active on insects, and studied its action and binding on both mammalian and insect sodium channels. Action of the alpha-like toxin on isolated cockroach axon is similar to that of an alpha-toxin, and the radioactive toxin binds with a high affinity to insect sodium channels. Other sodium channel neurotoxins interact competitively or allosterically with the insect alpha-like toxin receptor site, similarly to alpha-toxins, suggesting that the alpha-like toxin receptor site is closely related to receptor site 3. Conversely, on rat brain sodium channels, specific binding of 125I-alpha-like toxin could not be detected, although at high concentration it inhibits sodium current inactivation on rat brain sodium channels. The difficulty in measuring binding to rat brain channels may be attributed to low-affinity binding due to the acidic properties of the alpha-like toxins that also impair the interaction with receptor site 3. The results suggest that alpha-like toxins bind to a distinct receptor site on sodium channels that is differentially related to receptor site 3 on mammalian and insect sodium channels.

Animals↗

PubMed1998

Interaction of cysteine string proteins with the alpha1A subunit of the P/Q-type calcium channel.

Cysteine string proteins (Csps) are J-domain chaperone proteins anchored at the surface of synaptic vesicles. Csps are involved in neurotransmitter release and may modulate presynaptic calcium channel activity, although the molecular mechanisms are unknown. Interactions between Csps, proteins of the synaptic core (SNARE) complex, and P/Q-type calcium channels were therefore explored. Co-immunoprecipitation suggested that Csps occur in complexes containing synaptobrevin (VAMP), but not syntaxin 1, SNAP-25, nor P/Q-type calcium channels labeled with 125I-omega-conotoxin MVIIC. However binding experiments with 35S-labeled Csp1 demonstrated an interaction (apparent KD = 700 nM at pH 7.4 and 4 degreesC) with a fusion protein containing a segment of the cytoplasmic loop linking homologous domains II-III of the alpha1A calcium channel subunit (BI isoform, residues 780-969). Binding was specific as it was displaced by unlabeled Csp1, and no interactions were detected with fusion proteins containing other calcium channel domains, VAMP, or syntaxin 1A. A Csp binding site on the P/Q-type calcium channel is thus located within the 200 residue synaptic protein interaction site that can also bind syntaxin I, SNAP-25, and synaptotagmin I. Csp may act as a molecular chaperone to direct assembly or disassembly of exocytotic complexes at the calcium channel.

Animals↗

PubMed1998

A beta 4 isoform-specific interaction site in the carboxyl-terminal region of the voltage-dependent Ca2+ channel alpha 1A subunit.

The voltage-gated calcium channel beta subunit is a cytoplasmic protein that stimulates activity of the channel-forming subunit, alpha 1, in several ways. Complementary binding sites on alpha 1 and beta have been identified that are highly conserved among isoforms of the two subunits, but this interaction alone does not account for all of the functional effects of the beta subunit. We describe here the characterization in vitro of a second interaction, involving the carboxyl-terminal cytoplasmic domain of alpha 1A and showing specificity for the beta 4 (and to a lesser extent beta 2a) isoform. A deletion and chimera approach showed that the carboxyl-terminal region of beta 4, poorly conserved between beta isoforms, contains the interaction site and plays a role in the regulation of channel inactivation kinetics. This is the first demonstration of a molecular basis for the specificity of functional effects seen for different combinations of these two channel components.

Animals↗

PubMed1998

Subunit interaction sites in voltage-dependent Ca2+ channels: role in channel function.

Voltage-dependent Ca2+ channels are heteromeric complexes found in the plasma membrane of virtually all cell types and show a high level of electrophysiological and pharmacological diversity. Associated with the pore-forming alpha 1 subunit are the membrane anchored, largely extracellular alpha2-delta, the cytoplasmic beta and sometimes a transmembrane gamma subunit; these subunits dramatically influence the properties and surface expression of these channels. Effects vary depending on subunit isoforms, suggesting that functional diversity of native channels reflects heterogeneity of combinations. Interaction sites between subunits have been identified and advances have been made in our understanding of the molecular basis of functional effects of the auxiliary subunits, their capacity to be regulated by G proteins, and their interaction with related cellular systems.

Amino Acid Sequence↗

PubMed1997

Dissection of functional domains of the voltage-dependent Ca2+ channel alpha2delta subunit.

Coexpression of the cloned voltage-dependent Ca2+ channel alpha2delta subunit with the pore-forming alpha1 subunit results in a significant increase in macroscopic current amplitude. To gain insight into the mechanism underlying this interaction, we have examined the regulatory effect of either the alpha2delta complex or the delta subunit on the Ca2+ channel alpha1 subunit. Transient transfection of tsA201 cells with the cardiac L-type alpha1C subunit alone resulted in the expression of inward voltage-activated currents as well as measurable [3H]-PN200-110 binding to membranes from transfected cells. Coexpression of the alpha2delta subunit significantly increased the macroscopic current amplitude, altered the voltage dependence and the kinetics of the current, and enhanced [3H]-PN200-110 binding. Except for the increase in amplitude, coexpression of the delta subunit reproduced entirely the effects of the full-length alpha2delta subunit on the biophysical properties of the alpha1C currents. However, no effect on specific [3H]-PN200-110 binding was observed on delta subunit coexpression. Likewise, profound effects on current kinetics of the neuronal alpha1A subunit were observed on coexpression of the alpha2delta complex in Xenopus oocytes. Furthermore, by using a chimeric strategy, we localized the region involved in this regulation to the transmembrane domain of the delta subunit. These data strongly suggest that the molecular determinants involved in alpha2delta regulation are conserved across L-type and non-L type Ca2+ channels. Taken together, our results indicate that the region of the alpha2delta subunit involved in the modulation of the gating properties of the high voltage-activated calcium channels is localized in the delta domain of the protein. In contrast, the level of membrane expression of functional channels relies on the presence of the alpha2 domain of the alpha2delta complex.

Calcium Channels↗

PubMed1997

T-type Ca2+ current properties are not modified by Ca2+ channel beta subunit depletion in nodosus ganglion neurons.

At the molecular level, our knowledge of the low voltage-activated Ca2+ channel (T-type) has made little progress. Using an antisense strategy, we investigated the possibility that the T-type channels have a structure similar to high voltage-activated Ca2+ channels. It is assumed that high voltage-activated channels are made of at least three components: a pore forming alpha1 subunit combined with a cytoplasmic modulatory beta subunit and a primarily extracellular alpha2delta subunit. We have examined the effect of transfecting cranial primary sensory neurons with generic anti-beta antisense oligonucleotides. We show that in this cell type, blocking expression of all known beta gene products does not affect T-type current, although it greatly decreases the current amplitude of high voltage-activated channels and modifies their voltage dependence. This suggests that beta subunits are likely not constitutive of T-type Ca2+ channels in this cell type.

Calcium↗

PubMed1997

Direct interaction of the calcium sensor protein synaptotagmin I with a cytoplasmic domain of the alpha1A subunit of the P/Q-type calcium channel.

Synaptotagmins are synaptic vesicle proteins containing two calcium-binding C2 domains which are involved in coupling calcium influx through voltage-gated channels to vesicle fusion and exocytosis of neurotransmitters. The interaction of synaptotagmins with native P/Q-type calcium channels was studied in solubilized synaptosomes from rat cerebellum. Antibodies against synaptotagmins I and II, but not IV co-immunoprecipitated [125I]omega-conotoxin MVIIC-labelled calcium channels. Direct interactions were studied between in vitro-translated [35S]synaptotagmin I and fusion proteins containing cytoplasmic loops of the alpha1A subunit (BI isoform). Gel overlay revealed the association of synaptotagmin I with a single region (residues 780-969) located in the intracellular loop connecting homologous domains II and III. Saturable calcium-independent binding occurred with equilibrium dissociation constants of 70 nM and 340 nM at 4 degrees C and pH 7.4, and association was blocked by addition of excess recombinant synaptotagmin I. Direct synaptotagmin binding to the pore-forming subunit of the P/Q-type channel may optimally locate the calcium-binding sites that initiate exocytosis within a zone of voltage-gated calcium entry.

Amino Acid Sequence↗

PubMed1997

Direct binding of G-protein betagamma complex to voltage-dependent calcium channels.

Voltage-dependent Ca2+ channels play a central role in controlling neurotransmitter release at the synapse. They can be inhibited by certain G-protein-coupled receptors, acting by a pathway intrinsic to the membrane. Here we show that this inhibition results from a direct interaction between the G-protein betagamma complex and the pore-forming alpha1 subunits of several types of these channels. The interaction is mediated by the cytoplasmic linker connecting the first and second transmembrane repeats. Within this linker, binding occurs both in the alpha1 interaction domain (AID), which also mediates the interaction between the alpha1 and beta subunits of the channel, and in a second downstream sequence. Further analysis of the binding site showed that several amino-terminal residues in the AID are critical for Gbetagamma binding, defining a site distinct from the carboxy-terminal residues shown to be essential for binding the beta-subunit of the Ca2+ channel. Mutation of an arginine residue within the N-terminal motif abolished betagamma binding and rendered the channel refractory to G-protein modulation when expressed in Xenopus oocytes, showing that the interaction is indeed responsible for G-protein-dependent modulation of Ca2+ channel activity.

Amino Acid Sequence↗

PubMed1996

Expression and subunit interaction of voltage-dependent Ca2+ channels in PC12 cells.

Nerve growth factor (NGF)-induced differentiation in PC12 cells is accompanied by changes in the expression of voltage-dependent Ca2+ channels. Ca2+ channels are multimeric complexes composed of at least three subunits (alpha1, beta, and alpha2delta) and are involved in neuronal migration, gene expression, and neurotransmitter release. Although attempts have been undertaken to elucidate NGF regulation of Ca2+ channel expression, the changes in subunit composition of these channels during differentiation still remain uncertain. In the present study, patch-clamp recordings show that in addition to the previously documented L-type and N-type Ca2+ currents, undifferentiated PC12 cells also express an omega-agatoxin-IVA-sensitive (P/Q-type) component. In addition, the corresponding mRNA encoding the pore-forming alpha1 subunits for these channels (C, B, and A, respectively) was detected. Likewise, mRNA for three distinct auxiliary beta subunits (1, 2, 3) were also found, beta3 protein being dominantly expressed. Immunoprecipitation experiments show that the N-type Ca2+ channel is associated with either a beta2 or beta3 subunit and that NGF increases the channel expression without affecting its beta subunit association. These results (1) indicate that the diversity of Ca2+ currents in PC12 cells arise from the expression of three distinct alpha1 and three different beta subunit genes; (2) support a model for heterogenous beta subunit association of the N-type Ca2+ channel in a single cell type; and (3) suggest that the regulation of the N-type Ca2+ channel during NGF-mediated differentiation involves an increase in the number of functional channels with no apparent changes in subunit composition.

Animals↗

PubMed1996

Identification of three subunits of the high affinity omega-conotoxin MVIIC-sensitive Ca2+ channel.

N-, P- and Q-type voltage-dependent Ca2+ channels control neurotransmitter release in the nervous system and are blocked by omega-conotoxin MVIIC. In this study, both a high affinity and a low affinity binding site for omega-conotoxin MVIIC were detected in rabbit brain. The low affinity binding site is shown to be present on the N-type Ca2+ channel. Using optimized conditions for specific labeling of the high affinity omega-conotoxin MVIIC receptor and a panel of subunit specific antibodies, the molecular structure of the high affinity receptor was investigated. We demonstrate for the first time that this receptor is composed of at least alpha1A, alpha2delta, and any one of the four brain beta subunits. Such association of different beta subunits with alpha1A and alpha2delta components may produce Ca2+ channels with distinct functional properties, such as P- and Q-type.

Animals↗

PubMed1996

Identification of critical amino acids involved in alpha1-beta interaction in voltage-dependent Ca2+ channels.

In voltage-dependent Ca2+ channels, alpha1 and beta subunits interact via two cytoplasmic regions defined as Alpha Interaction Domain (AID) and Beta Interaction Domain (BID). Several novel amino acids for that interaction have now been mapped in both domains by point mutations. It was found that three of the nine amino acids in AID and four of the eight BID amino acids tested were essential for the interaction. Whereas the important AID amino acids were clustered around five residues, the important BID residues were more widely distributed within a larger 16 amino acid sequence. The affinity of the AIDA GST fusion protein for the four interacting beta 1b BID mutants was not significantly altered compared with the wild-type beta 1b despite the close localization of mutated residues to disruptive BID amino acids. Expression of these interactive beta mutants with the full-length alpha 1A subunit only slightly modified the stimulation efficiency when compared with the wild-type beta 1b subunit. Our data suggest that non-disruptive BID sequence alterations do not dramatically affect the beta subunit-induced current stimulation.

Amino Acid Sequence↗

PubMed1996

Beta subunit heterogeneity in N-type Ca2+ channels.

The beta subunit of the voltage-dependent Ca2+ channel is a cytoplasmic protein that interacts directly with an alpha1 subunit, thereby modulating the biophysical properties of the channel. Herein, we demonstrate that the alpha1B subunit of the N-type Ca2+ channel associates with several different beta subunits. Polyclonal antibodies specific for three different beta subunits immunoprecipitated 125I-omega-conotoxin GVIA binding from solubilized rabbit brain membranes. Enrichment of the N-type Ca2+ channels with an alpha1B subunit-specific monoclonal antibody showed the association of beta1b, beta3, and beta4 subunits. Protein sequencing of tryptic peptides of the 57-kDa component of the purified N-type Ca2+ channel confirmed the presence of the beta3 and beta4 subunits. Each of the beta subunits bound to the alpha1B subunit interaction domain with similar high affinity. Thus, our data demonstrate important heterogeneity in the beta subunit composition of the N-type Ca2+ channels, which may be responsible for some of the diverse kinetic properties recorded from neurons.

Amino Acid Sequence↗

PubMed1996

Structural and functional diversity of voltage-activated calcium channels.

Data gathered from the expression of cDNAs that encode the subunits of voltage-dependent Ca2+ channels have demonstrated important structural and functional similarities among these channels. Despite these convergences, there are also significant differences in the nature and functional importance of subunit-subunit and protein-Ca2+ channel interactions. There is evidence demonstrating that the functional differences between Ca2+ channel subtypes is due to several factors, including the expression of distinct alpha 1 subunit proteins, the selective association of structural subunits and modulatory proteins, and differences in posttranslational processing and cell regulation. We summarize several avenues of research that should provide significant clues about the structural features involved in the biophysical and functional diversity of voltage-dependent Ca2+ channels.

Amino Acid Sequence↗

PubMed1996

Dual function of the voltage-dependent Ca2+ channel alpha 2 delta subunit in current stimulation and subunit interaction.

Voltage-dependent Ca2+ channels are modulated by complex interactions with the alpha 2 delta subunit. In vitro translation was used to demonstrate a single transmembrane topology of the alpha 2 delta subunit in which all but the transmembrane sequence and 5 carboxy-terminal amino acids are extracellular. The glycosylated extra-cellular domain is required for current stimulation, as shown by coexpression of truncated alpha 2 delta subunits with alpha 1A and beta 4 subunits in Xenopus oocytes and deglycosylation with peptide-N-glycosidase F. However, coexpression of the transmembrane domain-containing delta subunit reduced the stimulatory effects of full-length alpha 2 delta subunits and substitution of a different transmembrane domain resulted in a loss of current stimulation. These results support a model whereby the alpha 2 delta transmembrane domain mediates subunit interactions and the glycosylated extracellular domain enhances current amplitude.

Animals↗

PubMed1995

Association of native Ca2+ channel beta subunits with the alpha 1 subunit interaction domain.

beta Subunits of voltage-dependent Ca2+ channels play an important role in regulating Ca2+ channel function. The sites of alpha 1-beta subunit interaction have been localized recently to cytoplasmic domains of both subunits. The alpha 1 subunit interaction domain (AID) is an 18-amino-acid conserved motif located between repeats I and II on all alpha 1 subunits which is essential for the binding of beta subunits. In order to further study the interaction of beta subunits with AID, we have expressed a 50-amino-acid glutathione S-transferase (GST) fusion protein from the alpha 1A subunit that contains the AID. Mutant GST fusion proteins that contain a single amino acid change (Y392S, Y392F, and Y392W) in the AIDA along with control GST were coupled to glutathione-Sepharose beads to form affinity beads. Binding assays using these affinity beads with in vitro synthesized 35S-labeled beta 2 and beta 3 subunits demonstrate that the hydroxyl group on tyrosine 392 of AIDA is critical for binding to beta subunits. The affinity bead assay was also used to identify and characterize native beta subunits from detergent extracts of different tissues. The AIDA affinity beads, but not the control or Y392S beads, specifically bind beta subunits from detergent extracts of skeletal muscle, cardiac muscle, and brain. Immunoblot analyses demonstrate the presence of beta 1a in skeletal muscle, beta 2 and beta 3 in cardiac muscle, and beta 1b, beta 3, and beta 4 in brain. The assays also demonstrate the AIDA beads bind to beta subunits from tissue homogenates extracted with low salt and no detergent suggesting the existence of a pool of beta subunits which is not always associated with alpha 1 subunits. Also, beta subunits from solubilized skeletal muscle triads can be affinity-purified using AIDA CNBr-Sepharose. Our data demonstrate that the AID binds to native beta subunits from detergent and non-detergent tissue extracts illustrating that this domain on the alpha 1 subunit is the major anchoring site for the beta subunit.

Amino Acid Sequence↗

PubMed1995

Subunit regulation of the neuronal alpha 1A Ca2+ channel expressed in Xenopus oocytes.

1. Voltage-dependent Ca2+ channels are multi-protein complexes composed of at least three subunits: alpha 1, alpha 2 delta and beta. Ba2+ currents were recorded in Xenopus oocytes expressing the neuronal alpha 1A Ca2+ channel, using the two-electrode voltage-clamp technique. Various subunit combinations were studied: alpha 1A, alpha 1A alpha 2 delta b, alpha 1A beta or alpha 1A alpha 2 delta b beta. 2. The alpha 1A subunit alone directs the expression of functional Ca2+ channels. It carries all the properties of the channel: gating, permeability, voltage dependence of activation and inactivation, and pharmacology. The alpha 1A channel is activated by low voltages when physiological concentrations of the permeant cation are used. Both ancillary subunits alpha 2 delta and beta induced considerable changes in the biophysical properties of the alpha 1A current. The subunit specificity of the changes in current properties was analysed for all four beta gene products by coexpressing beta 1b, beta 2a, beta 3 and beta 4. 3. All beta subunits induce a stimulation in the current amplitude, a change in inactivation kinetics, and two hyperpolarizing shifts--one in the voltage dependence of activation and a second in the voltage dependence of steady-state inactivation. The most significant difference in regulation among beta subunits is the induction of variable rate constants of current inactivation. Rates of inactivation were induced in the following order (fastest to slowest): beta 3 > beta 1b = beta 4 > beta 2a. 4. The alpha 2 delta b subunit does not modify the properties of alpha 1A Ca2+ channels in the absence of beta subunits. However, this subunit increases the beta-induced stimulation in current amplitude and also regulates the beta-induced change in inactivation kinetics. 5. Of all the subunit combinations tested, Ca2+ channels that included a beta subunit were the most prone to decrease in activity. It is concluded that beta subunits are the primary target for the inhibitory mechanisms involved in Ca2+ channel run-down. 6. Both alpha 2 delta b and beta 1 b subunits slightly modified the sensitivity of the alpha 1A subunit to the snail peptide omega-conotoxin MVIIC. 7. The subunit-induced changes in properties of the alpha 1A channel are surprisingly similar to changes reported for other alpha 1 subunits. These modifications in channel activity should therefore represent important functional landmarks in the on-going characterization of subunit-subunit interactions.

Animals↗