From the
Department of Physiology and Biophysics, Cellular and Molecular Neurobiology Research Group, University of Calgary, Calgary, T2N 4N1, Canada and
Department of Molecular and Cellular Neurobiology, Research Institute Neurosciences, Vrije Universiteit, 1081 HV Amsterdam, The Netherlands
Received for publication, March 3, 2003 , and in revised form, April 1, 2003.
![]() |
ABSTRACT |
---|
![]() ![]() ![]() ![]() ![]() ![]() |
---|
![]() |
INTRODUCTION |
---|
![]() ![]() ![]() ![]() ![]() ![]() |
---|
Cav2.1 (P/Q-type) and Cav2.2 (N-type) channels are densely localized at presynaptic zones of vertebrate neurons (8, 9, 10) where they are physically coupled to proteins of the synaptic vesicle release proteins such as syntaxin1, SNAP25, and synaptotagmin (1, 2, 7, 11). This possibly serves to optimize the efficiency of synaptic transmission and as a negative feedback mechanism allowing the regulation of calcium channel activity during various steps of exocytosis (7, 12). Mammalian N-type and P/Q-type calcium channels are differentially inhibited upon activation of G protein-coupled receptors (7, 13, 14, 15). This effect is mediated by G protein subunits and may serve to fine-tune synaptic activity.
Invertebrate species do not appear to contain the same structural diversity of vertebrate calcium channel genes in their genomes and, with one possible exception (16), possess only single homologs of the three major calcium channel families, Cav1, Cav2, and Cav3 (12, 17). Invertebrate Cav2 representatives are considered functional as well as structural correlates of both mammalian N- (Cav2.2) and P/Q-type (Cav2.1) calcium channels (12, 17). Cav2 homologs from Drosophila (DmCa1A/cac) (18, 19), Caenorhabditis elegans (unc-2) (20), and more recently, the freshwater mollusk Lymnaea stagnalis (LCav2a) (21), are required for invertebrate synaptic transmission, reminiscent of the roles of N- and P/Q-type channels in mammalian synapses (3, 22). The invertebrate Cav2 calcium channel 1 subunits, unlike mammalian N-type and P/Q-type calcium channels, do not display an elongated domain II-III linker region, which in mammalian synaptic channels characteristically contain interaction sites for syntaxin, SNAP-25, and synaptotagmin (19, 21). However, although full-length sequences of invertebrate presynaptic calcium channels have been in the public domain for years, there has not been a report describing the functional expression of any of the cloned invertebrate Cav2 synaptic calcium channels. An exception to this is a single report of expression of a squid Cav2 homolog in Xenopus oocytes (23), a system known to endogenously express calcium channels as well as low levels of synaptic proteins. This has precluded a detailed functional analysis of these channels and has been an obstacle to taking full advantage of invertebrate synaptic preparations to address fundamental aspects of synaptic transmission at the molecular level.
Here we report the expression and characterization of a L. stagnalis Cav2 calcium channel 1 subunit homolog (LCav2a). We show that LCav2a channels are ineffectively targeted to the plasma membrane but that this can be overcome by the replacement of a short stretch of amino acids at the N-terminal region of LCav2a (44 amino acids) with its counterpart from rat Cav2.1. When coexpressed with rat
1b and
2-
, the functional properties of the modified LCav2a-5'rbA calcium channel resemble in many ways those of mammalian N-type calcium channels showing similar activation and inactivation behavior, preferential permeation of barium over calcium, a lack of pronounced calcium-dependent inactivation, and complete block by cadmium ions. Consistent with what has been reported for native Lymnaea calcium currents in neurons (24, 25), the LCav2a-5'rbA channel is insensitive to micromolar concentrations of
-conotoxin GVIA and does not have characteristics of L-type channels with regard to nifedipine sensitivity. As expected from the lack of an identifiable syntaxin binding site (21), the coexpression of the channel with Lymnaea syntaxin1 did not affect channel function. Interestingly, the channel was irreversibly inhibited in the presence of GTP
S1 but not functionally affected by syntaxin1, suggesting that invertebrate calcium channels may display distinct regulation from their vertebrate counterparts.
![]() |
EXPERIMENTAL PROCEDURES |
---|
![]() ![]() ![]() ![]() ![]() ![]() |
---|
Sequence ComparisonsLymnaea genes and rat homologs were aligned using a progressive pairwise, multiple alignment in PILEUP (UNIX-based, GCG Wisconsin Package 2002, Accelrys, Madison, WI). Aligned rat genes included Cav2.1 (P/Q type) (GI:1705706), Cav2.2 (N-type) (GI:25453410), syntaxin1A (GI:417842), and syntaxin1B (GI:631888).
Preparation of LCav2 Polyclonal AntibodyThe antigen to make the calcium channel polyclonal antibody was derived from a 15-mer peptide, KAEDNENDSEQNDND (Henk Hilkman, Netherlands Cancer Institute, Amsterdam), corresponding to amino acids 418432 of LCav2a cytoplasmic I-II linker. The purity of the peptide was confirmed by matrix-assisted laser desorption ionization mass spectrometry and analytical high performance liquid chromatography. Rabbits were immunized for a 4-week period with adjuvants and antigen conjugated to the carrier protein KLH (Washington Biotechnology Inc., Baltimore, MD). IgG rabbit antiserum was tested for immunoreactivity with the antigen by spot blot.
Transient Transfection of Mammalian CellsHuman embryonic kidney tsA-201 cells were grown and transfected using standard calcium phosphate protocol (26). cDNAs transfected included a cDNA construct encoding for green fluorescent protein (Clontech), calcium channel subunits rat 2-
1 and rat
1b, and either LCav2a or LCav2a-5'rbA or rat Cav2.2, plus when stated, Lsytx1A, rat syntaxin1A, or C-terminal fragment of the
-adrenergic receptor kinase (
-ARKct). Cells slated for immunohistochemistry were left to incubate at 37 °C on poly-L-lysinecoated dishes, whereas those slated for electrophysiological experiments were incubated at 28 °C for 24 days before analysis.
LCav2 Immunostaining in Mammalian CellsMock-transfected tsA-201 cells or tsA-201 cells transfected with rat 2-
1, rat
1b, and either LCav2a or LCav2a-5'rbA were incubated at 37 °C for 4 days, fixed with 1% paraformaldehyde for 12 h at 4 °C, then permeabilized in 1% Nonidet-P40 (octylphenoxypolyethoxyethanol polyethyleneglycol-p-isooctylphenyl ether) for 1 h at room temperature. Afterward, cells were washed in TBS-BSA-Triton blocking agent (50 mM Tris, 150 mM NaCl, 1 g/liter bovine serum albumin; Sigma-Aldrich, A9647), 0.5% Triton-X100, pH 7.4, and then incubated with 1:1000 dilution, LCav2 anti-rabbit polyclonal antibody in TBS-BSA-Triton overnight at 4 °C. The next day cells were exposed to 10x washes in TBS-BSA-Triton and treated with a 1:500 dilution of Alexa Fluor 568 goat anti-rabbit IgG (Molecular Probes, Inc., Eugene OR) in TBS-BSA-Triton overnight at 4 °C. The next day, cells were washed 10x in TBS (50 mM Tris, 150 mM NaCl, pH 7.4), wet-dried, and mounted in fluorescence anti-fading media (ProLong AntiFade, Molecular Probes). Images were visualized and analyzed on an Olympus confocal microscope.
Immunoblot Analysis of LCav2 and Lsytx1 Protein Expression in tsA-201 CellsTransiently transfected or untransfected tsA-201 cells incubated for 4 days at 37 °C were harvested for Western blot analysis using a standard procedure (26). Proteins were detected with primary antibodies at 1:2000 dilution, raised in rabbits against LCav2a or Lsytx1 (ANR-002, Alomone Labs, Jerusalem, Israel). Color detection of gel blots were processed using previously described methods (21).
Functional Assessment and Analysis of LCav2 cDNA Expression Calcium channel activity in transiently transfected tsA-201 cells was characterized via whole cell patch clamp using an Axopatch 200B amplifier (Axon Instruments, Union City, CA), pCLAMP 9.0 software, and previously described recording procedures and solutions (26). Data analysis was carried out using Clampfit (pClamp 9, Axon Instruments) and SigmaPlot 2000 (Jandel Scientific, SPSS Science, Chicago, IL.). Steady state inactivation curves were fit with standard Boltzmann relations I = 1/(1 + exp(V - Vh/S)), where I is the normalized peak current amplitude, V is the holding potential, Vh is the half-inactivation potential, and S is a slope factor. Whole cell current voltage relations were fitted with the equation I = G(V - Erev)/(1 + exp(Va - V)/S), where G is the maximum slope conductance, I is the peak current amplitude, V is the test potential, Erev is the reversal potential, Va is the half-activation potential, and S is a slope factor inversely proportional the effective gating charge. Time constants for inactivation, , were determined from monoexponential fits to the raw data (Fig. 3A). Error bars reflect S.E., and numbers in parentheses reflect numbers of experiments. Statistical analysis was carried out using Sigmastat (Jandel Scientific). Differences between mean values from each experimental group were tested using paired and unpaired Student's t tests and were considered significant if p < 0.05.
|
![]() |
RESULTS AND DISCUSSION |
---|
![]() ![]() ![]() ![]() ![]() ![]() |
---|
|
|
We then attempted a strategy used previously to obtain expression of a mammalian Cav2.2 calcium channel variant in Xenopus oocytes (27, 28, 29) by replacing part of the N-terminal region of the LCav2a channel (44 amino acids) with the corresponding sequence from rat Cav2.1 (creating LCav2a-5'rbA). As shown in Fig. 2C, LCav2a-5'rbA is detected in the plasma membrane as rings of fluorescence of differing intensities around the cell edges, apparent in approximately one-third of all transfected cells, similar to the fraction of green fluorescent protein-positive cells in typical transfections. Single bands of predicted size (240 kDa) for LCav2a were detected in an immunoblot of LCav2a and LCav2a-5'rbA-transfected tsA-201 cells but not in mock-transfected cells (Fig. 2D). Together with the relatively low abundance of membrane staining and absence of observed barium currents in LCav2a-transfected cells, this indicates that the native N terminus of LCav2a either directly antagonizes plasma membrane trafficking in mammalian cells or that it might be missing an N-terminal domain required for efficient trafficking or stabilization of channels in mammalian cell membranes (see Fig. 2E for sequence alignment of the rat Cav2.2 and LCav2a N-terminal regions). It is interesting to note that among rat Cav2 channels, there is a common, almost identical 28-amino acid N-terminal sequence just upstream of where LCav2a differs with rat channels (see the boxed amino-acids, Fig. 2E), raising the possibility that this motif may perhaps be involved in membrane targeting. Further construction of chimeras will be needed to substantiate this possibility.
The Biophysical Properties of LCav2a-5'rbA Are Similar to Those of Mammalian N-type ChannelsWhen analyzed electrophysiologically, the LCav2a-5'rbA channel construct resulted in robust barium currents in 46 of 82 green fluorescent protein-positive cells when transiently expressed in tsA-201 cells. This clearly contrasts with the lack of detectable expression of the wild type LCav2a channel. We therefore carried out a detailed analysis of biophysical properties of the LCav2a-5'rbA calcium channel. A shown in Fig. 3A, the LCav2a-5'rbA channel supports barium currents with moderate inactivation kinetics. As seen from the ensemble current-voltage relations (Fig. 3B), currents first activate at about -20 mV and peak near +20 mV. The half-activation potential determined from the fit of the IV curve was +8.5 mV, consistent with LCav2a-5'rbA acting as a high voltage-activated calcium channel. A similar value was obtained from fits to individual IV relations (10.8 ± 1.9 mV, n = 26). The peak of the IV curve of LCav2a-5'rbA is shifted about 20 mV positive to the human Cav2.2 (N-type) calcium channel (30) and about 10 mV positive to the rat Cav2.2 isoform (31) under similar experimental conditions. Steady state inactivation of the LCav2a-5'rbA channels were examined using 5-s prepulses to various holding potentials before a +20-mV test depolarization. The ensemble of the steady state inactivation curves is well described by a Boltzmann relation (Fig. 3C). The half-inactivation potential from the ensemble fit (-39.9 mV) and from fits to individual steady state inactivation curves (-39.6 ± 6.4 mV, n = 14) is about 10 mV more positive than that of the human Cav2.2 calcium channel (30) and about 5 mV more depolarized than that seen with rat Cav2.2 (26, 32) under these experimental conditions. As seen with mammalian calcium channels, the voltage dependence of the time constant of inactivation slightly decreases at more depolarized potentials (Fig. 3D).
Fig. 4A depicts current records obtained from the same cell in 20 mM external barium and 20 mM external calcium. As evident from these current recordings and from the current voltage relations in Fig. 4B, replacement of barium with calcium resulted in a dramatic reduction in peak current amplitude as seen with many other types of vertebrate mammalian high voltage-activated calcium channels. In six such paired experiments, the maximum slope conductance underwent a statistically significant reduction (from 10.5 ± 1.8 nanosiemens to 3.95 ± 0.56 nanosiemens, n = 6, p < 0.05) when calcium replaced barium as the external charge carrier (see the inset to Fig. 4B). This effect may be due to a reduction in single channel conductance, an effect on maximum open probability, or a combination thereof and is seen with most types of mammalian high voltage-activated calcium channels with the exception of Cav2.3 (33). These data are consistent with the observation that the selectivity filter region in the four transmembrane domains of Cav2.1 and Cav2.2 calcium channels is conserved in LCav2a (see Fig. 4C). In addition, the half-activation potential in this set of experiments shifted from 8.1 ± 2.5 to 26.58 ± 1.8 mV (n = 6, p < 0.05), as reported for a number of voltage-gated calcium channels (see Refs. 33 and 34). It is interesting to note that half-activation potential shifts of this magnitude are more typical to L-type calcium channels, with N-type and P/Q-type channels showing a smaller (10 mV) effect (33).
|
We also examined the time course of inactivation in both barium and calcium. As shown in Fig. 4C, when the current records of Fig. 4A were superimposed to overlap at peak, a small degree of kinetic speeding could be observed in calcium, perhaps indicative of a small degree of calcium-sensitive inactivation. Examination of this effect at a number of different test potentials shows that calcium significantly speeds the rate of inactivation at more depolarized voltages (Fig. 4D). This small degree of speeding is comparable with that reported for rat P/Q-type calcium channels but much less pronounced than the calcium-dependent inactivation of L-type calcium channels. It is, however, important to note that the calcium-induced 20-mV shift in half-activation potential could potentially result in a similar shift in the voltage dependence of the time constant for voltage-dependent inactivation, thus attributing the observed effects purely to voltage-dependent rather than calcium-dependent inactivation (i.e. compare the inactivation time constants in barium and calcium at +40 and +60 mV, respectively). Taken together, our data thus indicate that LCav2a-5'rbA calcium channels undergo only little if any calcium-dependent inactivation.
LCav2a-5'rbA Channels Are -Conotoxin GVIA-resistantIt has been shown previously that native Lymnaea neuronal whole cell calcium currents are insensitive to classical blockers such as
-conotoxin GVIA and nifedipine (24, 25). Indeed, to date no selective blocker of these native currents has been identified. As shown in Fig. 5A, the LCav2a-5'rbA channel shows the predicted lack of nifedipine and
-conotoxin GVIA block. At concentrations (i.e. 3 µM GVIA and 5 µM nifedipine) at which mammalian N-type and L-type channels would, respectively, be completely blocked (35, 36), the LCav2a-5'rbA channel showed only about a 10% reduction in peak current amplitude in response to
-conotoxin GVIA and no detectable inhibition by nifedipine. In contrast, application of 100 µM cadmium completely abolished current activity within 10 s of application, as expected from a high voltage-activated calcium channel. The lack of conotoxin inhibition is interesting given the relatively high degree of sequence homology within the putative
-conotoxin GVIA binding region of the Cav2.2 calcium channel domain III S5-S6 region (Fig. 5B, see Refs. 27 and 36). By contrast, the inhibition by cadmium ions is consistent with the sequence conservation of the narrow region of the pore (Fig. 4E, see Refs. 37 and 38).
|
LCav2a-5'rbA Is Uniquely Regulated by G ProteinsBoth rat N-type and P/Q-type calcium channels are inhibited by direct interactions with G protein subunits (see Refs. 39, 40, 41). A hallmark of G protein inhibition of these channels is that it can be relieved after the application of a strong depolarizing prepulse (see 42, 43). Under control conditions, the application of such a prepulse resulted in a slight decrease in peak current amplitude by 13 ± 2.9% (n = 16), presumably due to a small degree of inactivation occurring during the prepulse and indicating a lack of tonic (background) G protein inhibition. To elicit a putative G protein inhibition of LCav2a-5'rbA channel activity, we added 100 µM GTP
S to the patch pipette but consistently failed to observe current activity upon cell rupture in seven experiments. In one additional experiment, however, current activity could be observed but was completely eliminated within 2 min of cell rupture and could not be recovered with application of prepulses. These data suggest the possibility that rapid dialysis of the cell with GTP
S may have resulted in an irreversible inhibition of LCav2a-5'rbA activity. To examine this possibility, the very tip of the recording pipette was filled with intracellular recording solution, and the rest of the pipette was back-filled with GTP
S-containing solution, thus allowing us to slow the dialysis of the cell with GTP
S. As shown in Fig. 6A, this resulted in the appearance of inward barium currents. However, unlike under control conditions, where currents remained stable over a time course of 1015 min (not shown), channel activity decayed rapidly, presumably due to activation of G proteins in response to dialysis with GTP
S. The application of prepulses had only little effect on current amplitude, indicating that membrane depolarization was ineffective in reversing the GTP
S-mediated inhibition. For comparison, rat N-type calcium channels would under these conditions display a
200300% increase in peak current amplitude in response to application of the prepulse (44).
|
This leaves two possibilities. First, as in mammalian Cav2.2 calcium channels, G subunits liberated by GTP
S-induced dissociation of the heterotrimeric G protein complex could perhaps directly bind to the channel to inhibit channel opening. If so, then binding would have to be unusually tight since prepulses were ineffective in relieving the GTP
S-mediated inhibition. As seen in Fig. 6B, the LCav2a-5'rbA channel and the rat Cav2.2 channel share a high degree of sequence homology in the first putative G protein binding motif in the domain I-II linker (which overlaps with the calcium channel
subunit interaction site, alpha interaction domain sequence), whereas in the second putative G protein region identified in rat Cav2.1 and Cav2.2 channels (7, 15), there is little sequence similarity to the LCav2a channel. It is, thus, conceivable that this results in a more stable G protein-channel interaction that is resistant to membrane depolarization.
Alternatively, it is possible that G subunits might be incapable of interacting with the LCav2a-5'rbA channel. Instead, GTP
S might trigger an intracellular signaling cascade that could secondarily inhibit channel activity. A number of electrophysiological studies in preparations of identified molluscan neurons (including Lymnaea) could be consistent with the existence of such a mechanism (24). In Helisoma, Phe-Met-Arg-Phe-NH2 triggers a PTX sensitive, Go-mediated inhibition of calcium currents, which results in a depression of neurotransmitter release (45) and inhibition of growth cone motility (46). This effect could be mimicked by GTP
S and was shown to be irreversible (45, 46). Similar observations with Phe-Met-Arg-Phe-NH2 inhibition of calcium currents have been reported for neurons of Helix aspera, (47), Aplysia californica (48), and Lymnaea (49). However, although there are some apparent parallels to our observations with transiently expressed LCav2a-5'rbA channels, it remains to be determined whether GTP
S similarly affects native Lymnaea synaptic calcium currents. Nonetheless, it seems clear that the regulation of transiently expressed LCav2a-5'rbA calcium channels appears to be fundamentally different from those of their mammalian counterparts, but additional studies will need to be conducted to pinpoint the precise signaling mechanism underlying the unique regulation of this Cav2 calcium channel homolog.
Syntaxin1 Regulation of the LCav2a-5'rbA ChannelWe have shown previously that unlike the rat N-type calcium channel (1, 2, 26, 28, 32, 50), the LCav2a calcium channel is incapable of directly interacting with syntaxin1 (21). This is due primarily to the absence of the synaptic protein interaction site in the LCav2a II-III linker region (Fig. 1B). Hence, one would predict that the coexpression of syntaxin1, which in rat N-type calcium channels results in a pronounced hyperpolarizing shift in half-inactivation potential (28, 32, 50), should not affect LCav2a-5'rbA channel gating. Rather than using rat syntaxin1A or 1B, which we have characterized previously, we used Lymnaea syntaxin1 (Lsytx1) for coexpression studies. To demonstrate expression of Lsytx1 in tsA-201 cells, we first generated a Western blot of tissue homogenated from mock- and Lsytx1-transfected cells probed with an antibody to rat syntaxin1 (Fig. 7A). We then coexpressed Lsytx1 with the rat N-type calcium channel to determine whether Lsytx1 mediated similar functional effects to those described for rat syntaxin1A, that is, a hyperpolarizing shift in the midpoint of the steady state inactivation curve, and a tonic G protein inhibition of the channel triggered by syntaxin binding to the channel, which can be assessed by application of strong depolarizing prepulse (26, 32, 50). Interestingly, unlike rat syntaxin1A and 1B, Lsytx1 did not affect the position of the steady state inactivation curve of the rat N-type channel (control, Vh = -44.8 ± 1.7 mV, n = 7; Lsytx1, Vh = -47 ± 2.4 mV, n = 10, see Fig. 7B) despite being able to bind to its domain II-III linker region as we showed recently (21). The lack of Lsytx1 expression on rat N-type channel inactivation is, however, somewhat surprising since Lsytx1 is highly homologous to both rat syntaxin1A and -1B (see Fig. 7C), both of which affect N-type channel gating (32). This result may, thus, provide novel clues about the syntaxin structural determinants that are required to affect N-type channel gating. In the presence of Lsytx1, we did, however, detect tonic G protein inhibition of the rat N-type channel (32 ± 7% current enhancement by the prepulse, n = 10, not shown), which differed significantly from background. Although this effect was not nearly as large at that described by us previously for rat syntaxin1A (80% enhancement after the prepulse; see Ref. 32), this observation together with the Western blot shown in Fig. 7A suggests that Lsytx1 was functionally expressed in tsA-201 cells and is active on the N-type channel.
|
We next examined the action of Lsytx1 on the functional properties of the LCav2a-5'rbA calcium channel. To eliminate any syntaxin-mediated G protein inhibition that might interfere with our ability to record from the channel, we also coexpressed the C-terminal fragment of the -adrenergic receptor kinase (
-ARKct), a known inhibitor of G
-mediated signaling events (26). Under these circumstances, the half-inactivation potential of the channel was not significantly affected by the presence of Lsytx1 (Fig. 7C), consistent with our previous report showing that Lsytx1A is incapable of binding to the channel in vitro (21). These data suggest that the modulation of presynaptic calcium channel gating by syntaxin1 is likely a vertebrate specialization.
Potential Limitations of Our StudyIn this study, the 5' end of rat Cav2.1 was necessary to promote the expression of this channel in mammalian cells. Without this modification, the functional description of the invertebrate channel would not have been possible, thus making use of a chimeric channel was a necessary evil. Based on previous structure function studies on mammalian calcium channels, many of the functional properties that we examined here (permeation, p loops (37, 38); voltage-dependent inactivation, I-II linker and S6 segments (51, 52, 53, 54, 55, 56); calcium-sensitive inactivation, C terminus (57, 58, 59, 60); dihydropyridine sensitivity, IIS5, IIIS6, and IVS6 segments (61, 62); conotoxin block, IIIS5-S6 region (27, 36); syntaxin regulation, II-III linker (1, 2, 7, 11, 26, 28, 32, 50)) are unlikely to be affected by the presence of the mammalian Cav2.1 N-terminal sequence. However, this may not be so with regard to G protein regulation of the channel, if it were to be mediated by a direct action of G subunits on the channel. Although the major G
interaction sites on the mammalian Cav2 channels are found in the domain I-II linker and the C-terminal regions (15, 40, 63), the N terminus has been implicated in regulating the functional effects of G protein
subunits on N-type channel activity (64). Thus, whereas mammalian Cav2.1 channels are typically only weakly regulated by direct action of G
(13, 39, 41), we cannot rule out the possibility that the presence of the Cav2.1 N terminus could enhance a putative direct G
modulation of LCav2a. However, as we discussed above, in light of previous literature, it appears more likely that the GTP
S-mediated inhibition is due to an indirect regulation by one or more second messengers. Future experiments will attempt to delineate the exact messenger pathway involved and whether the observed effects are affected by the presence of the Cav2.1 N terminus.
Novel Perspectives from Our StudiesHere we provide for the first time a functional description of an expressed invertebrate synaptic Cav2 calcium channel in a mammalian cell line.
First, we show here that properties of mammalian Cav2 channels cannot be universally applied to invertebrates, since toxin sensitivity and regulation of the channels by syntaxin1 appear to be unique to vertebrate channels. At the molecular level, such differences may in part reflect modifications or adaptations in the calcium-dependent neurotransmitter release process in either invertebrates or vertebrates.
Second, despite structural divergences, many of the characteristic biophysical features of mammalian P/Q- and N-type channels are found in the LCav2a-5'rbA channels. Selective permeability to ions (barium and calcium) and responsiveness to changes in membrane voltage, including the rate and threshold of activation and inactivation, are remarkably similar, suggesting that the fundamental biophysical characters responsible for calcium- and voltage-dependent synaptic transmission are conserved at the level of the calcium channel. This similarity is shared at a biophysical level of invertebrate synaptic transmission where kinetic features of calcium-dependent neurotransmitter release are comparable with mammalian ones.
Third, as we reported previously, the invertebrate channel LCav2a lacks an equivalent for the mammalian synaptic protein interaction (synprint) site in the II-III linker, enabling it to bind synaptic proteins such as Lymnaea syntaxin1 or even mammalian syntaxin1A. Conversely, there is a reverse compatibility from mammals to invertebrates, where mammalian synprint binds with high affinity to invertebrate synaptic proteins, including syntaxin1, SNAP-25, and synaptotagmin1 (21). A functional consequence is that application of a synprint peptide to an invertebrate synapse blocks synaptic transmission as has been reported in mammals (21). To our surprise however, the invertebrate syntaxin (Lsytx1) does not appear to be functionally compatible with mammalian Cav2 channels. Even though Lsytx1 is strikingly homologous to both mammalian syntaxin1A and syntaxin1B (see Fig. 7C) and capable of physically binding to Cav2.2 (21), we did not observe an Lsytx1-induced reduction in Cav2.2 channel availability. This brings a unique perspective to the relationship between synaptic calcium channels and synaptic proteins, suggesting that Cav2 channels and syntaxin1 may have coevolved to allow for syntaxin-mediated regulation of Cav2 channel activity. Indeed, minor residue changes in syntaxin1 and more profound adaptations such as the insertion of a 250300-amino acid synprint domain into vertebrate Cav2 calcium channels appear required to allow a functional regulation of mammalian Cav2 channels by syntaxin1. The lack of this interaction in invertebrates may perhaps provide an opportunity to reconstitute functional interactions between synaptic calcium channels and synaptic proteins and investigate their consequences on synaptic release, thus providing novel insights into the intricacies of synaptic transmission.
Finally, identified synapses between Lymnaea neurons are highly amenable for analysis using molecular and electrophysiological approaches and optical imaging (21, 65, 66, 67). A combined approach using in vitro expression and hypothesis testing in native invertebrate neurons may, thus, permit us to dissect the interplay between presynaptic calcium channels, second messenger systems, and synaptic proteins.
![]() |
FOOTNOTES |
---|
* This work was supported by an operating grant from the Canadian Institutes of Health Research (to G. W. Z.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
¶ This author holds a postdoctoral fellowship award from the Human Frontier Science Program.
|| This author previously held postdoctoral research fellowships from the Canadian Institutes of Health Research and the Heart and Stroke Foundation of Canada.
** This author holds a Senior Scholar Award from the Alberta Heritage Foundation for Medical Research and is a Canadian Institutes of Health Research Investigator. To whom correspondence should be addressed: Dept. of Physiology and Biophysics, University of Calgary, 3330 Hospital Dr. NW, Calgary, T2N 4N1, Canada. Tel.: 403-220-8687; Fax: 403-210-8106; E-mail: Zamponi{at}ucalgary.ca.
1 The abbreviations used are: GTPS, guanosine 5'-3-O-(thio)triphosphate; BSA, bovine serum albumin.
![]() |
ACKNOWLEDGMENTS |
---|
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() |
---|