Interactions between presynaptic Ca2+ channels, cytoplasmic messengers and proteins of the synaptic vesicle release complex.

Influx of Ca(2+) through presynaptic voltage-gated Ca(2+) channels is a key step in rapid neurotransmitter release. The amount of Ca(2+) entering through these channels is modulated by a plethora of intracellular messenger molecules, including betagamma-subunits of G proteins, and protein kinases. In addition, Ca(2+) channels bind physically to proteins of the vesicle-release machinery in a Ca(2+)-dependent manner, which can, in turn, regulate the activity of Ca(2+) channels. Recent evidence suggests that second messengers and presynaptic vesicle-release proteins do not regulate Ca(2+) channel activity as independent entities, but that there is extensive crosstalk between these two mechanisms. The complex interactions between second messengers, vesicle-release proteins and voltage-gated Ca(2+) channels might provide multiple avenues for fine-tuning Ca(2+) entry into the presynaptic terminal and, consequently, neurotransmission.

Distinct molecular determinants govern syntaxin 1A-mediated inactivation and G-protein inhibition of N-type calcium channels.

We have reported recently that syntaxin 1A mediates two effects on N-type channels transiently expressed in tsA-201 cells: a hyperpolarizing shift in the steady-state inactivation curve as well as a tonic inhibition of the channel by G-protein betagamma subunits (Jarvis et al., 2000). Here we have examined some of the molecular determinants and factors that modulate the action of syntaxin 1A on N-type calcium channels. With the additional coexpression of SNAP25, the syntaxin 1A-induced G-protein modulation of the channel became reduced in magnitude by approximately 50% but nonetheless remained significantly higher than the low levels of background inhibition seen with N-type channels alone. In contrast, coexpression of nSec-1 did not reduce the syntaxin 1A-mediated G-protein inhibition; however, interestingly, nSec-1 was able to induce tonic G-protein inhibition even in the absence of syntaxin 1A. Both SNAP25 and nSec-1 blocked the negative shift in half-inactivation potential that was induced by syntaxin 1A. Activation of protein kinase C via phorbol esters or site-directed mutagenesis of three putative PKC consensus sites in the syntaxin 1A binding region of the channel (S802, S896, S898) to glutamic acid (to mimic a permanently phosphorylated state) did not affect the syntaxin 1A-mediated G-protein modulation of the channel. However, in the S896E and S898E mutants, or after PKC-dependent phosphorylation of the wild-type channels, the susceptibility of the channel to undergo shifts in half-inactivation potential was removed. Thus, separate molecular determinants govern the ability of syntaxin 1A to affect N-type channel gating and its modulation by G-proteins.

Syntaxin 1A supports voltage-dependent inhibition of alpha1B Ca2+ channels by Gbetagamma in chick sensory neurons.

N-type Ca(2+) channels are modulated by a variety of G-protein-coupled pathways. Some pathways produce a transient, voltage-dependent (VD) inhibition of N channel function and involve direct binding of G-protein subunits; others require the activation of intermediate enzymes and produce a longer-lasting, voltage-independent (VI) form of inhibition. The ratio of VD:VI inhibition differs significantly among cell types, suggesting that the two forms of inhibition play unique physiological roles in the nervous system. In this study, we explored mechanisms capable of altering the balance of VD and VI inhibition in chick dorsal root ganglion neurons. We report that (1) VD:VI inhibition is critically dependent on the Gbetagamma concentration, with VI inhibition dominant at low Gbetagamma concentrations, and (2) syntaxin-1A (but not syntaxin-1B) shifts the ratio in favor of VD inhibition by potentiating the VD effects of Gbetagamma. Variations in expression levels of G-proteins and/or syntaxin provide the means to alter over a wide range both the extent and the rate of Ca(2+) influx through N channels.

Amino acid residues outside of the pore region contribute to N-type calcium channel permeation.

It is widely believed that the selectivity of voltage-dependent calcium channels is mainly controlled by amino acid residues contained within four p-loop motifs forming the pore of the channel. An examination of the amino acid sequences of high voltage-activated calcium channels reveals that their domain III S5-H5 regions contain a highly conserved motif with homology to known EF hand calcium binding proteins, hinting that this region may contribute to channel permeation. To test this hypothesis, we used site-directed mutagenesis to replace three conserved negatively charged residues in the N-type calcium channel alpha1B subunit (Glu-1321, Asp-1323, and Glu-1332) with positively charged amino acids (lysine and arginine) and studied their effect on ion selectivity using whole cell and single channel patch clamp recordings. Whereas the wild type channels conducted barium much more effectively than calcium, the mutant displayed nearly equal permeabilities for these two ions. Individual replacement of residue 1332 or a double substitution of residues 1321 and 1323 with lysine and arginine, respectively, were equally effective. Disruption of the putative EF hand motif through replacement of the central glycine residue (1326) with proline resulted in a similar effect, indicating that the responses observed with the triple mutant were not due to changes in the net charge of the channel. Overall, our data indicate that residues outside of the narrow region of the pore have the propensity to contribute to calcium channel permeation. They also raise the possibility that interactions of calcium ions with a putative calcium binding domain at the extracellular side of the channel may underlie the differential permeabilities of the channel for barium and calcium ions.

Cysteine string protein regulates G protein modulation of N-type calcium channels.

Cysteine string proteins (CSPs) are secretory vesicle proteins bearing a "J domain" and a palmitoylated cysteine-rich "string" region that are critical for neurotransmitter release. The precise role of CSP in neurotransmission is controversial. Here, we demonstrate a novel interaction between CSP, receptor-coupled trimeric GTP binding proteins (G proteins), and N-type Ca2+ channels. G. subunits interact with the J domain of CSP in an ATP-dependent manner; in contrast, Gbetagamma subunits interact with the C terminus of CSP in both the presence and absence of ATP. The interaction of CSP with both G proteins and N-type Ca2+ channels results in a tonic G protein inhibition of the channels. In view of the crucial importance of N-type Ca2+ channels in presynaptic vesicle release, our data attribute a key role to CSP in the fine tuning of neurotransmission.

Cross-talk between G-protein and protein kinase C modulation of N-type calcium channels is dependent on the G-protein beta subunit isoform.

The modulation of N-type calcium current by protein kinases and G-proteins is a factor in the fine tuning of neurotransmitter release. We have previously shown that phosphorylation of threonine 422 in the alpha(1B) calcium channel domain I-II linker region resulted in a dramatic reduction in somatostatin receptor-mediated G-protein inhibition of the channels and that the I-II linker consequently serves as an integration center for cross-talk between protein kinase C (PKC) and G-proteins (Hamid, J., Nelson, D., Spaetgens, R., Dubel, S. J., Snutch, T. P., and Zamponi, G. W. (1999) J. Biol. Chem. 274, 6195-6202). Here we show that opioid receptor-mediated inhibition of N-type channels is affected to a lesser extent compared with that seen with somatostatin receptors, hinting at the possibility that PKC/G-protein cross-talk might be dependent on the G-protein subtype. To address this issue, we have examined the effects of four different types of G-protein beta subunits on both wild type and mutant alpha(1B) calcium channels in which residue 422 has been replaced by glutamate to mimic PKC-dependent phosphorylation and on channels that have been directly phosphorylated by protein kinase C. Our data show that phosphorylation or mutation of residue 422 antagonizes the effect of Gbeta(1) on channel activity, whereas Gbeta(2), Gbeta(3), and Gbeta(4) are not affected. Our data therefore suggest that the observed cross-talk between G-proteins and protein kinase C modulation of N-type channels is a selective feature of the Gbeta(1) subunit.

Differential modulation of N-type 1B and P/Q-type 1A calcium channels by different G protein subunit isoforms.

Using transient calcium phosphate transfection into the human embryonic kidney tsa-201 cell line and subsequent whole-cell patch-clamp protocols, we examined the tonic modulation of cloned N- and P/Q-type calcium channels by five different G protein beta subunits via strong depolarizing voltage prepulses. For N- and P/Q-type channels, the magnitude of inhibition was dependent on the Gbeta subtype co-expressed. Both the absolute and relative magnitudes of Gbeta subunit-induced inhibition of P/Q-type channels differed from those observed with the N-type channel. For each calcium channel subtype, kinetics of both the prepulse-mediated recovery from inhibition and the re-inhibition following the prepulse were examined for each of the Gbeta subunits by varying either the duration between the pre- and the test pulse or the length of the prepulse. For each channel subtype, we observed a differential Gbeta subunit rank order with regard to the rates of re-inhibition and recovery from inhibition. On average, P/Q-type channels exhibited more rapid rates of recovery from inhibition than those observed with N-type channels. Different Gbeta subtypes mediated different degrees of slowing of activation kinetics. The differential modulation of P/Q- and N-type channels by various Gbeta subtypes may provide a mechanism for fine tuning the amount of calcium entering the presynaptic nerve termini.

Fast inactivation of voltage-dependent calcium channels. A hinged-lid mechanism?

We recently described domains II and III as important determinants of fast, voltage-dependent inactivation of R-type calcium channels (Spaetgens, R. L., and Zamponi, G. W. (1999) J. Biol. Chem. 274, 22428-22438). Here we examine in greater detail the structural determinants of inactivation using a series of chimeras comprising various regions of wild type alpha(1C) and alpha(1E) calcium channels. Substitution of the II S6 and/or III S6 segments of alpha(1E) into the alpha(1C) backbone resulted in rapid inactivation rates that closely approximated those of wild type alpha(1E) channels. However, neither individual or combined substitution of the II S6 and III S6 segments could account for the 60 mV more negative half-inactivation potential seen with wild type alpha(1E) channels, indicating that the S6 regions contribute only partially to the voltage dependence of inactivation. Interestingly, the converse replacement of alpha(1E) S6 segments of domains II, III, or II+III with those of alpha(1C) was insufficient to significantly slow inactivation rates. Only when the I-II linker region and the domain II and III S6 regions of alpha(1E) were concomitantly replaced with alpha(1C) sequence could inactivation be abolished. Conversely, introduction of the alpha(1E) domain I-II linker sequence into alpha(1C) conferred alpha(1E)-like inactivation rates, indicating that the domain I-II linker is a key contributor to calcium channel inactivation. Overall, our data are consistent with a mechanism in which inactivation of voltage-dependent calcium channels may occur via docking of the I-II linker region to a site comprising, at least in part, the domain II and III S6 segments.

G protein modulation of N-type calcium channels is facilitated by physical interactions between syntaxin 1A and Gbetagamma.

The direct modulation of N-type calcium channels by G protein betagamma subunits is considered a key factor in the regulation of neurotransmission. Some of the molecular determinants that govern the binding interaction of N-type channels and Gbetagamma have recently been identified (see, i.e., Zamponi, G. W., Bourinet, E., Nelson, D., Nargeot, J., and Snutch, T. P. (1997) Nature 385, 442-446); however, little is known about cellular mechanisms that modulate this interaction. Here we report that a protein of the presynaptic vesicle release complex, syntaxin 1A, mediates a crucial role in the tonic inhibition of N-type channels by Gbetagamma. When syntaxin 1A was coexpressed with (N-type) alpha(1B) + alpha(2)-delta + beta(1b) channels in tsA-201 cells, the channels underwent a 18 mV negative shift in half-inactivation potential, as well as a pronounced tonic G protein inhibition as assessed by its reversal by strong membrane depolarizations. This tonic inhibition was dramatically attenuated following incubation with botulinum toxin C, indicating that syntaxin 1A expression was indeed responsible for the enhanced G protein modulation. However, when G protein betagamma subunits were concomitantly coexpressed, the toxin became ineffective in removing G protein inhibition, suggesting that syntaxin 1A optimizes, rather than being required for G protein modulation of N-type channels. We also demonstrate that Gbetagamma physically binds to syntaxin 1A, and that syntaxin 1A can simultaneously interact with Gbetagamma and the synprint motif of the N-type channel II-III linker. Taken together, our experiments suggest a mechanism by which syntaxin 1A mediates a colocalization of G protein betagamma subunits and N-type calcium channels, thus resulting in more effective G protein coupling to, and regulation of, the channel. Thus, the interactions between syntaxin, G proteins, and N-type calcium channels are part of the structural specialization of the presynaptic terminal.

Characterization of human recombinant annexin II tetramer purified from bacteria: role of N-terminal acetylation.

Annexin II tetramer (AIIt) is a Ca2+-dependent, phosphatidylserine-binding, and F-actin-bundling phosphoprotein which is localized to both the extracellular and cytoplasmic surfaces of the plasma membrane. The tetramer is composed of two p36 heavy chains and two p11 light chains. We have produced prokaryotic cDNA expression constructs for both p36 and p11. Both proteins were expressed in large amounts in Escherichia coli upon induction with IPTG. Electrospray ionization mass spectrometry and amino acid sequence analysis of purified recombinant p36 (rp36) and recombinant p11 (rp11) suggested that the recombinant proteins were identical to their native counterparts except for the lack of N-terminal acetylation of rp36. Furthermore, the non-acetylated rp36 bound rp11 and formed AIIt. The circular dichroism spectra and urea denaturation profiles of acetylated AIIt and non-acetylated rAIIt were identical. In addition, both the acetylated AIIt and non-acetylated rAIIt were similar in their Ca2+ dependence and concentration dependence of phospholipid liposome aggregation, chromaffin granule aggregation, and F-actin bundling. These results suggest that N-terminal acetylation of p36 is not in fact necessary for binding of the protein to p11 and that N-terminal acetylation does not affect the conformational stability of AIIt or the in vitro activities of AIIt. The availability of large amounts of rAIIt will facilitate further characterization of the structure-function relationships of the protein.

t-Haplotypes of the mouse may involve a change in intercalary DNA.

The naturally occurring t-haplotypes of the mouse exhibit a set of peculiar genetic properties, including strong suppression of crossing over in the segment of chromosome 17 between the loci of T and H--2. Study of the genetics of mutant haplotypes suggests that the observed effects on meiosis and embryonic development may be due to an altered form of intercalary DNA (iDNA) in the relevant chromosomal region (band 17B).