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.     

Molecular determinants of syntaxin 1 modulation of N-type calcium channels

We have previously reported that syntaxin 1A, a component of the presynaptic SNARE complex, directly modulates N-type calcium channel gating in addition to promoting tonic G-protein inhibition of the channels, whereas syntaxin 1B affects channel gating but does not support G-protein modulation (Jarvis, S. E., and Zamponi, G. W. (2001) J. Neurosci. 21, 2939-2948). Here, we have investigated the molecular determinants that govern the action of syntaxin 1 isoforms on N-type calcium channel function. In vitro evidence shows that both syntaxin 1 isoforms physically interact with the G-protein beta subunit and the synaptic protein interaction (synprint) site contained within the N-type calcium channel domain II-III linker region. Moreover, in vitro evidence suggests that distinct domains of syntaxin participate in each interaction, with the COOH-terminal SNARE domain (residues 183-230) binding to Gbeta and the N-terminal (residues 1-69) binding to the synprint motif of the channel. Electrophysiological analysis of chimeric syntaxin 1A/1B constructs reveals that the variable NH(2)-terminal domains of syntaxin 1 are responsible for the differential effects of syntaxin 1A and 1B on N-type calcium channel function. Because syntaxin 1 exists in both "open" and "closed" conformations during exocytosis, we produced a constitutively open form of syntaxin 1A and found that it still promoted G-protein inhibition of the channels, but it did not affect N-type channel availability. This state dependence of the ability of syntaxin 1 to mediate N-type calcium channel availability suggests that syntaxin 1 dynamically regulates N-type channel function during various steps of exocytosis. Finally, syntaxin 1A appeared to compete with Ggamma for the Gbeta subunit both in vitro and under physiological conditions, suggesting that syntaxin 1A may contain a G-protein gamma subunit-like domain.     

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.     

Selective inhibition of Cav3.3 T-type calcium channels by Galphaq/11-coupled muscarinic acetylcholine receptors

T-type calcium channels play critical roles in controlling neuronal excitability, including the generation of complex spiking patterns and the modulation of synaptic plasticity, although the mechanisms and extent to which T-type Ca(2+) channels are modulated by G-protein-coupled receptors (GPCRs) remain largely unexplored. To examine specific interactions between T-type Ca(2+) channel subtypes and muscarinic acetylcholine receptors (mAChRS), the Cav3.1 (alpha(1G)), Cav3.2 (alpha(1H)), and Cav3.3 (alpha) T-type Ca(2+)(1I)channels were co-expressed with the M1 Galpha(q/11)-coupled mAChR. Perforated patch recordings demonstrate that activation of M1 receptors has a strong inhibitory effect on Cav3.3 T-type Ca(2+) currents but either no effect or a moderate stimulating effect on Cav3.1 and Cav3.2 peak current amplitudes. This differential modulation was observed for both rat and human T-type Ca(2+) channel variants. The inhibition of Cav3.3 channels by M1 receptors is reversible, use-independent, and associated with a concomitant increase in inactivation kinetics. Loss-of-function experiments with genetically encoded antagonists of Galpha and Gbetagamma proteins and gain-of-function experiments with genetically encoded Galpha subtypes indicate that M1 receptor-mediated inhibition of Cav3.3 occurs through Galpha(q/11). This is supported by experiments showing that activation of the M3 and M5 Galpha(q/11)-coupled mAChRs also causes inhibition of Cav3.3 currents, although Galpha(i)-coupled mAChRs (M2 and M4) have no effect. Examining Cav3.1-Cav3.3 chimeric channels demonstrates that two distinct regions of the Cav3.3 channel are necessary and sufficient for complete M1 receptor-mediated channel inhibition and represent novel sites not previously implicated in T-type channel modulation.     

Taphonomy of modern communal burrow systems of the Plains vizcacha (Lagostomus maximus,Chinchillidae) in the Pampas region of Argentina: implications for the fossil record

The Plains vizcacha (Lagostomus maximus) is one of the largest rodents in South America. They live in communal burrow systems (vizcacheras) shaped by complex subterranean galleries which produce a strong impact on the local landscape. This paper presents the results of an actualistic study conducted with abandoned vizcacheras from the Pampas region of Argentina. The main objective is to evaluate the role of this rodent in the formation of the fossil record. Results indicate that the Plains vizcacha is responsible for the mixing, accumulation, and transport of materials; such as sticks, caliche, dung, feces, and abundant bone remains. Their burrowing activity and the accumulating habits, modifies the landscape, creating environments conducive to the buildup of objects and the reuse by different animals. These characteristics result in very complex associations of materials of different origins; making this rodent an important taphonomic agent with the potential to significantly impact the fossil record.     

Evolutionary and structural constraints influencing apolipoprotein A-I amyloid behavior

Apolipoprotein A-I (apoA-I) has a key function in the reverse cholesterol transport. However, aggregation of apoA-I single point mutants can lead to hereditary amyloid pathology. Although several studies have tackled the biophysical and structural consequences introduced by these mutations, there is little information addressing the relationship between the evolutionary and structural features that contribute to the amyloid behavior of apoA-I. We combined evolutionary studies, in silico mutagenesis and molecular dynamics (MD) simulations to provide a comprehensive analysis of the conservation and pathogenic role of the aggregation-prone regions (APRs) present in apoA-I. Sequence analysis demonstrated that among the four amyloidogenic regions described for human apoA-I, only two (APR1 and APR4) are evolutionary conserved across different species of Sarcopterygii. Moreover, stability analysis carried out with the FoldX engine showed that APR1 contributes to the marginal stability of apoA-I. Structural properties of full-length apoA-I models suggest that aggregation is avoided by placing APRs into highly packed and rigid portions of its native fold. Compared to silent variants extracted from the gnomAD database, the thermodynamic and pathogenic impact of amyloid mutations showed evidence of a higher destabilizing effect. MD simulations of the amyloid variant G26R evidenced the partial unfolding of the alpha-helix bundle with the concomitant exposure of APR1 to the solvent, suggesting an insight into the early steps involved in its aggregation. Our findings highlight APR1 as a relevant component for apoA-I structural integrity and emphasize a destabilizing effect of amyloid variants that leads to the exposure of this region.     

Complex Population Structure and Virulence Differences among Serotype 2 Streptococcus suis Strains Belonging to Sequence Type 28

Streptococcus suis is a major swine pathogen and a zoonotic agent. Serotype 2 strains are the most frequently associated with disease. However, not all serotype 2 lineages are considered virulent. Indeed, sequence type (ST) 28 serotype 2 S. suis strains have been described as a homogeneous group of low virulence. However, ST28 strains are often isolated from diseased swine in some countries, and at least four human ST28 cases have been reported. Here, we used whole-genome sequencing and animal infection models to test the hypothesis that the ST28 lineage comprises strains of different genetic backgrounds and different virulence. We used 50 S. suis ST28 strains isolated in Canada, the United States and Japan from diseased pigs, and one ST28 strain from a human case isolated in Thailand. We report a complex population structure among the 51 ST28 strains. Diversity resulted from variable gene content, recombination events and numerous genome-wide polymorphisms not attributable to recombination. Phylogenetic analysis using core genome single-nucleotide polymorphisms revealed four discrete clades with strong geographic structure, and a fifth clade formed by US, Thai and Japanese strains. When tested in experimental animal models, strains from this latter clade were significantly more virulent than a Canadian ST28 reference strain, and a closely related Canadian strain. Our results highlight the limitations of MLST for both phylogenetic analysis and virulence prediction and raise concerns about the possible emergence of ST28 strains in human clinical cases.     

Use 'em and lose 'em-activity-induced removal of calcium channels from the plasma membrane

Calcium influx via L-type (Cav1.2 and Cav1.3) calcium channels is tightly regulated to ensure optimal intracellular calcium levels. Although much is known about acute modulation of these channels by second messengers, the mechanisms that control their trafficking to and from the plasma membrane remain poorly understood. In this issue of Neuron, Green and colleagues demonstrate that the opening of L-type calcium channels results in negative feedback regulation due to their calcium-dependent internalization.     

Trace metal contents in water and sediments in Samborombón Bay wetland,Argentina

Samborombón Bay is the most extensive myxohaline wetland of Argentina. This Ramsar site is considered a priority area for the conservation of biodiversity in the country. Numerous rivers and channels that drain the Pampean plain, such as the Salado River and the channels Lower Salado, 15, 9, A, 1, and 2, cross this wetland and flow into Samborombón Bay. Trace metal concentration (As, Cd, Cr, Cu, Mn, Ni, Pb, and Zn) in water and sediments were determined in low and high water periods. In the first case, contents were higher than the Guide Levels for the Protection of the Aquatic Biota (GLPAB) in most of the sampling stations. In high water period, only As and Zn were detected. Relevant physico-chemical parameters were analyzed by a principal component analysis (PCA). Trace metal concentrations in sediments suggest, as a whole, that this wetland has been so far exposed to low to moderate levels of anthropic influence. It must be emphasized however, that the presence of metals in both the water and sediments of this bay might have negative effects on the biota and on the regional trophic web.