Trypsin cleaves acid-sensing ion channel 1a in a domain that is critical for channel gating

Acid-sensing ion channels (ASICs) are neuronal Na(+) channels that are members of the epithelial Na(+) channel/degenerin family and are transiently activated by extracellular acidification. ASICs in the central nervous system have a modulatory role in synaptic transmission and are involved in cell injury induced by acidosis. We have recently demonstrated that ASIC function is regulated by serine proteases. We provide here evidence that this regulation of ASIC function is tightly linked to channel cleavage. Trypsin cleaves ASIC1a with a similar time course as it changes ASIC1a function, whereas ASIC1b, whose function is not modified by trypsin, is not cleaved. Trypsin cleaves ASIC1a at Arg-145, in the N-terminal part of the extracellular loop, between a highly conserved sequence and a sequence that is critical for ASIC1a inhibition by the venom of the tarantula Psalmopoeus cambridgei. This channel domain controls the inactivation kinetics and co-determines the pH dependence of ASIC gating. It undergoes a conformational change during inactivation, which renders the cleavage site inaccessible to trypsin in inactivated channels.     

Structural analysis of the voltage-dependent calcium channel beta subunit functional core and its complex with the alpha 1 interaction domain

Voltage-dependent calcium channels (VDCC) are multiprotein assemblies that regulate the entry of extracellular calcium into electrically excitable cells and serve as signal transduction centers. The alpha1 subunit forms the membrane pore while the intracellular beta subunit is responsible for trafficking of the channel to the plasma membrane and modulation of its electrophysiological properties. Crystallographic analyses of a beta subunit functional core alone and in complex with a alpha1 interaction domain (AID) peptide, the primary binding site of beta to the alpha1 subunit, reveal that beta represents a novel member of the MAGUK protein family. The findings illustrate how the guanylate kinase fold has been fashioned into a protein-protein interaction module by alteration of one of its substrate sites. Combined results indicate that the AID peptide undergoes a helical transition in binding to beta. We outline the mechanistic implications for understanding the beta subunit's broad regulatory role of the VDCC, particularly via the AID.     

The N-terminal extracellular domain is required for polycystin-1-dependent channel activity

Autosomal dominant polycystic kidney disease (PKD) is caused by mutation of polycystin-1 or polycystin-2. Polycystin-2 is a Ca(2+)-permeable cation channel. Polycystin-1 is an integral membrane protein of less defined function. The N-terminal extracellular region of polycystin-1 contains potential motifs for protein and carbohydrate interaction. We now report that expression of polycystin-1 alone in Chinese hamster ovary (CHO) cells and in PKD2-null cells can confer Ca(2+)-permeable non-selective cation currents. Co-expression of a loss-of-function mutant of polycystin-2 in CHO cells does not reduce polycystin-1-dependent channel activity. A polycystin-1 mutant lacking approximately 2900 amino acids of the extracellular region is targeted to the cell surface but does not produce current. Extracellular application of antibodies against the immunoglobulin-like PKD domains reduces polycystin-1-dependent current. These results support the hypothesis that polycystin-1 is a surface membrane receptor that transduces the signal via changes in ionic currents.     

The N-terminal zinc binding domain of ClpX is a dimerization domain that modulates the chaperone function

Clp ATPases are unique chaperones that promote protein unfolding and subsequent degradation by proteases. The mechanism by which this occurs is poorly understood. Here we demonstrate that the N-terminal domain of ClpX is a C4-type zinc binding domain (ZBD) involved in substrate recognition. ZBD forms a very stable dimer that is essential for promoting the degradation of some typical ClpXP substrates such as lambdaO and MuA but not GFP-SsrA. Furthermore, experiments indicate that ZBD contains a primary binding site for the lambdaO substrate and for the cofactor SspB. Removal of ZBD from the ClpX sequence renders the ATPase activity of ClpX largely insensitive to the presence of ClpP, substrates, or the SspB cofactor. All these results indicate that ZBD plays an important role in the ClpX mechanism of function and that ATP binding and/or hydrolysis drives a conformational change in ClpX involving ZBD.     

A Ca2+-binding domain in RyR1 that interacts with the calmodulin binding site and modulates channel activity

A fragment of RyR1 (amino acids 4064-4210) is predicted to fold to at least one lobe of calmodulin and to bind Ca(2+). This fragment of RyR1 (R4064-4210) was subcloned, expressed, refolded, and purified. Consistent with the predicted folding pattern, R4064-4210 was found to bind two molecules of Ca(2+) and undergo a structural change upon binding Ca(2+) that exposes hydrophobic amino acids. R4064-4210 also binds to RyR1, the L-type Ca(2+) channel (Cav(1.1)), and several synthetic calmodulin binding peptides. Both R4064-4210 and a peptide representing the calmodulin-binding region of RyR1 (R3614-3643) alter the Ca(2+) dependence of ((3)H)ryanodine binding to RyR1, suggesting that they may both be interfering with an intramolecular interaction between amino acids 4064-4210 and amino acids 3614-3643 in the native RyR1 to alter or regulate the response of the channel to changes in Ca(2+) concentration. The finding that a domain within RyR1 binds Ca(2+) and interacts with calmodulin-binding motifs may provide insights into the mechanism for calcium- and calmodulin-dependent regulation of this channel and perhaps for its regulation by the L-type Ca(2+) channel.     

Ebp1 is a dsRNA-binding protein associated with ribosomes that modulates eIF2alpha phosphorylation

dsRNA-binding domains (dsRBDs) characterize an expanding family of proteins involved in different cellular processes, ranging from RNA editing and processing to translational control. Here we present evidence that Ebp1, a cell growth regulating protein that is part of ribonucleoprotein (RNP) complexes, contains a dsRBD and that this domain mediates its interaction with dsRNA. Deletion of Ebp1's dsRBD impairs its localization to the nucleolus and its ability to form RNP complexes. We show that in the cytoplasm, Ebp1 is associated with mature ribosomes and that it is able to inhibit the phosphorylation of serine 51 in the eukaryotic initiation factor 2 alpha (eIF2alpha). In response to various cellular stress, eIF2alpha is phosphorylated by distinct protein kinases (PKR, PERK, GCN2, and HRI), and this event results in protein translation shut-down. Ebp1 overexpression in HeLa cells is able to protect eIF2alpha from phosphorylation at steady state and also in response to various treatments. We demonstrate that Ebp1 interacts with and is phosphorylated by the PKR protein kinase. Our results demonstrate that Ebp1 is a new dsRNA-binding protein that acts as a cellular inhibitor of eIF2alpha phosphorylation suggesting that it could be involved in protein translation control.     

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.     

The GK domain of the voltage-dependent calcium channel beta subunit is essential for binding to the alpha subunit

The β subunits of voltage-dependent calcium channels bind the pore-forming α₁ subunit and play an important role in the regulation of calcium channel function. Recently, we have identified a new splice variant of the β₄ subunit, which we have termed the β_4d subunit. The β_4d subunit is a truncated splice variant of the β_4b subunit and lacks parts of the guanylate kinase (GK) domain and the C-terminus. The calcium current in BHK cells expressing α_1C and α₂δ with the β_4d subunit was as small as that without the β_4d subunit. Western blot analysis revealed that β_4d protein was expressed to a lesser extent that the β_4b protein. In addition, a GST pull down assay showed that the β_4d subunit could not interact with the α₁ subunit of the calcium channel. Collectively, our results suggest that the GK domain of the β subunit is essential for the expression of the functional calcium channel.     

Schizosaccharomyces pombe Pxl1 is a paxillin homologue that modulates Rho1 activity and participates in cytokinesis

Schizosaccharomyces pombe Rho GTPases regulate actin cytoskeleton organization and cell integrity. We studied the fission yeast gene SPBC4F6.12 based on its ability to suppress the thermosensitivity of cdc42-1625 mutant strain. This gene, named pxl1(+), encodes a protein with three LIM domains that is similar to paxillin. Pxl1 does not interact with Cdc42 but it interacts with Rho1, and it negatively regulates this GTPase. Fission yeast Pxl1 forms a contractile ring in the cell division region and deletion of pxl1(+) causes a delay in cell-cell separation, suggesting that it has a function in cytokinesis. Pxl1 N-terminal region is required and sufficient for its localization to the medial ring, whereas the LIM domains are necessary for its function. Pxl1 localization requires actin polymerization and the actomyosin ring, but it is independent of the septation initiation network (SIN) function. Moreover, Pxl1 colocalizes and interacts with Myo2, and Cdc15, suggesting that it is part of the actomyosin ring. Here, we show that in cells lacking Pxl1, the myosin ring is not correctly assembled and that actomyosin ring contraction is delayed. Together, these data suggest that Pxl1 modulates Rho1 GTPase signaling and plays a role in the formation and contraction of the actomyosin ring during cytokinesis.     

Mutations of nonconserved residues within the calcium channel alpha1-interaction domain inhibit beta-subunit potentiation

Voltage-dependent calcium channels consist of a pore-forming subunit (Ca(V)alpha(1)) that includes all the molecular determinants of a voltage-gated channel, and several accessory subunits. The ancillary beta-subunit (Ca(V)beta) is a potent activator of voltage-dependent calcium channels, but the mechanisms and structural bases of this regulation remain elusive. Ca(V)beta binds reversibly to a conserved consensus sequence in Ca(V)alpha(1), the alpha(1)-interaction domain (AID), which forms an alpha-helix when complexed with Ca(V)beta. Conserved aromatic residues face to one side of the helix and strongly interact with a hydrophobic pocket on Ca(V)beta. Here, we studied the effect of mutating residues located opposite to the AID-Ca(V)beta contact surface in Ca(V)1.2. Substitution of AID-exposed residues by the corresponding amino acids present in other Ca(V)alpha(1) subunits (E462R, K465N, D469S, and Q473K) hinders Ca(V)beta's ability to increase ionic-current to charge-movement ratio (I/Q) without changing the apparent affinity for Ca(V)beta. At the single channel level, these Ca(V)1.2 mutants coexpressed with Ca(V)beta(2a) visit high open probability mode less frequently than wild-type channels. On the other hand, Ca(V)1.2 carrying either a mutation in the conserved tryptophan residue (W470S, which impairs Ca(V)beta binding), or a deletion of the whole AID sequence, does not exhibit Ca(V)beta-induced increase in I/Q. In addition, we observed a shift in the voltage dependence of activation by +12 mV in the AID-deleted channel in the absence of Ca(V)beta, suggesting a direct participation of these residues in the modulation of channel activation. Our results show that Ca(V)beta-dependent potentiation arises primarily from changes in the modal gating behavior. We envision that Ca(V)beta spatially reorients AID residues that influence the channel gate. These findings provide a new framework for understanding modulation of VDCC gating by Ca(V)beta.     

Photoaffinity probes for the alpha 1-adrenergic receptor and the calcium channel bind to a common domain in P-glycoprotein

P-glycoprotein is a 130-180-kDa integral membrane protein that is overproduced in multidrug-resistant cells. The protein appears to act as an energy-dependent drug efflux pump that has broad specificity for structurally diverse hydrophobic antitumor drugs. Many agents, such as the calcium channel blocker verapamil, reverse multidrug resistance and also interact with P-glycoprotein. The goal of this work was to determine if a common binding site participates in the transport of antitumor drugs and/or the reversal of drug resistance. This was done by comparing the peptide maps of P-glycoprotein (encoded by mdr1b) after it was labeled with a photoactive calcium channel blocker, [3H]azidopine, and a newly identified photoaffinity analog for P-glycoprotein 2-[4-(4-azido-3-[125I]iodobenzoyl) piperazin-1-yl]-4-amino-6,7-dimethoxyquinazoline [( 125I]iodoaryl azidoprazosin). [125I] Iodoaryl azidoprazosin, which classically has been used to identify the alpha 1-adrenergic receptor, bound to P-glycoprotein and was preferentially competed by vinblastine greater than actinomycin D greater than doxorubicin greater than colchicine. Peptide maps derived from P-glycoprotein labeled with [3H]azidopine or [125I]iodoaryl azidoprazosin were identical. After maximal digestion under conditions for Cleveland mapping, a single major 6-kDa fragment was obtained after digestion with V8 protease, whereas two major fragments, 6.5 and 5.5 kDa, were detected after digestion with chymotrypsin. The 6.0-kDa V8 fragment and the 6.5-kDa chymotrypsin fragment were both found when P-glycoprotein encoded by mdr1a and mdr1b was compared. Despite its specific interaction with P-glycoprotein, neither iodoaryl azidoprazosin nor prazosin markedly reversed resistance compared with verapamil or azidopine. Further, multidrug-resistant cells were 900-fold resistant to vinblastine but only 5-fold resistant to prazosin. These data demonstrate that structurally diverse reversal and/or antitumor agents are likely to have differential affinity for a small common domain of P-glycoprotein.     

The cation channel mucolipin-1 is a bifunctional protein that facilitates membrane remodeling via its serine lipase domain

Phospholipase modulators have been shown to affect the topology of lipid bilayers and the formation of tubulo-vesicular structures, but the specific endogenous phospholipases involved have yet to be identified. Here we show that TRPML1 (MLN1), a Ca(2+)-permeable channel, contributes to membrane remodeling through a serine lipase consensus domain, and thus represents a novel type of bifunctional protein. Remarkably, this serine lipase active site determines the ability of MLN1 to generate tubulo-vesicular extensions in mucolipin-1-expressing oocytes, human fibroblasts and model membrane vesicles. Our demonstration that MLN1 is involved in membrane remodeling and the formation of extensions suggests that it may play a role in the formation of cellular processes linked to the late endosome/lysosome (LE/L) pathway. MLN1 is absent or mutated in patients with mucolipidosis IV (MLIV), a lysosomal disorder with devastating neurological and other consequences. This study provides potential insight into the pathophysiology of MLIV.     

The TPR domain of PgaA is a multifunctional scaffold that binds PNAG and modulates PgaB-dependent polymer processing

The synthesis of exopolysaccharides as biofilm matrix components by pathogens is a crucial factor for chronic infections and antibiotic resistance. Many periplasmic proteins involved in polymer processing and secretion in Gram-negative synthase dependent exopolysaccharide biosynthetic systems have been individually characterized. The operons responsible for the production of PNAG, alginate, cellulose and the Pel polysaccharide each contain a gene that encodes an outer membrane associated tetratricopeptide repeat (TPR) domain containing protein. While the TPR domain has been shown to bind other periplasmic proteins, the functional consequences of these interactions for the polymer remain poorly understood. Herein, we show that the C-terminal TPR region of PgaA interacts with the de-N-acetylase domain of PgaB, and increases its deacetylase activity. Additionally, we found that when the two proteins form a complex, the glycoside hydrolase activity of PgaB is also increased. To better understand structure-function relationships we determined the crystal structure of a stable TPR module, which has a conserved groove formed by three repeat motifs. Tryptophan quenching, mass spectrometry analysis and molecular dynamics simulation studies suggest that the crystallized TPR module can bind PNAG/dPNAG via its electronegative groove on the concave surface, and potentially guide the polymer through the periplasm towards the porin for export. Our results suggest a scaffolding role for the TPR domain that combines PNAG/dPNAG translocation with the modulation of its chemical structure by PgaB.     

Regulation of the L-type calcium channel by alpha 5beta 1 integrin requires signaling between focal adhesion proteins

The L-type calcium channel is the major calcium influx pathway in vascular smooth muscle and is regulated by integrin ligands, suggesting an important link between extracellular matrix and vascular tone regulation in tissue injury and remodeling. We examined the role of integrin-linked tyrosine kinases and focal adhesion proteins in regulation of L-type calcium current in single vascular myocytes. Soluble tyrosine kinase inhibitors blocked the increase in current produced by alpha(5) integrin antibody or fibronectin, whereas tyrosine phosphatase inhibition enhanced the effect. Cell dialysis with an antibody to focal adhesion kinase or with FRNK, the C-terminal noncatalytic domain of focal adhesion kinase, produced moderate (24 or 18%, respectively) inhibition of basal current but much greater inhibition (63 or 68%, respectively) of integrin-enhanced current. A c-Src antibody and peptide inhibitors of the Src homology-2 domain or a putative Src tyrosine phosphorylation site on the channel produced similar inhibition. Antibodies to the cytoskeletal proteins paxillin and vinculin, but not alpha-actinin, inhibited integrin-dependent current by 65-80%. Therefore, alpha(5)beta(1) integrin appears to regulate a tyrosine phosphorylation cascade involving Src and various focal adhesion proteins that control the function of the L-type calcium channel. This interaction may represent a novel mechanism for control of calcium influx in vascular smooth muscle and other cell types.     

straightjacket is required for the synaptic stabilization of cacophony, a voltage-gated calcium channel alpha1 subunit

In a screen to identify genes involved in synaptic function, we isolated mutations in Drosophila melanogaster straightjacket (stj), an alpha(2)delta subunit of the voltage-gated calcium channel. stj mutant photoreceptors develop normal synaptic connections but display reduced "on-off" transients in electroretinogram recordings, indicating a failure to evoke postsynaptic responses and, thus, a defect in neurotransmission. stj is expressed in neurons but excluded from glia. Mutants exhibit endogenous seizure-like activity, indicating altered neuronal excitability. However, at the synaptic level, stj larval neuromuscular junctions exhibit approximately fourfold reduction in synaptic release compared with controls stemming from a reduced release probability at these synapses. These defects likely stem from destabilization of Cacophony (Cac), the primary presynaptic alpha(1) subunit in D. melanogaster. Interestingly, neuronal overexpression of cac partially rescues the viability and physiological defects in stj mutants, indicating a role for the alpha(2)delta Ca(2+) channel subunit in mediating the proper localization of an alpha(1) subunit at synapses.     

Monkey rotavirus binding to alpha2beta1 integrin requires the alpha2 I domain and is facilitated by the homologous beta1 subunit

Rotaviruses utilize integrins during virus-cell interactions that lead to infection. Cell binding and infection by simian rotavirus SA11 were inhibited by antibodies (Abs) to the inserted (I) domain of the alpha2 integrin subunit. To determine directly which integrins or other proteins bind rotaviruses, cell surface proteins precipitated by rotaviruses were compared with those precipitated by anti-alpha2beta1 Abs. Two proteins precipitated by SA11 and rhesus rotavirus RRV from MA104 and Caco-2 cells migrated indistinguishably from alpha2beta1 integrin, and SA11 precipitated beta1 from alpha2beta1-transfected CHO cells. These viruses specifically precipitated two MA104 cell proteins only, but an additional 160- to 165-kDa protein was precipitated by SA11 from Caco-2 cells. The role of the alpha2 I domain in rotavirus binding, infection, and growth was examined using CHO cell lines expressing wild-type or mutated human alpha2 or alpha2beta1. Infectious SA11 and RRV, but not human rotavirus Wa, specifically bound CHO cell-expressed human alpha2beta1 and, to a lesser extent, human alpha2 combined with hamster beta1. Binding was inhibited by anti-alpha2 I domain monoclonal Abs (MAbs), but not by non-I domain MAbs to alpha2, and required the presence of the alpha2 I domain. Amino acid residues 151, 221, and 254 in the metal ion-dependent adhesion site of the alpha2 I domain that are necessary for type I collagen binding to alpha2beta1 were not essential for rotavirus binding. Rotavirus-alpha2beta1 binding led to increased virus infection and RRV growth. SA11 and RRV require the alpha2 I domain for binding to alpha2beta1, and their binding to this integrin is distinguishable from that of collagen.