Role of Repeat I in the fast inactivation kinetics of the Ca(V)2.3 channel

The molecular basis for inactivation in Ca(V)2.3 (alpha 1E) channels was studied after expression of alpha 1E/alpha 1C (Ca(V)2.3/Ca(V)1.2) chimeras in Xenopus oocytes. In the presence of 10 mM Ba(2+), the CEEE chimera (Repeat I+part of the I-II linker from Ca(V)1.2) displayed inactivation properties similar to Ca(V)1.2 despite being more than 90% homologous to Ca(V)2.3. The transmembrane segments of Repeat I did not appear to be crucial as inactivation of EC(IS1-6)EEE was not significantly different than Ca(V)2.3. In contrast, EC(AID)EEE, with the beta-subunit binding domain from Ca(V)1.2, tended to behave like Ca(V)1.2 in terms of inactivation kinetics and voltage dependence. A detailed kinetic analysis revealed nonetheless that CEEE and EC(AID)EEE retained the fast inactivation time constant (tau(fast) approximately equal to 20-30 ms) that is a distinctive feature of Ca(V)2.3. Altogether, these data suggest that the region surrounding the AID binding site plays a pivotal albeit not exclusive role in determining the inactivation properties of Ca(V)2.3.     

Specificity of plasma membrane targeting by the rous sarcoma virus gag protein

Budding of C-type retroviruses begins when the viral Gag polyprotein is directed to the plasma membrane by an N-terminal membrane-binding (M) domain. While dispersed basic amino acids within the M domain are critical for stable membrane association and consequent particle assembly, additional residues or motifs may be required for specific plasma membrane targeting and binding. We have identified an assembly-defective Rous sarcoma virus (RSV) Gag mutant that retains significant membrane affinity despite having a deletion of the fourth alpha-helix of the M domain. Examination of the mutant protein's subcellular distribution revealed that it was not localized to the plasma membrane but instead was mistargeted to intracytoplasmic membranes. Specific plasma membrane targeting was restored by the addition of myristate plus a single basic residue, by multiple basic residues, or by the heterologous hydrophobic membrane-binding domain from the cellular Fyn protein. These results suggest that the fourth alpha-helix of the RSV M domain promotes specific targeting of Gag to the plasma membrane, either through a direct interaction with plasma membrane phospholipids or a membrane-associated cellular factor or by maintaining the conformation of Gag to expose specific plasma membrane targeting sequences.     

Contribution of cytosolic cysteine residues to the gating properties of the Kir2.1 inward rectifier

The topological model proposed for the Kir2.1 inward rectifier predicts that seven of the channel 13 cysteine residues are distributed along the N- and C-terminus regions, with some of the residues comprised within highly conserved domains involved in channel gating. To determine if cytosolic cysteine residues contribute to the gating properties of Kir2.1, each of the N- and C-terminus cysteines was mutated into either a polar (S, D, N), an aliphatic (A,V, L), or an aromatic (W) residue. Our patch-clamp measurements show that with the exception of C76 and C311, the mutation of individual cytosolic cysteine to serine (S) did not significantly affect the single-channel conductance nor the channel open probability. However, mutating C76 to a charged or polar residue resulted either in an absence of channel activity or a decrease in open probability. In turn, the mutations C311S (polar), C311R (charged), and to a lesser degree C311A (aliphatic) led to an increase of the channel mean closed time due to the appearance of long closed time intervals (T(c) >or= 500 ms) and to a reduction of the reactivation by ATP of rundown Kir2.1 channels. These changes could be correlated with a weakening of the interaction between Kir2.1 and PIP(2), with C311R and C311S being more potent at modulating the Kir2.1-PIP(2) interaction than C311A. The present work supports, therefore, molecular models whereby the gating properties of Kir2.1 depend on the presence of nonpolar or neutral residues at positions 76 and 311, with C311 modulating the interaction between Kir2.1 and PIP(2).     

Galphaq-coupled receptor internalization specifically induced by Galphaq signaling. Regulation by EBP50

In the present report, we investigated the effect of ezrin-radixin-moesin-binding phosphoprotein 50 (EBP50) expression on the agonist-induced internalization of the thromboxane A(2) beta receptor (TPbeta receptor). Interestingly, we found that EBP50 almost completely blocked TPbeta receptor internalization, which could not be reversed by overexpression of G protein-coupled receptor (GPCR) kinases and arrestins. Because we recently demonstrated that EBP50 can bind to and inhibit Galpha(q), we next studied whether Galpha(q) signaling could induce TPbeta receptor internalization, addressing the long standing question about the relationship between GPCR signaling and their internalization. Expression of a constitutively active Galpha(q) mutant (Galpha(q)-R183C) resulted in a robust internalization of the TPbeta receptor, which was unaffected by expression of dominant negative mutants of arrestin-2 and -3, but inhibited by expression of EBP50 or dynamin-K44A, a dominant negative mutant of dynamin. Phospholipase Cbeta and protein kinase C did not appear to significantly contribute to internalization of the TPbeta receptor, suggesting that Galpha(q) induces receptor internalization through a phospholipase Cbeta- and protein kinase C-independent pathway. Surprisingly, there appears to be specificity in Galpha protein-mediated GPCR internalization. Galpha(q)-R183C also induced the internalization of CXCR4 (Galpha(q)-coupled), whereas it failed to do so for the beta(2)-adrenergic receptor (Galpha(s)-coupled). Moreover, Galpha(s)-R201C, a constitutively active form of Galpha(s), had no effect on internalization of the TPbeta, CXCR4, and beta(2)-adrenergic receptors. Thus, we showed that Galpha protein signaling can lead to internalization of GPCRs, with specificity in both the Galpha proteins and GPCRs that are involved. Furthermore, a new function has been described for EBP50 in its capacity to inhibit receptor endocytosis.     

Residues 293 and 294 are ligand contact points of the human angiotensin type 1 receptor

The human angiotensin II type 1 receptor (hAT(1)) was photolabeled with a high-affinity radiolabeled photoreactive analogue of AngII, (125)I-[Sar(1), Val(5), p-Benzoyl-L-phenylalanine(8)]AngII ((125)I-[Sar(1),Bpa(8)]AngII). Chemical cleavage with CNBr produced a 7 kDa fragment (285-334) of the C-terminal portion of the hAT(1). Manual Edman radiosequencing of photolabeled, per-acetylated, and CNBr-fragmented receptor showed that ligand incorporation occurred through Phe(293) and Asn(294) within the seventh transmembrane domain of the hAT(1). Receptor mutants with Met introduced at the presumed contact residues, F293M and N294M, were photolabeled and then digested with CNBr. SDS-PAGE analysis of those digested mutant receptors confirmed the contact positions 293 and 294 through ligand release induced by CNBr digestion. Additional receptor mutants with Met residues introduced into the N- and C-terminal proximity of those residues 293 and 294 of the hAT(1) produced, upon photolabeling and CNBr digestion, fragmentation patterns compatible only with the above contact residues. These data indicate that the C-terminal residue of AngII interacts with residues 293 and 294 of the seventh transmembrane domain of the human AT(1) receptor. Taking into account a second receptor-ligand contact at the second extracellular loop and residue 3 of AngII (Boucard, A. A., Wilkes, B. C., Laporte, S. A., Escher, E., Guillemette, G., and Leduc, R. (2000) Biochemistry 39, 9662-70) the Ang II molecule must adopt an extended structure in the AngII binding pocket.     

Acute G-CSF therapy is not protective during lethal E. coli sepsis

We investigated whether decreases in circulating polymorphonuclear neutrophils (PMN) during lethal Escherichia coli (E. coli) sepsis in canines are related to insufficient host granulocyte colony-stimulating factor (G-CSF). Two-year-old purpose-bred beagles had intraperitoneal E. coli-infected or -noninfected fibrin clots surgically placed. By 10 to 12 h following clot, both infected survivors and nonsurvivors had marked increases (P = 0.001) in serum G-CSF levels (mean peak G-CSF ng/ml +/- SE, 1,931 +/- 364 and 2,779 +/- 681, respectively) compared with noninfected controls (134 +/- 79), which decreased at 24 to 48 h. Despite increases in G-CSF, infected clot placement caused delayed (P = 0.06) increases in PMN (mean +/- SE change from baseline in cells x 10(3)/mm(3) at 24 and 48 h) in survivors (+3.9 +/- 3.9 and +13.8 +/- 3.6) compared with noninfected controls (+13.1 +/- 2.8 and +9.1 +/- 2.5). Furthermore, infected nonsurvivors had decreases in PMN (-1.4 +/- 1.0 and -1.1 +/- 2.3, P = 0.006 compared with the other groups). We next investigated whether administration of G-CSF immediately after clot placement and continued for 96 h to produce more rapid and prolonged high levels of G-CSF after infection would alter PMN levels. Although G-CSF caused large increases in PMN compared with control protein from 2 to 48 h following clot in noninfected controls, it caused much smaller increases in infected survivors and decreases in infected nonsurvivors (P = 0.03 for the ordered effect of G-CSF comparing the three groups). Thus insufficient host G-CSF is unlikely the cause of decreased circulating PMN in this canine model of sepsis. Other factors associated with sepsis either alone or in combination with G-CSF itself may reduce increases or cause decreases in circulating PMN.     

Nedd8 modification of cul-1 activates SCF(beta(TrCP))-dependent ubiquitination of IkappaBalpha

Regulation of NF-kappaB occurs through phosphorylation-dependent ubiquitination of IkappaBalpha, which is degraded by the 26S proteasome. Recent studies have shown that ubiquitination of IkappaBalpha is carried out by a ubiquitin-ligase enzyme complex called SCF(beta(TrCP)). Here we show that Nedd8 modification of the Cul-1 component of SCF(beta(TrCP)) is important for function of SCF(beta(TrCP)) in ubiquitination of IkappaBalpha. In cells, Nedd8-conjugated Cul-1 was complexed with two substrates of SCF(beta(TrCP)), phosphorylated IkappaBalpha and beta-catenin, indicating that Nedd8-Cul-1 conjugates are part of SCF(beta(TrCP)) in vivo. Although only a minute fraction of total cellular Cul-1 is modified by Nedd8, the Cul-1 associated with ectopically expressed betaTrCP was highly enriched for the Nedd8-conjugated form. Moreover, optimal ubiquitination of IkappaBalpha required Nedd8 and the Nedd8-conjugating enzyme, Ubc12. The site of Nedd8 ligation to Cul-1 is essential, as SCF(beta(TrCP)) containing a K720R mutant of Cul-1 only weakly supported IkappaBalpha ubiquitination compared to SCF(beta(TrCP)) containing WT Cul-1, suggesting that the Nedd8 ligation of Cul-1 affects the ubiquitination activity of SCF(beta(TrCP)). These observations provide a functional link between the highly related ubiquitin and Nedd8 pathways of protein modification and show how they operate together to selectively target the signal-dependent degradation of IkappaBalpha.     

pH and external Ca(2+) regulation of a small conductance Cl(-) channel in kidney distal tubule

A single channel characterization of the Cl(-) channels in distal nephron was undertaken using vesicles prepared from plasma membranes of isolated rabbit distal tubules. The presence in this vesicle preparation of ClC-K type Cl(-) channels was first established by immunodetection using an antibody raised against ClC-K isoforms. A ClC-K1 based functional characterization was next performed by investigating the pH and external Ca(2+) regulation of a small conductance Cl(-) channel which we identified previously by channel incorporation experiments. Acidification of the cis (external) solution from pH 7.4 to 6.5 led to a dose-dependent inhibition of the channel open probability P(O). Similarly, changing the trans pH from 7.4 to 6.8 resulted in a 4-fold decrease of the channel P(O) with no effect on the channel conductance. Channel activity also appeared to be regulated by cis (external) Ca(2+) concentration, with a dose-dependent increase in channel activity as a function of the cis Ca(2+) concentration. It is concluded on the basis of these results that the small conductance Cl(-) channel present in rabbit distal tubules is functionally equivalent to the ClC-K1 channel in the rat. In addition, the present work constitutes the first single channel evidence for a chloride channel regulated by external Ca(2+).     

Mutations in the EF-hand motif impair the inactivation of barium currents of the cardiac alpha1C channel.

Calcium-dependent inactivation has been described as a negative feedback mechanism for regulating voltage-dependent calcium influx in cardiac cells. Most recent evidence points to the C-terminus of the alpha1C subunit, with its EF-hand binding motif, as being critical in this process. The EF-hand binding motif is mostly conserved between the C-termini of six of the seven alpha1 subunit Ca2+ channel genes. The role of E1537 in the C-terminus of the alpha1C calcium channel inactivation was investigated here after expression in Xenopus laevis oocytes. Whole-cell currents were measured in the presence of 10 mM Ba2+ or 10 mM Ca2+ after intracellular injection of 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid. Against all expectations, our results showed a significant reduction in the rate of voltage-dependent inactivation as measured in Ba2+ solutions for all E1537 mutants, whereas calcium-dependent inactivation appeared unscathed. Replacing the negatively charged glutamate residue by neutral glutamine, glycine, serine, or alanine significantly reduced the rate of Ba2+-dependent inactivation by 1.5-fold (glutamine) to 3.5-fold (alanine). The overall rate of macroscopic inactivation measured in Ca2+ solutions was also reduced, although a careful examination of the distribution of the fast and slow time constants suggests that only the slow time constant was significantly reduced in the mutant channels. The fast time constant, the hallmark of Ca2+-dependent inactivation, remained remarkably constant among wild-type and mutant channels. Moreover, inactivation of E1537A channels, in both Ca2+ and Ba2+ solutions, appeared to decrease with membrane depolarization, whereas inactivation of wild-type channels became faster with positive voltages. All together, our results showed that E1537 mutations impaired voltage-dependent inactivation and suggest that the proximal part of the C-terminus may play a role in voltage-dependent inactivation in L-type alpha1C channels.