Mark Hnatowich

STRUCTURE-FUNCTION ANALYSIS OF CALX1.1, A NA+-CA2+EXCHANGER FROM DROSOPHILA : MUTAGENESIS OF IONIC REGULATORY SITES

Cytoplasmic Na+ and Ca2+ regulate the activity of Na+-Ca2+ exchange proteins, in addition to serving as the transported ions, and protein regions involved in these processes have been identified for the canine cardiac Na+-Ca2+ exchanger, NCX1.1. Although protein regions associated with Na+ i - and Ca2+ i -dependent regulation are highly conserved among cloned Na+-Ca2+ exchangers, it is unknown whether or not the structure-function relationships characteristic of NCX1.1 apply to any other exchangers. Therefore, we studied structure-function relationships in a Na+-Ca2+ exchanger from Drosophila, CALX1.1, which is unique among characterized members of this family of proteins in that μm levels of Ca2+ i inhibit exchange current. Wild-type and mutant CALX1.1 exchangers were expressed in Xenopus oocytes and characterized electrophysiologically using the giant excised patch technique. Mutations within the putative regulatory Ca2+ i binding site of CALX1.1, like corresponding alterations in NCX1.1, led to reduced ability (i.e. D516V and D550I) or inability (i.e. G555P) of Ca2+ i to inhibit Na+-Ca2+exchange activity. Similarly, mutations within the putative XIP region of CALX1.1, as in NCX1.1, led to two distinct phenotypes: acceleration (i.e. K306Q) and elimination (i.e. Δ310–313) of Na+ i -dependent inactivation. These results indicate that the respective regulatory roles of the Ca2+ i binding site and XIP region are conserved between CALX1.1 and NCX1.1, despite opposite responses to Ca2+ i . We extended these findings using chimeric constructs of CALX1.1 and NCX1.1 to determine whether or not functional interconversion of Ca2+ i regulatory phenotypes was feasible. With one chimera (i.e. CALX:NCX:CALX), substitution of a 193-amino acid segment, from the large intracellular loop of NCX1.1, for the corresponding 177-amino acid segment of CALX1.1 led to an exchanger that was stimulated by Ca2+ i . This result indicates that the regulatory Ca2+ i binding site of NCX1.1 retains function in a CALX1.1 parent transporter and that the substituted segment contains some of the amino acid sequence(s) required for transduction of the Ca2+ i binding signal.

Crystal structures of progressive Ca2+ binding states of the Ca2+ sensor Ca2+ binding domain 1 (CBD1) from the CALX Na+/Ca2+ exchanger reveal incremental conformational transitions.

Na+/Ca2+ exchangers (NCX) constitute a major Ca2+ export system that facilitates the re-establishment of cytosolic Ca2+ levels in many tissues. Ca2+ interactions at its Ca2+ binding domains (CBD1 and CBD2) are essential for the allosteric regulation of Na+/Ca2+ exchange activity. The structure of the Ca2+-bound form of CBD1, the primary Ca2+ sensor from canine NCX1, but not the Ca2+-free form, has been reported, although the molecular mechanism of Ca2+ regulation remains unclear. Here, we report crystal structures for three distinct Ca2+ binding states of CBD1 from CALX, a Na+/Ca2+ exchanger found in Drosophila sensory neurons. The fully Ca2+-bound CALX-CBD1 structure shows that four Ca2+ atoms bind at identical Ca2+ binding sites as those found in NCX1 and that the partial Ca2+ occupancy and apoform structures exhibit progressive conformational transitions, indicating incremental regulation of CALX exchange by successive Ca2+ binding at CBD1. The structures also predict that the primary Ca2+ pair plays the main role in triggering functional conformational changes. Confirming this prediction, mutagenesis of Glu455, which coordinates the primary Ca2+ pair, produces dramatic reductions of the regulatory Ca2+ affinity for exchange current, whereas mutagenesis of Glu520, which coordinates the secondary Ca2+ pair, has much smaller effects. Furthermore, our structures indicate that Ca2+ binding only enhances the stability of the Ca2+ binding site of CBD1 near the hinge region while the overall structure of CBD1 remains largely unaffected, implying that the Ca2+ regulatory function of CBD1, and possibly that for the entire NCX family, is mediated through domain interactions between CBD1 and the adjacent CBD2 at this hinge.

Endogenous digitalis-like factors: in vitro comparison of biological and immunological activities of peptide and steroid candidates.

Endogenous substances that modulate the activity of (Na+ + K+)-ATPase through interaction at the cardiac glycoside site have been postulated. Reports of digitalis-like biological and immunological activity exhibited by certain ACTH/MSH peptides and 14-OH steroids make these compounds potential candidates as endogenous digitalis-like factors. We tested several ACTH/MSH peptides and 14 alpha-OH steroids in four in vitro assays and detected no significant cardiac glycoside-like activity. On the other hand, chlormadinone acetate, a progesterone derivative shown to bind with high affinity to the digitalis receptor, was nearly equipotent to digoxigenin in a [3H]ouabain radioreceptor assay. In a [3H]digoxin radioimmunoassay, however, digoxigenin and digoxin were equipotent but chlormadinone acetate was inactive. A clear dissociation between radioreceptor assay and radioimmunoassay activity was also observed using 15 beta-OH-progesterone. Our findings indicate that (a) ACTH/MSH peptides and 14 alpha-OH steroids are not viable candidates as endogenous digitalis-like factors, (b) digoxin antibodies are not necessarily directed at molecular determinants critical for biological activity, and (c) among the compounds reported to exhibit digitalis-like activity and postulated to share structural features with an endogenous steroidal digitalis-like factor, only chlormadinone acetate and its congeners appear to constitute tenable models.

Cold-restraint stress reduces [3H]etorphine binding to rat brain membranes: Influence of acute and chronic morphine and naloxone

[3H]Etorphine binding was characterized in rat brain homogenates depleted of endogenous opioids from animals acutely and chronically treated with morphine or naloxone and either unstressed or subjected to a 3-h restraint period in the cold. There was significant reduction in the number of high-affinity opiate binding sites in brain tissue from stressed as compared to unstressed animals. Despite the fact that the opiate drug regimens used produce marked behavioral and physiological effects, stress-induced opiate receptor depletion was not influenced by the drugs or by withdrawal. The various drug treatments also failed to produce significant changes in opiate receptor site densities or affinities in either stressed or unstressed animals. We propose that persistent activation of opiate receptors by endogenous opioids released during restraint stress leads to receptor down-regulation.

Effect of binding protein surface charge on palmitate uptake by hepatocyte suspensions

Studies were directed at determining whether hepatocytes, isolated from female Sprague‐Dawley rats, facilitate the uptake of protein‐bound long‐chain fatty acids. We postulated one form of facilitated uptake may occur through an ionic interaction between the protein‐ligand complex and the cell surface. These interactions are expected to supply additional ligand to the cell for uptake. The clearance rate of [3H]‐palmitate in the presence of α1‐acid‐glycoprotein (pI=2.7), albumin (pI=4.9) and lysozyme (pI=11.0) was investigated. Palmitate uptake was determined in the presence of protein concentrations that resulted in similar unbound ligand fractions (=0.03). The experimental clearance rates were compared to the theoretical predictions based upon the diffusion‐reaction model. By use of our experimentally determined equilibrium binding and dissociation rate constants for the various protein‐palmitate complexes, the diffusion‐reaction model predicted clearance rates were 4.9 μl s−1/106 cells, 4.8 μl s−1/106 cells and 5.5 μl s−1/106 cells for α1‐acid‐glycoprotein, albumin and lysozyme, respectively; whereas the measured hepatocyte palmitate clearance rates were 1.2±0.1 μl s−1/106 cells, 2.3±0.3 μl s−1/106 cells and 7.1±0.7 μl s−1/106, respectively. Hepatocyte palmitate clearance was significantly faster (P<0.01) in the presence of lysozyme than albumin which was significantly faster than α1‐acid‐glycoprotein (P<0.01). The marked difference in clearance rates could not be explained by considering differences in solution viscosity. Our results are consistent with the notion that ionic interactions between protein‐ligand complexes and the cell surface facilitate the ligand uptake by decreasing the diffusional distance of the unbound ligand and/or by facilitating the protein‐ligand dissociation rate.

Apparent Competitive Inhibition of Radioligand Binding to Receptors: Experimental and Theoretical Considerations in the Analysis of Equilibrium Binding Data

Radioligand binding and displacement experiments are often interpreted in terms of simple competition between two ligands for occupancy of a single binding site on a receptor. Given our current understanding of the complexities of receptor structure and function, it is probable that more complex interactions occur in many cases. By analysis of a hypothetical two-site receptor model, we show that apparent competitive inhibition can arise in several ways, depending on the specificities of the two sites and the interactions between them. We show that binding experiments can in some cases be used to rule out certain models from among a group of apparently plausible ones, provided that experimental criteria are met which permit a meaningful statistical comparison of models to be made. Ideally, these should include: i) an independent study of ligand and inhibitor binding in the absence of each other; ii) carrying out saturation binding and displacement experiments over as wide a range of ligand and inhibitor concentrations as possible; iii) computerized curve-fitting and statistical analysis as a tool for model-testing. While practical limitations may restrict the attainment of such goals, a thorough study of the equilibrium binding properties of a particular receptor system provides the foundation for the design of more definitive experiments at the molecular level, upon which the proof of any binding model ultimately must rest.

Effect of nitric oxide on albumin-palmitate binding

Bovine serum albumin (albumin) was modified by treatment with nitric oxide (NO) to form S-nitrosoalbumin. Analysis of the reduced sulfhydryl groups showed that more than 99% of the albumin was converted to S-nitrosoalbumin. Using a 1:1 molar ratio of protein:palmitate, the unbound palmitate fraction in the presence of S-nitrosoalbumin was determined to be greater (28%) than in the presence of albumin as determined by heptane: water partitioning. NO degradation products neither affected the palmitate heptane:water partition ratio in the absence of binding protein nor the hepatocyte uptake of [3H]palmitic acid. The equilibrium association constants (Ka) for albumin-palmitate and S-nitrosoalbumin-palmitate complexes were determined using the stepwise equilibrium model. The Ka for the first and second palmitate binding sites were (4.6 +/- 1.2) x 10(8) M-1 and (3.3 +/- 0.5) x 10(7) M-1 and (3.1 +/- 0.9) x 10(8) M-1 and (1.3 +/- 0.8) x 10(8) M-1 for albumin and S-nitrosoalbumin, respectively. Thus, the increased unbound fraction of palmitate in the presence of S-nitrosoalbumin was apparently due to a decreased binding affinity at the first high-affinity binding site. Palmitate uptake by hepatocyte suspensions was 27% higher in the presence of S-nitrosoalbumin as compared with albumin. This increase paralleled the increased unbound palmitate fraction. When the albumin concentration was adjusted to account for the increased unbound fraction, there was no difference in the palmitate uptake rates between albumin and S-nitrosoalbumin. Our findings indicate that under conditions where NO concentrations are high (e.g. cirrhosis) and extensive S-nitrosylation of serum albumin occurs, the decreased ligand binding ability of S-nitrosoalbumin may be an important consideration when modeling drug uptake in pathological states.

TGFβ1 regulates scleraxis expression in primary cardiac myofibroblasts by a Smad-independent mechanism.

In cardiac wound healing following myocardial infarction (MI), relatively inactive resident cardiac fibroblasts phenoconvert to hypersynthetic/secretory myofibroblasts that produce large quantities of extracellular matrix (ECM) and fibrillar collagen proteins. Our laboratory and others have identified TGFβ1 as being a persistent stimulus in the chronic and inappropriate wound healing phase that is marked by hypertrophic scarring and eventual stiffening of the entire myocardium, ultimately leading to the pathogenesis of heart failure following MI. Ski is a potent negative regulator of TGFβ/Smad signaling with known antifibrotic effects. Conversely, Scleraxis is a potent profibrotic basic helix-loop-helix transcription factor that stimulates fibrillar collagen expression. We hypothesize that TGFβ1 induces Scleraxis expression by a novel Smad-independent pathway. Our data support the hypothesis that Scleraxis expression is induced by TGFβ1 through a Smad-independent pathway in the cardiac myofibroblast. Specifically, we demonstrate that TGFβ1 stimulates p42/44 (Erk1/2) kinases, which leads to increased Scleraxis expression. Inhibition of MEK1/2 using U0126 led to a sequential temporal reduction of phospho-p42/44 and subsequent Scleraxis expression. We also found that adenoviral Ski expression in primary myofibroblasts caused a significant repression of endogenous Scleraxis expression at both the mRNA and protein levels. Thus we have identified a novel TGFβ1-driven, Smad-independent, signaling cascade that may play an important role in regulating the fibrotic response in activated cardiac myofibroblasts following cardiac injury.

SnoN as a novel negative regulator of TGF-β/Smad signaling: a target for tailoring organ fibrosis.

Remodeling of the extracellular matrix is beneficial during the acute wound healing stage following tissue injury. In the short term, resident fibroblasts and myofibroblasts regulate the matrix remodeling process through production of matricellular protein components that provide structural support to the damaged tissue. This process is largely governed by the transforming growth factor-β1 (TGF-β1) pathway, a critical mediator of the remodeling process. In the long term, chronic activation of the TGF-β1 pathway promotes excessive synthesis and deposition of matrix proteins, including fibrillar collagens, which ultimately leads to organ failure. SnoN (and its alternatively-spliced isoforms SnoN2, SnoA, and SnoI) is one of four members of a family of negative regulators of TGF-β1 signaling that includes Ski and functional Smad-suppressing elements on chromosomes 15 and 18. SnoN has been shown to be structurally and functionally similar to Ski and has been demonstrated to directly interact with Ski to abrogate gene expression. Despite this, little progress has been made in delineating a specific role for SnoN in the regulation of myofibroblast phenotype and function. This review outlines the current body of knowledge of what we refer to as the Ski-Sno superfamily, with a focus on the structural and functional importance of SnoN in mediating the fibrotic response by myofibroblasts following tissue injury.

μ-Calpain-mediated deregulation of cardiac, brain, and kidney NCX1 splice variants

μ-Calpain is a Ca(2+)-activated protease abundant in mammalian tissues. Here, we examined the effects of μ-calpain on three alternatively spliced variants of NCX1 using the giant, excised patch technique. Membrane patches from Xenopus oocytes expressing either heart (NCX1.1), kidney (NCX1.3), or brain (NCX1.4) variants of NCX1 were exposed to μ-calpain and their Na(+)-dependent (I(1)) and Ca(2+)-dependent (I(2)) regulatory phenotypes were assessed. For these exchangers, I(1) inactivation is evident as a Na(+)(i)-dependent decay of peak outward currents whereas I(2) regulation manifests as outward current activation by micromolar Ca(2+)(i) concentrations. Notably, with NCX1.1 and NCX1.4 but not in NCX1.3, higher Ca(2+)(i) levels alleviate I(1) inactivation. Our results show that (i) μ-calpain selectively ablates Ca(2+)-dependent (I(2)) regulation leading to a constitutive activation of exchange current, (ii) μ-calpain has much smaller effects on Na(+)-dependent (I(1)) regulation, produced by a slight destabilization of the I(1) state, and (iii) Ca(2+)-dependent regulation (I(2)) and Ca(2+)-mediated alleviation of I(1) appear to be functionally distinct mechanisms, the latter of which is left largely intact after μ-calpain treatment. The ability of μ-calpain to selectively and constitutively activate Na(+)-Ca(2+) exchange currents may have important pathophysiological implications in tissue where these splice variants are expressed.