Frédéric Jaisser

Role of beta-subunit domains in the assembly, stable expression, intracellular routing, and functional properties of Na,K-ATPase.

The β-subunit of Na,K-ATPase (βNK) interacts with the catalytic α-subunit (αNK) in the ectodomain, the transmembrane, and the cytoplasmic domain. The functional significance of these different interactions was studied by expressing αNK inXenopus oocytes along with N-terminally modified βNK or with chimeric βNK/βH,K-ATPase (βHK). Complete truncation of the βNK N terminus allows for cell surface-expressed, functional Na,K-pumps that exhibit, however, reduced apparent K+ and Na+ affinities as assessed by electrophysiological measurements. A mutational analysis suggests that these functional effects are not related to a direct interaction of the β N terminus with the αNK but rather that N-terminal truncation induces a conformational change in another functionally relevant β domain. Comparison of the functional properties of αNK·βNK, αNK·βHK, or αNK·βNK/βHK complexes shows that the effect of the βNK on K+ binding is mainly mediated by its ectodomain. Finally, βHK/NK containing the transmembrane domain of βHK produces stable but endoplasmic reticulum-retained αNK·β complexes, while αNK/βHK complexes can leave the ER but exhibit reduced ouabain binding capacity and transport function. Thus, interactions of both the transmembrane and the ectodomain of βNK with αNK are necessary to form correctly folded Na,K-ATPase complexes that can be targeted to the plasma membrane and/or become functionally competent. Furthermore, the β N terminus plays a role in the β-subunit’s folding necessary for correct interactions with the α-subunit.

A direct relationship between plasma aldosterone and cardiac L-type Ca2+ current in mice

Aldosterone is involved in a variety of pathophysiological processes that ultimately cause cardiovascular diseases. Despite this, the physiological role of aldosterone in heart function remains elusive. We took advantage of transgenic mouse models characterized by a renal salt‐losing (SL) or salt‐retaining (SR) phenotype, thus exhibiting chronically high or low plasma aldosterone levels, respectively, to investigate the chronic effects of aldosterone in cardiomyocytes devoid of pathology. On a diet containing normal levels of salt, these animals do not develop any evidence of cardiovascular disease. Using the whole cell patch‐clamp technique on freshly isolated adult ventricular cardiomyocytes, we observed that the amplitude of L‐type Ca2+ currents (ICa) correlates with plasma aldosterone levels. Larger values of ICa are associated with high aldosterone concentrations in SL models, whereas smaller values of ICa were observed in the SR model. Neither the time‐ nor the voltage‐dependent properties of ICa varied measurably. In parallel, we determined whether modulation of ICa by blood concentration of aldosterone has a major physiological impact on the excitation–contraction coupling of the cardiomyocytes. Action potential duration, [Ca2+]i transient amplitude and contraction are increased in the SL model and decreased in the SR model. In conclusion, we demonstrate that the blood concentration of aldosterone exerts chronic regulation of ICa in mouse cardiomyocytes. This regulation has important consequences for excitation–contraction coupling and, potentially, for other Ca2+‐regulated functions in cardiomyocytes.

Molecular Consequences of a Frameshifted DLX3 Mutant Leading to Tricho-Dento-Osseous Syndrome

The homeodomain protein Distal-less-3 (Dlx3) plays a crucial role during embryonic development. This transcription factor is known to be essential for placental formation and to be involved in skin and skeletal organogenesis. In humans, a frameshift mutation in the coding sequence of the DLX3 gene results in an ectodermal dysplasia called Tricho-Dento-Osseous syndrome (TDO). The main features of this autosomal dominant disorder are defects in hair, teeth, and bone. To investigate the functional alterations caused by the mutated DLX3TDO isoform ex vivo, we used tetracycline-inducible osteoblastic and keratinocyte cell lines and calvarial derived osteoblasts in which the expression of DLX3WT and/or DLX3TDO could be regulated and monitored. Immunocytochemical analysis revealed that both DLX3WT and DLX3TDO recombinant proteins are targeted to the nucleus. However, as demonstrated by electrophoresis mobility shift assay, DLX3TDO is not able to bind to the canonical Dlx3 binding site. Furthermore, we demonstrate that the frameshifted C-terminal domain in DLX3TDO is accountable for the loss of DNA binding activity because the C-terminal domain in DLX3WT is not required for DNA binding activity. Although DLX3TDO alone cannot bind to a Dlx3 responsive element, when DLX3WT and DLX3TDO are co-expressed they form a complex that can bind DNA. Concomitant with the inability to bind DNA, DLX3TDO has a defective transcriptional activity. Moreover, the transcriptional activity of DLX3WT is significantly reduced in the presence of the mutated isoform, indicating that DLX3TDO has a dominant negative effect on DLX3WT transcriptional activity.

The β Subunit Modulates Potassium Activation of the Na-K Pumpa

We recently cloned the alpha 1 and the beta 1 and beta 3 subunits of the Na,K-ATPase of the toad Bufo marinus. To investigate possible functional differences between beta 1 and beta 3, we studied the potassium activation of Na-K pumps expressed in the oocyte of Xenopus laevis. Na-K pump activity was measured as K(+)-induced current in voltage-clamped oocytes. We could take advantage of the relative resistance to ouabain conferred by the Bufo alpha subunit to study specifically the exogenously expressed Na-K pumps after inhibition of the ouabain-sensitive endogenous Xenopus Na-K pumps. Coinjection of Bufo alpha 1 subunit cRNA with either beta 1 or beta 3 cRNAs results in the expression of functional Na-K pumps that share similar low ouabain sensitivity but differ in their K+ half activation constant (K1/2). Similar results were obtained with Xenopus alpha 1 and beta 1 or beta 3 subunits and with Bufo/Xenopus heterodimers. We conclude that some specific sequence of the beta subunit can influence the activation of the Na,K pump by extracellular K+ ions.

Chapter 4 Structure–Function Relationship of Na,K-ATPase: The Digitalis Receptor

Publisher Summary It has been postulated that the digitalis receptor, that is, Na,K-ATPase activity, can be regulated by circulating endogenous ligands generally termed endo-ouabain. Therefore, the digitalis receptor may be important not only for the control of heart function, but also for a number of cellular processes in brain, kidney, and skeletal and smooth muscle. This chapter discusses the general properties of the ligand, the cardiac glycosides, the structure, and the heterogeneity of the digitalis receptor— that is, the Na,K-ATPase and the structure–function relationship of the drug-binding site. The first approach to the study of structure–function relationship of the digitalis receptor used site-specific ligands— that is, photoactivable ouabain analogues. The second approach uses the expression of α-subunit, from ouabain-resistant species (rat or mouse), into ouabain-sensitive cells. The second approach consists in site-directed mutagenesis of the amino acid residues that are thought to be significant in determining the contact site, with a ligand, for instance, the amino acids with hydrogen-bounding potential. The difficulty of this approach is that a large number of random mutations must be introduced. Many of them are likely to be either silent or nonfunctional. Using this approach, an aromatic amino acid in the first transmembrane domain has been shown to be a determinant of ouabain affinity. The third approach takes advantage of the fact that cardiac glycosides can be used positively to select cells that have been mutated in their ouabain-binding site to confer ouabain resistance. Many cell lines with such a phenotype have been described in the chapter. This approach has the significant advantage of allowing only functional mutants to be selected. The functional assay allows measurements of the binding parameters of wild type and mutated subunits.