James W. Van Huysse

Cation and Cardiac Glycoside Binding Sites of the Na,K-ATPasea

From the structural data obtained by systematically altering residues of the Na,K-ATPase, we are beginning to understand portions of how this active cation transporter couples hydrolysis of ATP with the vectorial movement of cations against their ionic gradients. In addition, the inhibitory action of cardiac glycosides and their interaction sites on the protein has focused our attentions on a catalytic core of the protein involving the H5-H6 transmembrane segment. In future investigations, both the ATP and the Na+ sites of the Na,K-ATPase must be uncovered to refine the structural picture of this complex transporter.

Enhanced pressor response to increased CSF sodium concentration and to central ANG I in heterozygous α2 Na+-K+-ATPase knockout mice

Intracerebroventricular (ICV) infusion of NaCl mimics the effects of a high-salt diet in salt-sensitive hypertension, raising the sodium concentration in the cerebrospinal fluid (CSF [Na]) and subsequently increasing the concentration of an endogenous ouabain-like substance (OLS) in the brain. The OLS, in turn, inhibits the brain Na(+)-K(+)-ATPase, causing increases in the activity of the brain renin-angiotensin system (RAS) and blood pressure. The Na(+)-K(+)-ATPase alpha (catalytic)-isoform(s) that mediates the pressor response to increased CSF [Na] is unknown, but it is likely that one or more isoforms that bind ouabain with high affinity are involved (e.g., the Na(+)-K(+)-ATPase alpha(2)- and/or alpha(3)-subunits). We hypothesize that OLS-induced inhibition of the alpha(2)-subunit mediates this response. Therefore, a chronic reduction in alpha(2) expression via a heterozygous gene knockout (alpha(2) +/-) should enhance the pressor response to increased CSF [Na]. Intracerebroventricular (ICV) infusion of artificial CSF containing 0.225 M NaCl increased mean arterial pressure (MAP) in both wild-type (+/+) and alpha(2) +/- mice, but to a greater extent in alpha(2) +/-. Likewise, the pressor response to ICV ouabain was enhanced in alpha(2) +/- mice, demonstrating enhanced sensitivity to brain Na(+)-K(+)-ATPase inhibition per se. The pressor response to ICV ANG I but not ANG II was also enhanced in alpha(2) +/- vs. alpha(2)+/+ mice, suggesting an enhanced brain RAS activity that may be mediated by increased brain angiotensin converting enzyme (ACE). The latter hypothesis is supported by enhanced ACE ligand binding in the organum vasculosum laminae terminalis. These studies demonstrate that chronic downregulation of Na(+)-K(+)-ATPase alpha(2)-isoform expression by heterozygous knockout increases the pressor response to increased CSF [Na] and activates the brain RAS. Since these changes mimic those produced by the endogenous brain OLS, the brain alpha(2)-isoform may be a target for the brain OLS during increases in CSF [Na], such as in salt-dependent hypertension.

Hypertension from chronic central sodium chloride in mice is mediated by the ouabain-binding site on the Na,K-ATPase α2-isoform

A chronic increase in the concentration of sodium chloride in the cerebrospinal fluid (CSF) (↑CSF [NaCl]) appears to be critically important for the development of salt-dependent hypertension. In agreement with this concept, increasing CSF [NaCl] chronically by intracerebroventricular (icv) infusion of NaCl-rich artificial CSF (aCSF-HiNaCl) in rats produces hypertension by the same mechanisms (i.e., aldosterone-ouabain pathway in the brain) as that produced by dietary sodium in salt-sensitive strains. We first demonstrate here that icv aCSF-HiNaCl for 10 days also causes hypertension in wild-type (WT) mice. We then used both WT and gene-targeted mice to explore the mechanisms. In WT mice with a ouabain-sensitive Na,K-ATPase α(2)-isoform (α2(S/S)), mean arterial pressure rose by ~25 mmHg within 2 days of starting aCSF-HiNaCl (0.6 nmol Na/min) and remained elevated throughout the study. Ouabain (171 pmol/day icv) increased blood pressure to a similar extent. aCSF-HiNaCl or ouabain given at the same rates subcutaneously instead of intracerebroventricularly had no effect on blood pressure. The pressor response to icv aCSF-HiNaCl was abolished by an anti-ouabain antibody given intracerebroventricularly but not subcutaneously, indicating that it is mediated by an endogenous ouabain-like substance in the brain. We compared the effects of icv aCSF-HiNaCl or icv ouabain on blood pressure in α2(S/S) versus knockout/knockin mice with a ouabain-resistant endogenous α(2)-subunit (α2(R/R)). In α2(R/R), there was no pressor response to icv aCSF-HiNaCl in contrast to WT mice. The α2(R/R) genotype also lacked a pressor response to icv ouabain. These data demonstrate that chronic ↑CSF [NaCl] causes hypertension in mice and that the blood pressure response is mediated by the ouabain-like substance in the brain, specifically by its binding to the α(2)-isoform of the Na,K-ATPase.

Critical effects on catalytic function produced by amino acid substitutions at Asp804 and Asp808 of the al isoform of Na,K‐ATPase

At two intramembrane carboxyl‐containing amino acids of the sheep al isoform of Na,K‐ATPase (Asp 804 and Asp808) both charge‐conserving (Asp to Glu) and charge‐deleting (Asp to Asn, Leu and Ala) replacements were made and the altered enzymes studied. Nucleotide changes encoding the amino acid substitutions were placed in a cDNA encoding a ouabainresistant enzyme (sheep α1 RD) and the encoded enzymes were expressed in ouabain‐sensitive HeLa cells. Transfections with cDNAs carrying all Asp 804 substitutions, along with those carrying As808Ala, Asp808 Asn, and Asps808Leu replacements failed to confer ouabain resistance to the cells, indicating critical roles for Asp804 and Asps808. Only the expression of the Asp 808Glu enzyme produced ouabain‐resistant HeLa cells, demonstrating that the altered protein was functional. When the inactive proteins Asp804Ala and Asp080Ala were expressed using an alternative selection system (the protein carrying the amino acid substitution was the ouabain‐sensitive wild‐type sheep αl Na,K‐ATPase, which was expressed in ouabain‐resistant 3T3 cells), intact cells were able to bind extracellular ouabain with high affinity (K d =1–30 nM), indicating that the inactive proteins were synthesized and folded properly in the plasma membrane. The results demonstrate that carboxyl side chains at positions 804 and 808 are critical for enzyme catalytic function.

Na,K-ATPase: Cardiac Glycoside Binding and Functional Importance of Negatively Charged Amino Acids of Transmembrane Regions

Na+/K+-ATPase is the receptor for cardiac glycosides, a class of drugs used to treat congestive heart failure and arrhythymias. Binding of these compounds to the enzyme is antagonized by K+ ions suggesting that the binding sites for these ligands may either overlap or that binding of one may effect the other. Thus, defining the binding site for this class of drugs may help in understanding how Na+/K+-ATPase transports cations. An approach for defining the cardiac glycoside binding site is to use site-directed mutagenesis coupled with expression and selection systems. In addition, these techniques can be used to determine the role of specific amino acid residues in catalytic functions of the enzyme such as cation binding. Utilizing this approach we have identified amino acid residues which act as determinants of ouabain sensitivity as well as investigated the role specific transmembrane amino acids play in the catalytic activity of the enzyme. A functional approach is also being developed to determine if a naturally occurring ligand for Na+/K+-ATPase exists and whether it is physiologically significant.

Amino Acid Residues Involved in Ouabain Sensitivity and Cation Binding

The Na, K-ATPase, which is found in the cells of all higher eukaryotes, utilizes ATP to transport Na+ and K+ across the cell membrane. For every three sodium ions transported out of the cell two potassium ions are transported in. The enzyme is composed of two subunits, a larger a subunit, which is thought to contain most of the catalytic sites, and a smaller a subunit which is required for the proper processing and maturation of the enzyme. Three isoforms exist for the a subunit (α1, α2 and α3) and two isoforms exist for the β subunit (β1 and β2) in mammalian cells (Lingrel et al., 1990; Sweadner, 1989; Takeyasu et al, 1989). An additional isoform, β3, exists in Xenopous (Good et al., 1990). The a1 isoform is found in all cells while α2 is the primary isoform found in skeletal muscle, but is also present in the heart and nervous system. Expression of the α3 isoform is limited to the heart and nervous system (Orlowski and Lingrel, 1988). The cDNAs and genes corresponding to these isoforms have been isolated and the mechanisms responsible for their differential expression are being investigated (Lingrel et al., 1990). The Na,K-ATPase is a member of the P-type family of ATPases, which also include the Ca-ATPases and the H,K-ATPases. These transport proteins are similar in structure and have in common an aspart/l phosphate intermediate during their catalytic cycle. Site-directed mutagenesis and expression (MacLennan, 1990; Clarke et al., 1990; Vilsen and Andersen, 1992; Andersen and Vilsen, 1992) studies have been carried out to define cation binding sites in the Ca-ATPase but, less information is available using this approach with the Na.K-ATPase. The Na,K- ATPase differs from the other P-type ATPases in that it is sensitive to cardiac glycosides, a class of drugs which are used in the treatment of congestive heart failure and certain arrhythmias. The sensitivity of the enzyme to these drugs provides a useful tool for investigating structure-function relationships. In this report we describe progress made toward identifying potential cation binding sites as well as amino acid residues that are involved in determining cardiac glycoside sensitivity.