Thomas A. Pressley

Structural determinants for the ouabain-stimulated increase in Na–K ATPase activity

Recent studies suggest that at low concentrations, ouabain increases Na-K ATPase and NHE1 activity and activates the Src signaling cascade in proximal tubule cells. Our laboratory demonstrated that low concentrations of ouabain increase blood pressure in rats. We hypothesize that ouabain-induced increase in blood pressure and Na-K ATPase activity requires NHE1 activity and association. To test this hypothesis we treated rats with ouabain (1μgkg body wt(-1)day(-1)) for 9days in the presence or absence of the NHE1 inhibitor, zoniporide. Ouabain stimulated a significant increase in blood pressure which was prevented by zoniporide. Using NHE1-expressing Human Kidney cells 2 (HK2), 8 (HK8) and 11 (HK11) and Mouse Kidney cells from Wild type (WT) and NHE1 knock-out mice (SWE) cell lines, we show that ouabain stimulated Na-K ATPase activity and surface expression in a Src-dependent manner in NHE1-expressing cells but not in NHE1-deplete cells. Zoniporide prevented ouabain-induced stimulation of (86)Rb uptake in the NHE1-expressing cells. FRET and TIRF microscopy showed that ouabain increased association between GFP-NHE1 and mCherry-Na-K ATPase transfected into NHE1-deficient SWE cells. Mutational analysis demonstrated that the caveolin binding motif (CBM) of Na-K ATPase α1 is required for translocation of both Na-K ATPase α1 and NHE1 to the basolateral membrane. Mutations in activity or scaffold domains of NHE1 resulted in loss of ouabain-mediated regulation of Na-K ATPase. These results support that NHE1 is required for the ouabain-induced increase in blood pressure, and that the caveolin binding motif of Na-K ATPase α1 as well as the activity and scaffolding domains of NHE1 are required for their functional association.

A perchlorate sensitive iodide transporter in frogs

Nucleotide sequence comparisons have identified a gene product in the genome database of African clawed frogs (Xenopus laevis) as a probable member of the solute carrier family of membrane transporters. To confirm its identity as a putative iodide transporter, we examined the function of this sequence after heterologous expression in mammalian cells. A green monkey kidney cell line transfected with the Xenopus nucleotide sequence had significantly greater (125)I uptake than sham-transfected control cells. The uptake in carrier-transfected cells was significantly inhibited in the presence of perchlorate, a competitive inhibitor of mammalian Na(+)/iodide symporter. Tissue distributions of the sequence were also consistent with a role in iodide uptake. The mRNA encoding the carrier was found to be expressed in the thyroid gland, stomach, and kidney of tadpoles from X. laevis, as well as the bullfrog Rana catesbeiana. The ovaries of adult X. laevis also were found to express the carrier. Phylogenetic analysis suggested that the putative X. laevis iodide transporter is orthologous to vertebrate Na(+)-dependent iodide symporters. We conclude that the amphibian sequence encodes a protein that is indeed a functional Na(+)/iodide symporter in X. laevis, as well as R. catesbeiana.

Critical Role of the Isoform-Specific Region in α1-Na,K-ATPase Trafficking and Protein Kinase C-Dependent Regulation

The isoform-specific region (ISR) is a region of structural heterogeneity among the four isoforms of the catalytic alpha-subunit of the Na,K-ATPase and an important structural determinant for isoform-specific functions. In the present study, we examined the role of a potential dileucine clathrin adaptor recognition motif [DE]XXXL[LI] embedded within the alpha1-ISR. To this end, a rat alpha1 construct where leucine 499 was replaced by a valine (as found in the alpha2 isoform sequence) was compared to wild-type rat alpha1 after stable expression in opossum kidney cells. Total Na,K-ATPase expression, activity, or in situ (86)Rb(+) transport was not affected by the L499V mutation. However, surface Na,K-ATPase expression was nearly doubled. This increase was associated with a reduced rate of internalization from the cell surface of about 50% after a 4 h chase and became undetectable if clathrin-coated pit-mediated trafficking was blocked with chlorpromazine. Further, PKC-induced stimulation of Na,K-ATPase-mediated (86)Rb(+) uptake was doubled in mutant-expressing cells, comparable to the chimera containing the intact alpha2-ISR. Similar results were observed when the potential motif was disrupted by means of an E495S mutation. These findings suggest that a dileucine motif embedded within the Na,K-ATPase alpha1-ISR plays a critical role in the surface expression of Na,K-ATPase alpha1 polypeptides at steady state and in the response to PKC activation.

The insulation bag: learning thermoregulation through a "hands-in" activity

the extensive diversity of habitats to which animals have adapted necessitated the evolution of a number of different anatomic and behavioral mechanisms to tolerate the extremes in ambient temperatures to which these animals are exposed. Among these anatomic solutions to the seemingly compromising

A role for uteroglobin in glomerulopathy: Knockout mice as a model system

F OR THE TYPICAL nephrologist, the effort necessary to understand the details of molecular engineering must be as great as that required by a basic scientist like me during the average grand rounds case: each is daunting from the other’s point of view. Most intimidating of all to someone outside the field of molecular biology may be the subject of transgenic animals. If one can put aside momentarily that anxiety, however, a recent report from Zhang et al’ provides a fine example of how transgenic technology can be used to address fundamental questions of protein function. As a bonus, their work also suggests a mechanism underlying some forms of glomerular disease characterized by deposits of fibronectin (Fn) and collagen. The story begins with uteroglobin (UG), a secreted protein of approximately 16,000 daltons searching for a function.’ The number of names by which UG is known is exceeded only by the number of suggestions for its physiological relevance. It was first discovered in the rabbit uterus, but has since been observed in many nonreproductive tissues.3’4 Endocrinologists have used it as a model system because it is regulated by both steroid and polypeptide hormones. There is also a clear involvement of UG with the immune system, and it often acts as an immunosuppressant. Moreover, after binding to plasma membrane receptors, it seems to decrease cell interactions with the extracellular matrix. Despite the host of enzymes and processes with which UG may be involved, its biological importance at the systemic level was unknown. There was certainly little reason to suspect that its elimination would produce renal pathology. Indeed, it is not even expressed in the kidney.

Educational leadership: benefits of stepping outside the classroom

Although most educators have their greatest impact in the classroom, the increased need for diverse learning activities has created new opportunities for leadership. Moreover, many Tenure and Promotion Committees are finding that it is no longer sufficient to consider only lecture hours when evaluating a faculty members contributions to the teaching mission of an institution. Accordingly, the career path for an educator in a college or professional school is evolving. A newly recruited faculty member may start out with traditional classroom responsibilities, but activities other than lecture, such as flipped classrooms, online resources, and peer-to-peer teaching, may be quickly added to the mix. As faculty members gain experience, they often progress to positions of curriculum design or program review within an institution. Similarly, there is a need for administrators who have participated in a variety of learning activities, and schools frequently recruit for these positions from faculty with such exposure. Many senior faculty members leverage this expertise to regional or national levels by authoring textbooks and online materials or serving on advisory boards, review committees, and governance in professional societies and funding agencies. Excelling in these leadership opportunities can have a profound effect on the success of promotion and tenure applications, and they reward a skill set that extends beyond the teaching and organization needed in the classroom.

The stoichiometry of the Na-K pump : one plus one doesn't equal one

pick up nearly any textbook of cell physiology, and you will read that the constituent subunits of the Na-K pump exist in equimolar amounts. As a central player in Na+ and K+ transport, this protein complex has been the subject of intense biochemical scrutiny for decades, and the argument for a 1:1

Posttranslational Processing of the Catalytic Subunit from Na+/K+‐ATPasea

The catalytic a subunit of the Na,K-ATPase exists as three distinct isoforms (a 1, a2, and a3) whose primary structures are nearly identical. One exception is the amino terminus, and this divergence in sequence may be related to differences in posttranslational processing. The first five amino acids of the a 1 isoform predicted from its cDNA are not found in enzyme purified from enriched tissues such as renal outer medulla.’ This implies that a1 is cleaved enzymatically at some point during or after translation. By contrast, a3 is not thought to undergo this proteolysis. To facilitate evaluation of amino terminal structure and posttranslational cleavage, we developed site-directed antibodies specific for the amino termini of nascent (Y isoforms from rat.’ These reagents were used to test explicitly the extent of posttranslational processing undergone by a 1 and a3.