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A Na⁺/K⁺ ATPase Pump Regulates Chondrocyte Differentiation and Bone Length Variation in Mice.

ABSTRACT: The genetic and developmental mechanisms involved in limb formation are relatively well documented, but how these mechanisms are modulated by changes in chondrocyte physiology to produce differences in limb bone length remains unclear. Here, we used high throughput RNA sequencing (RNAseq) to probe the developmental genetic basis of variation in limb bone length in Longshanks, a mouse model of experimental evolution. We find that increased tibia length in Longshanks is associated with altered expression of a few key endochondral ossification genes such as Npr3, Dlk1, Sox9, and Sfrp1, as well reduced expression of Fxyd2, a facultative subunit of the cell membrane-bound Na⁺/K⁺ ATPase pump (NKA). Next, using murine tibia and cell cultures, we show a dynamic role for NKA in chondrocyte differentiation and in bone length regulation. Specifically, we show that pharmacological inhibition of NKA disrupts chondrocyte differentiation, by upregulating expression of mesenchymal stem cell markers (Prrx1, Serpina3n), downregulation of chondrogenesis marker Sox9, and altered expression of extracellular matrix genes (e.g., collagens) associated with proliferative and hypertrophic chondrocytes. Together, Longshanks and in vitro data suggest a broader developmental and evolutionary role of NKA in regulating limb length diversity.

SUBMITTER: Marchini M

PROVIDER: S-EPMC8712571 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:The Na(+),K(+)-ATPase pump achieves thermodynamically uphill exchange of cytoplasmic Na(+) ions for extracellular K(+) ions by using ATP-mediated phosphorylation, followed by autodephosphorylation, to power conformational changes that allow ion access to the pump's binding sites from only one side of the membrane at a time. Formally, the pump behaves like an ion channel with two tightly coupled gates that are constrained to open and close alternately. The marine agent palytoxin disrupts this coupling, allowing both gates to sometimes be open, so temporarily transforming a pump into an ion channel. We made a cysteine scan of Na(+),K(+)-ATPase transmembrane (TM) segments TM1 to TM6, and used recordings of Na(+) current flow through palytoxin-bound pump-channels to monitor accessibility of introduced cysteine residues via their reaction with hydrophilic methanethiosulfonate (MTS) reagents. To visualize the open-channel pathway, the reactive positions were mapped onto a homology model of Na(+),K(+)-ATPase based on the structure of the related sarcoplasmicand endoplasmic-reticulum (SERCA) Ca(2+)-ATPase in a BeF(3)(-)-trapped state,(1,2) in which the extra-cytoplasmic gate is wide open (although the cytoplasmic access pathway is firmly shut). The results revealed a single unbroken chain of reactive positions that traverses the pump from the extracellular surface to the cytoplasm, comprises residues from TM1, TM2, TM4 and TM6, and passes through the equivalent of cation binding site II in SERCA, but not through site I. Cavity search analysis of the homology model validated its use for mapping the data by yielding a calculated extra-cytoplasmic pathway surrounded by MTS-reactive residues. As predicted by previous experimental results, that calculated extra-cytoplasmic pathway abruptly broadens above residue T806, at the outermost end of TM6 that forms the floor of the extracellular-facing vestibule. These findings provide a structural basis for further understanding cation translocation by the Na(+),K(+)-ATPase and by other P-type pumps like the Ca(2+)- and H(+),K(+)-ATPases.

Project description:Na/K pumps build essential ion gradients across the plasmalemma of animal cells by coupling the extrusion of three Na+, with the import of two K+ and the hydrolysis of one ATP molecule. The mechanisms of selectivity and competition between Na+, K+, and inhibitory amines remain unclear. We measured the effects of external tetrapropylammonium (TPA+) and ethylenediamine (EDA2+) on three different Na/K pump transport modes in voltage-clamped Xenopus oocytes: 1) outward pump current (IP), 2) passive inward H+ current at negative voltages without Na+ or K+ (IH), and 3) transient charge movement reporting the voltage-dependent extracellular binding/release of Na+ (QNa). Both amines competed with K+ to inhibit IP. TPA+ inhibited IH without competing with H+, whereas EDA2+ did not alter IH at pH 7.6. TPA+ competed with Na+ in QNa measurements, reducing Na+-apparent affinity, evidenced by a ?-75 mV shift in the charge-voltage curve (at 20 mM TPA+) without reduction of the total charge moved (Qtot). In contrast, EDA2+ and K+ did not compete with Na+ to inhibit QNa; both reduced Qtot without decreasing Na+-apparent affinity. EDA2+ (15 mM) right-shifted the charge-voltage curve by ?+50 mV. Simultaneous occlusion of EDA2+ and Na+ by an E2P conformation unable to reach E1P was demonstrated by voltage-clamp fluorometry. Trypsinolysis experiments showed that EDA2+-bound pumps are much more proteolysis-resistant than Na+-, K+-, or TPA+-bound pumps, therefore uncovering unique EDA2+-bound conformations. K+ effects on QNa and IH were also evaluated in pumps inhibited with beryllium fluoride, a phosphate mimic. K+ reduced Qtot without shifting the charge-voltage curve, indicating noncompetitive effects, and partially inhibited IH to the same extent as TPA+ in non-beryllium-fluorinated pumps. These results demonstrate that K+ interacts with beryllium-fluorinated pumps inducing conformational changes that alter QNa and IH, suggesting that there are two external access pathways for proton transport by IH.

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A Na⁺/K⁺ ATPase Pump Regulates Chondrocyte Differentiation and Bone Length Variation in Mice.

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A Na+/K+ ATPase Pump Regulates Chondrocyte Differentiation and Bone Length Variation in Mice.

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OmicsDI is part of the ELIXIR infrastructure

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A Na⁺/K⁺ ATPase Pump Regulates Chondrocyte Differentiation and Bone Length Variation in Mice.