Importance of Conserved N-domain Residues Thr 441 , Glu 442 , Lys 515 , Arg 560 , and Leu 562 of Sarcoplasmic Reticulum Ca 2+ -ATPase for MgATP Binding and Subsequent Catalytic Steps: PLASTICITY OF THE NUCLEOTIDE-BINDING SITE

Nine single mutations were introduced to amino acid residues Thr441, Glu442, Lys515, Arg560, Cys561, and Leu562 located in the nucleotide-binding domain of sarcoplasmic reticulum Ca2+-ATPase, and the functional consequences were studied in a direct nucleotide binding assay, as well as by steady-state and transient kinetic measurements of the overall and partial reactions of the transport cycle. Some partial reaction steps were also examined in mutants with alterations to Phe487, Arg489, and Lys492. The results implicate all these residues, except Cys561, in high affinity nucleotide binding at the substrate site. Mutations Thr441 --> Ala, Glu442 --> Ala, and Leu562 --> Phe were more detrimental to MgATP binding than to ATP binding, thus pointing to a role for these residues in the binding of Mg2+ or to a difference between the interactions with MgATP and ATP. Subsequent catalytic steps were also selectively affected by the mutations, showing the involvement of the nucleotide-binding domain in these reactions. Mutation of Arg560 inhibited phosphoryl transfer but enhanced the E1PCa2 --> E2P conformational transition, whereas mutations Thr441 --> Ala, Glu442 --> Ala, Lys492 --> Leu, and Lys515 --> Ala inhibited the E1PCa2 --> E2P transition. Hydrolysis of the E2P phosphoenzyme intermediate was enhanced in Glu442 --> Ala, Lys492 --> Leu, Lys515 --> Ala, and Arg560 --> Glu. None of the mutations affected the low affinity activation by nucleotide of the phosphoenzyme-processing steps, indicating that modulatory nucleotide interacts differently from substrate nucleotide. Mutation Glu442 --> Ala greatly enhanced reaction of Lys515 with fluorescein isothiocyanate, indicating that the two residues form a salt link in the native protein

Disease mutations reveal residues critical to the interaction of P4-ATPases with lipid substrates

Abstract Phospholipid flippases (P4-ATPases) translocate specific phospholipids from the exoplasmic to the cytoplasmic leaflet of membranes. While there is good evidence that the overall molecular structure of flippases is similar to that of P-type ATPase ion-pumps, the transport pathway for the “giant” lipid substrate has not been determined. ATP8A2 is a flippase with selectivity toward phosphatidylserine (PS), possessing a net negatively charged head group, whereas ATP8B1 exhibits selectivity toward the electrically neutral phosphatidylcholine (PC). Setting out to elucidate the functional consequences of flippase disease mutations, we have identified residues of ATP8A2 that are critical to the interaction with the lipid substrate during the translocation process. Among the residues pinpointed are I91 and L308, which are positioned near proposed translocation routes through the protein. In addition we pinpoint two juxtaposed oppositely charged residues, E897 and R898, in the exoplasmic loop between transmembrane helices 5 and 6. The glutamate is conserved between PS and PC flippases, whereas the arginine is replaced by a negatively charged aspartate in ATP8B1. Our mutational analysis suggests that the glutamate repels the PS head group, whereas the arginine minimizes this repulsion in ATP8A2, thereby contributing to control the entry of the phospholipid substrate into the translocation pathway

Glutamate transporter activity promotes enhanced Na+/K+-ATPase-mediated extracellular K+ management during neuronal activity

KEY POINTS: Management of glutamate and K(+) in brain extracellular space is of critical importance to neuronal function. The astrocytic α2β2 Na(+)/K(+)‐ATPase isoform combination is activated by the K(+) transients occurring during neuronal activity. In the present study, we report that glutamate transporter‐mediated astrocytic Na(+) transients stimulate the Na(+)/K(+)‐ATPase and thus the clearance of extracellular K(+). Specifically, the astrocytic α2β1 Na(+)/K(+)‐ATPase subunit combination displays an apparent Na(+) affinity primed to react to physiological changes in intracellular Na(+). Accordingly, we demonstrate a distinct physiological role in K(+) management for each of the two astrocytic Na(+)/K(+)‐ATPase β‐subunits. ABSTRACT: Neuronal activity is associated with transient [K(+)](o) increases. The excess K(+) is cleared by surrounding astrocytes, partly by the Na(+)/K(+)‐ATPase of which several subunit isoform combinations exist. The astrocytic Na(+)/K(+)‐ATPase α2β2 isoform constellation responds directly to increased [K(+)](o) but, in addition, Na(+)/K(+)‐ATPase‐mediated K(+) clearance could be governed by astrocytic [Na(+)](i). During most neuronal activity, glutamate is released in the synaptic cleft and is re‐absorbed by astrocytic Na(+)‐coupled glutamate transporters, thereby elevating [Na(+)](i). It thus remains unresolved whether the different Na(+)/K(+)‐ATPase isoforms are controlled by [K(+)](o) or [Na(+)](i) during neuronal activity. Hippocampal slice recordings of stimulus‐induced [K(+)](o) transients with ion‐sensitive microelectrodes revealed reduced Na(+)/K(+)‐ATPase‐mediated K(+) management upon parallel inhibition of the glutamate transporter. The apparent intracellular Na(+) affinity of isoform constellations involving the astrocytic β2 has remained elusive as a result of inherent expression of β1 in most cell systems, as well as technical challenges involved in measuring intracellular affinity in intact cells. We therefore expressed the different astrocytic isoform constellations in Xenopus oocytes and determined their apparent Na(+) affinity in intact oocytes and isolated membranes. The Na(+)/K(+)‐ATPase was not fully saturated at basal astrocytic [Na(+)](i), irrespective of isoform constellation, although the β1 subunit conferred lower apparent Na(+) affinity to the α1 and α2 isoforms than the β2 isoform. In summary, enhanced astrocytic Na(+)/K(+)‐ATPase‐dependent K(+) clearance was obtained with parallel glutamate transport activity. The astrocytic Na(+)/K(+)‐ATPase isoform constellation α2β1 appeared to be specifically geared to respond to the [Na(+)](i) transients associated with activity‐induced glutamate transporter activity