44 research outputs found
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 āgiantā 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
Site-Directed Mutagenesis Analysis of the Role of the M5S5 Sector of the Sarcoplasmic Reticulum Ca 2+
Mutational Effects on Conformational Changes of the Dephospho- and Phospho-forms of the Na +
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 Ī±2Ī²2 Na(+)/K(+)āATPase isoform combination is activated by the K(+) transients occurring during neuronal activity. In the present study, we report that glutamate transporterāmediated astrocytic Na(+) transients stimulate the Na(+)/K(+)āATPase and thus the clearance of extracellular K(+). Specifically, the astrocytic Ī±2Ī²1 Na(+)/K(+)āATPase 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(+)āATPase Ī²āsubunits. ABSTRACT: Neuronal activity is associated with transient [K(+)](o) increases. The excess K(+) is cleared by surrounding astrocytes, partly by the Na(+)/K(+)āATPase of which several subunit isoform combinations exist. The astrocytic Na(+)/K(+)āATPase Ī±2Ī²2 isoform constellation responds directly to increased [K(+)](o) but, in addition, Na(+)/K(+)āATPaseāmediated K(+) clearance could be governed by astrocytic [Na(+)](i). During most neuronal activity, glutamate is released in the synaptic cleft and is reāabsorbed by astrocytic Na(+)ācoupled glutamate transporters, thereby elevating [Na(+)](i). It thus remains unresolved whether the different Na(+)/K(+)āATPase isoforms are controlled by [K(+)](o) or [Na(+)](i) during neuronal activity. Hippocampal slice recordings of stimulusāinduced [K(+)](o) transients with ionāsensitive microelectrodes revealed reduced Na(+)/K(+)āATPaseāmediated K(+) management upon parallel inhibition of the glutamate transporter. The apparent intracellular Na(+) affinity of isoform constellations involving the astrocytic Ī²2 has remained elusive as a result of inherent expression of Ī²1 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(+)āATPase was not fully saturated at basal astrocytic [Na(+)](i), irrespective of isoform constellation, although the Ī²1 subunit conferred lower apparent Na(+) affinity to the Ī±1 and Ī±2 isoforms than the Ī²2 isoform. In summary, enhanced astrocytic Na(+)/K(+)āATPaseādependent K(+) clearance was obtained with parallel glutamate transport activity. The astrocytic Na(+)/K(+)āATPase isoform constellation Ī±2Ī²1 appeared to be specifically geared to respond to the [Na(+)](i) transients associated with activityāinduced glutamate transporter activity