Voltage- and substrate-dependent interactions between sites in putative re-entrant domains of a Na+-coupled phosphate cotransporter

Ghezzi, Chiara; Meinild, Anne‐Kristine; Murer, Heini; Forster, Ian C.

doi:10.1007/s00424-011-0948-z

Cited by 18 publications

(29 citation statements)

References 47 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…(15). We made Cys-substitutions in AAD-IIc and investigated the constructs by means of conventional TEVC (steady-state kinetics) and voltage clamp fluorometry (VCF) (7,19,20,34,35). We chose sites in AAD-IIc that corresponded to those previously characterised in other SLC34 isoforms (Fig 6, Table 1).…”

Section: Cysteine Substitutions In Aad-iicmentioning

confidence: 99%

“…The substituted Cys were labelled with fluorophores to act as reporters of local conformational changes (19,20,34,35). The behavior of labelled oocytes expressing AAD-IIc-437 in response to changing the superfusate from 100Na to 100Ch was qualitatively the same as we previously reported for the NaPi-IIc mutant S437C (20): in the steady-state (V h =-60 mV), addition of Na + quenched the fluorescence.…”

Section: Fluorometric Assays Complement Presteady-state Relaxation Anmentioning

confidence: 99%

“…The electrogenic and electroneutral cotransport mechanisms can be understood in terms of a kinetic scheme (Fig 1A) that describes their respective transport cycles as an ordered sequence of partial reactions, some of which have been identified experimentally. For example, electrogenicity can be accounted for by assigning charge displacement to the empty carrier (01) together with extra-and intracellular interactions of one Na + ion (12, 09) (2,13,19). This cation is postulated to account for the uncoupled leak (2).…”

mentioning

confidence: 99%

“…This is even greater in the predicted transmembrane domain regions (36) and therefore all isoforms most likely share a common 3-D structure. The current lack of a 3-D structure and apparent absence of homology with membrane transport proteins with resolved structures has necessitated using indirect approaches (mutagenesis, cysteine scanning, crosslinking) to determine the structure-function relationships (16,19,20).…”

mentioning

confidence: 99%

“…However, the molecular engineering of NaPi-IIc resulted in compromised cotransport kinetics. The modified protein (hereafter referred to as AAD-IIc) showed a >200-fold reduced apparent affinity for P i : the Michaelis constant (K 0.5 P i ) was >1 mM (compared with 0.06 mM for NaPi-IIc), whereas for Na + activation (1 mM P i ), the apparent affinity constant (K 0.5 Na ) 80 mM was only marginally affected (40 mM for NaPi-IIc) (19). Furthermore, compared with other electrogenic isoforms, the presteady-state charge movements appeared to show little dependence on external [Na + ] (e.g.…”

mentioning

confidence: 99%

See 4 more Smart Citations

Conferring electrogenicity to the electroneutral phosphate cotransporter NaPi-IIc (SLC34A3) reveals an internal cation release step

Patti

Ghezzi

Forster

2013

Pflugers Arch - Eur J Physiol

Self Cite

View full text Add to dashboard Cite

The SLC34 family of Na(+)-dependent inorganic phosphate cotransporters comprises two electrogenic isoforms (NaPi-IIa, NaPi-IIb) and an electroneutral isoform (NaPi-IIc). Both fulfill essential physiological roles in mammalian phosphate homeostasis. By substitution of three conserved amino acids, found in all electrogenic isoforms, at corresponding sites in NaPi-IIc, electrogenicity was re-established and the Na(+)/P i stoichiometry increased from 2:1 to 3:1. However, this engineered electrogenic construct (AAD-IIc) had a reduced apparent P i affinity and different presteady-state kinetics from the wildtype NaPi-IIa/b. We investigated AAD-IIc using electrophysiology and voltage clamp fluorometry to elucidate the compromised behavior. The activation energy for cotransport was threefold higher than for NaPi-IIc and 1.5-fold higher than for NaPi-IIa and the temperature dependence of presteady-state charge displacements suggested that the large activation energy was associated with the empty carrier reorientation. AAD-IIc shows a weak interaction of external Na(+) ions with the electric field, and thus retains the electroneutral cooperative interaction of two Na(+) ions preceding external P i binding of NaPi-IIc. Most of the presteady-state charge movement was accounted for by the empty carrier (in the absence of external P i ), and the cytosolic release of one Na(+) ion (in the presence of P i ). Simulations using a kinetic model recapitulated the presteady-state and steady-state behavior and allowed identification of two critical partial reactions: the final release of Na(+) to the cytosol and external P i binding. Fluorometric recordings from AAD-IIc mutants with Cys substituted at functionally important sites established that AAD-IIc undergoes substrate-and voltage-dependent conformational changes that correlated qualitatively with its presteady-state kinetics. Manuscript-revised Click here to download Manuscript: AAD-EJP 2nd sub-final.docx Click here to view linked References 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65 2 AbstractThe SLC34 family of Na + -dependent inorganic phosphate cotransporters comprises two electrogenic isoforms (NaPi-IIa, NaPi-IIb) and an electroneutral isoform (NaPi-IIc). Both fulfil essential physiological roles in mammalian phosphate homeostasis. By substitution of 3 conserved amino acids, found in all electrogenic isoforms, at corresponding sites in NaPi-IIc, electrogenicity was re-established and the Na + :P i stoichiometry increased from 2:1 to 3:1. However, this engineered electrogenic construct (AAD-IIc) had a reduced apparent P i affinity and different presteady-state kinetics from the wild-type NaPi-IIa/b. We investigated AAD-IIc using electrophysiology and voltage clamp fluorometry to elucidate the compromised behavior. The activation energy for cotransport was 3-f...

show abstract

Section: Cysteine Substitutions In Aad-iicmentioning

confidence: 99%

Section: Fluorometric Assays Complement Presteady-state Relaxation Anmentioning

confidence: 99%

mentioning

confidence: 99%

mentioning

confidence: 99%

mentioning

confidence: 99%

See 3 more Smart Citations

Conferring electrogenicity to the electroneutral phosphate cotransporter NaPi-IIc (SLC34A3) reveals an internal cation release step

Patti

Ghezzi

Forster

2013

Pflugers Arch - Eur J Physiol

Self Cite

View full text Add to dashboard Cite

show abstract

Mechanisms and Regulation of Intestinal Phosphate Absorption

Hernando¹,

Wagner²

2018

Comprehensive Physiology

View full text Add to dashboard Cite

States of hypo- and hyperphosphatemia have deleterious consequences including rickets/osteomalacia and renal/cardiovascular disease, respectively. Therefore, the maintenance of appropriate plasma levels of phosphate is an essential requirement for health. This control is executed by the collaborative action of intestine and kidney whose capacities to (re)absorb phosphate are regulated by a number of hormonal and metabolic factors, among them parathyroid hormone, fibroblast growth factor 23, 1,25(OH) vitamin D , and dietary phosphate. The molecular mechanisms responsible for the transepithelial transport of phosphate across enterocytes are only partially understood. Indeed, whereas renal reabsorption entirely relies on well-characterized active transport mechanisms of phosphate across the renal proximal epithelia, intestinal absorption proceeds via active and passive mechanisms, with the molecular identity of the passive component still unknown. The active absorption of phosphate depends mostly on the activity and expression of the sodium-dependent phosphate cotransporter NaPi-IIb (SLC34A2), which is highly regulated by many of the factors, mentioned earlier. Physiologically, the contribution of NaPi-IIb to the maintenance of phosphate balance appears to be mostly relevant during periods of low phosphate availability. Therefore, its role in individuals living in industrialized societies with high phosphate intake is probably less relevant. Importantly, small increases in plasma phosphate, even within normal range, associate with higher risk of cardiovascular disease. Therefore, therapeutic approaches to treat hyperphosphatemia, including dietary phosphate restriction and phosphate binders, aim at reducing intestinal absorption. Here we review the current state of research in the field. © 2017 American Physiological Society. Compr Physiol 8:1065-1090, 2018.

show abstract

The SLC34 family of sodium-dependent phosphate transporters

Wagner

Hernando

Forster

et al. 2013

Pflugers Arch - Eur J Physiol

Self Cite

127

117

View full text Add to dashboard Cite

The SLC34 family of sodium-driven phosphate cotransporters comprises three members: NaPiIIa (SLC34A1), NaPi-IIb (SLC34A2), and NaPi-IIc (SLC34A3). These transporters mediate the translocation of divalent inorganic phosphate (HPO4 (2-)) together with two (NaPi-IIc) or three sodium ions (NaPi-IIa and NaPi-IIb), respectively. Consequently, phosphate transport by NaPi-IIa and NaPi-IIb is electrogenic. NaPi-IIa and NaPi-IIc are predominantly expressed in the brush border membrane of the proximal tubule, whereas NaPi-IIb is found in many more organs including the small intestine, lung, liver, and testis. The abundance and activity of these transporters are mostly regulated by changes in their expression at the cell surface and are determined by interactions with proteins involved in scaffolding, trafficking, or intracellular signaling. All three transporters are highly regulated by factors including dietary phosphate status, hormones like parathyroid hormone, 1,25-OH2 vitamin D3 or FGF23, electrolyte, and acid-base status. The physiological relevance of the three members of the SLC34 family is underlined by rare Mendelian disorders causing phosphaturia, hypophosphatemia, or ectopic organ calcifications. ABSTRACTThe SLC34 family of sodium-driven phosphate cotransporters comprises three

show abstract

Voltage- and substrate-dependent interactions between sites in putative re-entrant domains of a Na+-coupled phosphate cotransporter

Cited by 18 publications

References 47 publications

Conferring electrogenicity to the electroneutral phosphate cotransporter NaPi-IIc (SLC34A3) reveals an internal cation release step

Conferring electrogenicity to the electroneutral phosphate cotransporter NaPi-IIc (SLC34A3) reveals an internal cation release step

Mechanisms and Regulation of Intestinal Phosphate Absorption

The SLC34 family of sodium-dependent phosphate transporters

Contact Info

Product

Resources

About