Zurich Open Repository and Archive University of Zurich Main Library Strickhofstrasse 39 CH-8057 Zurich www.zora.uzh.ch Year: 2013 Phosphate transporters of the SLC20 and SLC34 families Forster, Ian C ; Hernando, Nati ; Biber, Jürg ; Murer, Heini Abstract: Transport of inorganic phosphate (Pi) across the plasma membrane is essential for normal cellular function. Members of two families of SLC proteins (SLC20 and SLC34) act as Na(+)-dependent, secondary-active cotransporters to transport Pi across cell membranes. The SLC34 proteins are ex- pressed in specific organs important for Pi homeostasis: NaPi-IIa (SLC34A1) and NaPi-IIc (SLC34A3) fulfill essential roles in Pi reabsorption in the kidney proximal tubule and NaPi-IIb (SLC34A2) medi- ates Pi absorption in the gut. The SLC20 proteins, PiT-1 (SLC20A1), PiT-2 (SLC20A2) are expressed ubiquitously in all tissues and although generally considered as ”housekeeping” transport proteins, the discovery of tissue-specific activity, regulatory pathways and gene-related pathophysiologies, is redefining their importance. This review summarizes our current knowledge of SLC20 and SLC34 proteins in terms of their basic molecular characteristics, physiological roles, known pathophysiology and pharmacology. DOI: https://doi.org/10.1016/j.mam.2012.07.007 Posted at the Zurich Open Repository and Archive, University of Zurich ZORA URL: https://doi.org/10.5167/uzh-79439 Journal Article Accepted Version Originally published at: Forster, Ian C; Hernando, Nati; Biber, Jürg; Murer, Heini (2013). Phosphate transporters of the SLC20 and SLC34 families. Molecular Aspects of Medicine, 34(2-3):386-395. DOI: https://doi.org/10.1016/j.mam.2012.07.007 1 Mini-reviews Series-Molecular Aspects of Medicine (JMAM) Phosphate transporters of the SLC20 and SLC34 families Ian C. Forster, Nati Hernando, Jürg Biber and Heini Murer Institute of Physiology and Zurich Center for Integrated Human Physiology, University of Zurich, Winterthurerstrasse 190,CH-8057 Zurich, Switzerland Contents 1 Introduction 2 The SLC34 family (NaPi-IIa, NaPi-IIb, NaPi-IIc) 2.1 NaPi-IIa 2.2 NaPi-IIb 2.3 NaPi-IIc 2.4 Kinetics and structure-function relationships of SLC34 proteins 2.4.1 Kinetics of SLC34 proteins 2.4.2 Structure-function relationships of SLC34 proteins 2.5 Physiological, pathophysiological and pharmaceutical aspects of SLC34 proteins 2.5.1 Physiological role and regulation 2.5.2 Pathophysiology and effect of naturally occurring mutations 2.5.3 Pharmacology 3 The SLC20 family (PiT-1, PiT-2) 3.1 PiT-1, PiT-2 3.2 Kinetics and structure-function relationships of SLC20 proteins 3.2.1 Kinetics 3.2.2 Structure-function relationships 3.3 Physiological, pathophysiological and pharmaceutical aspects of SLC20 proteins 3.3.1 Physiological role and regulation 3.3.2 Pathophysiology and effect of naturally occurring mutations 3.3.3 Pharmacology Keywords: Phosphate, cotransport Corresponding author: PD Dr Ian C Forster Email: [email protected] 2 Abstract Transport of inorganic phosphate (Pi) across the plasma membrane is essential for normal cellular function. Members of two families of SLC proteins (SLC20 and SLC34) act as Na+- dependent, secondary-active cotransporters to transport Pi across cell membranes. The SLC34 proteins are expressed in specific organs important for Pi homeostasis: NaPi-IIa (SLC34A1) and NaPi-IIc (SLC34A3) fulfill essential roles in Pi reabsorption in the kidney proximal tubule and NaPi-IIb (SLC34A2) mediates Pi absorption in the gut. The SLC20 proteins, PiT-1 (SLC20A1), PiT-2 (SLC20A2) are expressed ubiquitously in all tissues and although generally considered as “housekeeping” transport proteins, the discovery of tissue-specific activity,regulatory pathways and gene-related pathophysiologies, is redefining their importance. This review summarises our current knowledge of SLC20 and SLC34 proteins in terms of their basic molecular characteristics, physiological roles, known pathophysiologies and pharmacology. 3 1. Introduction In mammals, phosphorus is an essential element for cellular signaling, metabolic and synthetic pathways as well as fulfilling a necessary structural role in bone and phospholipid membranes. It is obtained from the diet as anionic inorganic phosphate (Pi) and requires active transport to traverse the cell membrane. Moreover, transepithelial transport of Pi in different epithelia (specifically in kidney and small intestine) contributes fundamentally to overall Pi homeostasis (Fig 1A). This is particularly evident in kidney where Pi reabsorption in the proximal tubule is regulated by a number of hormones and metabolic factors thereby adusting excretion of Pi according to the organism’s Pi requirements. Any defects in the regulation of cotransporter proteins or the expression of mutated proteins can lead to severe pathophysiological conditions. Two genetically distinct families of sodium-coupled cotransporters mediate transport of Pi in mammals: SLC20 comprising SLC20A1 (PiT1), SLC20A2 (PiT2) and SLC34 comprising SLC34A1 (NaPi-IIa), SLC34A2 (NaPi-IIb) and SLC34A3 (NaPi-IIc) (see Tables 1, 2). Whereas the physiological roles of SLC34 proteins, with their well-defined tissue distribution, has been extensively investigated and characterized, the novel roles of the more ubiquitously expressed SLC20 proteins are only beginning to emerge in addition to that to which they were originally ascribed, as housekeeping proteins. + 2. The SLC34 family (type II Na -coupled Pi transporters: NaPi-IIa, NaPi-IIb, NaPi-IIc) 2.1 NaPi-IIa NaPi-IIa (SLC34A1) was identified by functional expression cloning using X. laevis oocytes and rat and human kidney cDNA library (Magagnin et al., 1993). The major site of expression is the renal proximal tubule where the 80-90kDa protein is localized in the microvilli that form the brush border membrane (BBM). In human tissue, significant mRNA expression is detected only in the kidney (Fig 1B) (Nishimura and Naito, 2008). Under normal physiological conditions, the abundance of NaPi-IIa is highest in the S1 proximal tubule segments of juxtamedullary nephrons (Custer M, 1994). 2.2 NaPi-IIb NaPi-IIb (SLC34A2) was identified from a mouse embryo EST clone and was specifically localized to enterocytes of the small intestine (Hilfiker et al., 1998). In humans mRNA is detected in lungs, testis, salivary gland, thyroid gland, small intestine, liver, mammary gland, uterus but not in renal tissue (Fig 1B) (Nishimura and Naito, 2008). 2.3 NaPi-IIc NaPi-IIc (SLC34A3) was identified from human EST clones (Segawa et al., 2002). Expression of NaPi-IIc was found exclusively in the kidney and proposed to be growth related based on animal studies: in weaning rats it is expressed in the BBM of S1 segments of proximal tubules, but is strongly down-regulated in adult animals (Miyamoto et al.; Segawa et al., 2002). The 75kDa protein is localized at the apical membranes of proximal tubule epithelia of juxtamedullary nephrons (Segawa et al., 2002). Like NaPi-IIa, the distribution of mRNA in human tissue is also renal specific (Fig 1B) (Nishimura and Naito, 2008). 2.4 Kinetics and structure-function relationships of SLC34 proteins 4 2.4.1 Kinetics of SLC34 proteins All 3 isoforms preferentially transport divalent Pi (Bacconi et al., 2005; Forster et al., 1999) and are exclusively Na+-dependent, although Li+ ions (Andrini et al.) can partly replace Na+ to drive transport. The transport capacity of all SLC34 proteins is strongly dependent on pH, which defines the monovalent/divalent Pi distribution in the extracellular compartment and protons also directly modulate the transport kinetics e.g.(Forster et al., 2000). At pH 7.4, the 3 isoforms display similar apparent substrate affinities of approximately 100 µM and 40 + mM for divalent Pi and Na respectively. Functionally, they can be distinguished as electrogenic (NaPi-IIa,b) and electroneutral (NaPi-IIc) cotransporters (Fig 2A). NaPi-IIa/b translocate one net positive charge per transport cycle and their transport rate is a function of + membrane potential. They mediate transport with a 3:1 Na :Pi stoichiometry (Forster et al., 1999). The transport kinetics of NaPi-IIc are insensitive to membrane potential, no net charge is translocated and transport is mediated with a 2:1 stoichiometry (Bacconi et al., 2005; Segawa et al., 2002). The stoichiometry difference means that the theoretical Pi concentrating capacity is approximately 100-fold higher for NaPi-IIa/b, at the cost of a 10-fold greater inward flux of Na+ ions compared to NaPi-IIc, together with net charge movement, both of which must be compensated by the cell through the action of the Na+/K+-ATPase. Kinetic studies have established that for NaPi-IIa/b, 2 Na+ ions bind sequentially and cooperatively before Pi (Virkki et al., 2006). A 3rd Na+ binding transition precedes a rate- limiting reorientation of the fully loaded carrier. Li+ ions can compete for occupancy of the the 1st Na+ binding site (Andrini et al.).The order of release of substrates to the cytosol is unknown. For NaPi-IIc, the 1st Na+, which confers electrogenicity to NaPi-IIa/b, still interacts but is not cotransported (Ghezzi et al., 2009). Electrogenic SLC34 proteins exhibit an + uncoupled leak that is most likely mediated by Na ions and is active only in the absence of Pi (Andrini et al., 2008). The Na+-leak is not found for the electroneutral NaPi-IIc (Bacconi et al., 2005), nevertheless, naturally occurring mutations in NaPi-IIc have been reported to result in a significant Na+-leak (Jaureguiberry et al., 2008). 2.4.2 Structure-function relationships of SLC34 proteins. SLC34 proteins belong to a unique class of membrane proteins and share no homology with the other main transporter families, even at the bacterial level. The current lack of a 3-D structure of the mammalian SLC34 proteins or their bacterial homologs, means that structural information is based on indirect biophysical and biochemical studies on wild-type and engineered mutations, e.g.(Forster et al., 2002; Virkki et al., 2007). SLC34 proteins are all assumed to be functional monomers (Kohler et al., 2000), although indirect evidence suggests they may exist as dimers at the BBM (Forster et al., 2006; Gisler et al., 2007).