Amount Of NHE3 Research Articles

31 – Endosomal Snx17 Sets the Plasma Membrane Amount of Nhe3 and Prevents Its Degradation by Binding to the Nhe3 Cterminus

31 – Endosomal Snx17 Sets the Plasma Membrane Amount of Nhe3 and Prevents Its Degradation by Binding to the Nhe3 Cterminus

Mechanisms underlying the inhibitory effects of uroguanylin on NHE3 transport activity in renal proximal tubule

We previously demonstrated that uroguanylin (UGN) significantly inhibits Na(+)/H(+) exchanger (NHE)3-mediated bicarbonate reabsorption. In the present study, we aimed to elucidate the molecular mechanisms underlying the action of UGN on NHE3 in rat renal proximal tubules and in a proximal tubule cell line (LLC-PK(1)). The in vivo studies were performed by the stationary microperfusion technique, in which we measured H(+) secretion in rat renal proximal segments, through a H(+)-sensitive microelectrode. UGN (1 μM) significantly inhibited the net of proximal bicarbonate reabsorption. The inhibitory effect of UGN was completely abolished by either the protein kinase G (PKG) inhibitor KT5823 or by the protein kinase A (PKA) inhibitor H-89. The effects of UGN in vitro were found to be similar to those obtained by microperfusion. Indeed, we observed that incubation of LLC-PK(1) cells with UGN induced an increase in the intracellular levels of cAMP and cGMP, as well as activation of both PKA and PKG. Furthermore, we found that UGN can increase the levels of NHE3 phosphorylation at the PKA consensus sites 552 and 605 in LLC-PK(1) cells. Finally, treatment of LLC-PK(1) cells with UGN reduced the amount of NHE3 at the cell surface. Overall, our data suggest that the inhibitory effect of UGN on NHE3 transport activity in proximal tubule is mediated by activation of both cGMP/PKG and cAMP/PKA signaling pathways which in turn leads to NHE3 phosphorylation and reduced NHE3 surface expression. Moreover, this study sheds light on mechanisms by which guanylin peptides are intricately involved in the maintenance of salt and water homeostasis.

Translational Approaches for Pharmacotherapy Development for Acute Diarrhea

Areport is presented of a National Institute of Diabetes and Digestive and Kidney Diseases–sponsored single topic conference designed to determine scientific advances needed to encourage development of pharmacotherapy for diarrheal diseases. Acute diarrheal diseases are a global public health problem in developing and developed countries. They are a leading cause of mortality and morbidity for children under the age of 5 worldwide and have a major economic impact in developed countries. Although oral rehydration solution (ORS) along with oral zinc therapy has reduced mortality owing to acute diarrheal diseases, there are essentially no other approved, safe, and effective drugs to decrease stool volume and prevent fluid loss. On September 25–27, 2011, the National Institute of Diabetes and Digestive and Kidney Diseases convened a workshop on Translational Approaches for Pharmacotherapy Development for Acute Diarrhea to assess current treatments for acute diarrhea, to identify common host pathways targeted by infectious agents associated with acute diarrhea, to evaluate which pathophysiologic mechanisms are most clinically relevant as potential targets, and to identify promising areas for translational research.

NHE3 mobility in brush borders increases upon NHERF2-dependent stimulation by lyophosphatidic acid

The epithelial brush border (BB) Na(+)/H(+) exchanger NHE3 is associated with the actin cytoskeleton by binding both directly and indirectly to ezrin; indirect binding is via attachment to NHERF family proteins. NHE3 mobility in polarized epithelial cell BBs is restricted by the actin cytoskeleton and NHERF binding such that only approximately 30% of NHE3 in the apical domain of an OK cell line stably expressing NHERF2 is mobile, as judged by FRAP analysis. Given that levels of NHE3 are partially regulated by changes in trafficking, we investigated whether the cytoskeleton association of NHE3 was dynamic and changed as part of acute regulation to allow NHE3 trafficking. The agonist studied was lysophosphatidic acid (LPA), an inflammatory mediator, which acutely stimulates NHE3 activity by increasing the amount of NHE3 on the BBs by stimulated exocytosis. LPA acutely stimulated NHE3 activity in OK cells stably expressing NHERF2. Two conditions that totally prevented LPA stimulation of NHE3 activity only partially prevented stimulation of NHE3 mobility: the phosphoinositide 3-kinase (PI3K) inhibitor LY294002, and the NHE3F1 double mutant which has minimal direct binding of NHE3 to ezrin. These results show that LPA stimulation of NHE3 mobility occurs in two parts: (1) PI3K-dependent exocytic trafficking to the BB and (2) an increase in surface mobility of NHE3 in BBs under basal conditions. Moreover, the LPA stimulatory effect on NHE3 mobility required NHERF2. Although NHE3 and NHERF2 co-precipitated under basal conditions, they failed to co-precipitate 30 minutes after addition of LPA, whereas the physical association was re-established by 50-60 minutes. This dynamic interaction between NHERF2 and NHE3 was confirmed by acceptor photobleaching Förster Resonance energy Transfer (FRET). The restricted mobility of NHE3 in BBs under basal conditions as a result of cytoskeleton association is therefore dynamic and is reversed as part of acute LPA stimulation of NHE3. We suggest that this acute but transient increase in NHE3 mobility induced by LPA occurs via two processes: addition of NHE3 to the BB by exocytosis, a process which precedes binding of NHE3 to the actin cytoskeleton via NHERF2-ezrin, and by release of NHERF2 from the NHE3 already localized in the apical membrane, enabling NHE3 to distribute throughout the microvilli. These fractions of NHE3 make up a newly identified pool of NHE3 called the 'transit pool'. Moreover, our results show that there are two aspects of LPA signaling involved in stimulation of NHE3 activity: PI3K-dependent stimulated NHE3 exocytosis and the newly described, PI3K-independent dissociation of microvillar NHE3 from NHERF2.

NHERF3 (PDZK1) Contributes to Basal and Calcium Inhibition of NHE3 Activity in Caco-2BBe Cells

Elevated intracellular Ca(2+) ([Ca(2+)](i)) inhibition of NHE3 is reconstituted by NHERF2, but not NHERF1, by a mechanism involving the formation of multiprotein signaling complexes. To further evaluate the specificity of the NHERF family in calcium regulation of NHE3 activity, the current study determined whether NHERF3 reconstitutes elevated [Ca(2+)](i) regulation of NHE3. In vitro, NHERF3 bound the NHE3 C terminus between amino acids 588 and 667. In vivo, NHE3 and NHERF3 associate under basal conditions as indicated by co-immunoprecipitation, confocal microscopy, and fluorescence resonance energy transfer. Treatment of PS120/NHE3/NHERF3 cells, but not PS120/NHE3 cells, with the Ca(2+) ionophore, 4-bromo-A23187 (0.5 mum): 1) inhibited NHE3 V(max) activity; 2) decreased NHE3 surface amount; 3) dissociated NHE3 and NHERF3 at the plasma membrane by confocal immunofluorescence and fluorescence resonance energy transfer. Similarly, in Caco-2BBe cells, NHERF3 and NHE3 colocalized in the BB under basal conditions but after elevation of [Ca(2+)](i) by carbachol, this overlap was abolished. NHERF3 short hairpin RNA knockdown (>50%) in Caco-2BBe cells significantly reduced basal NHE3 activity by decreasing BB NHE3 amount. Also, carbachol-mediated inhibition of NHE3 activity was abolished in Caco-2BBe cells in which NHERF3 protein expression was significantly reduced. In summary: 1) NHERF3 colocalizes and directly binds NHE3 at the plasma membrane under basal conditions; 2) NHERF3 reconstitutes [Ca(2+)](i) inhibition of NHE3 activity and dissociates from NHE3 in fibroblasts and polarized intestinal epithelial cells with elevated [Ca(2+)](i); 3) NHERF3 short hairpin RNA significantly reduced NHE3 basal activity and brush border expression in Caco-2BBe cells. These results demonstrate that NHERF3 reconstitutes calcium inhibition of NHE3 activity by anchoring NHE3 basally and releasing it with elevated Ca(2+).

Na+/H+ Exchanger NHE3 Activity and Trafficking Are Lipid Raft-dependent

A previous study showed that approximately 25-50% of rabbit ileal brush border (BB) Na(+)/H(+) exchanger NHE3 is in lipid rafts (LR) (Li, X., Galli, T., Leu, S., Wade, J. B., Weinman E. J., Leung, G., Cheong, A., Louvard, D., and Donowitz, M. (2001) J. Physiol. (Lond.) 537, 537-552). Here, we examined the role of LR in NHE3 transport activity using a simpler system: opossum kidney (OK) cells (a renal proximal tubule epithelial cell line) containing NHE3. approximately 50% of surface (biotinylated) NHE3 in OK cells distributed in LR by density gradient centrifugation. Disruption of LR with methyl-beta-cyclodextrin (MbetaCD) decreased NHE3 activity and increased K'(H+)(i), but K(m)((Na+)) was not affected. The MbetaCD effect was completely reversed by repletion of cholesterol, but not by an inactive analog of cholesterol (cholestane-3beta,5alpha,6beta-triol). The MbetaCD effect was specific for NHE3 activity because it did not alter Na(+)-dependent l-Ala uptake. MbetaCD did not alter OK cell BB topology and did not change the surface amount of NHE3, but greatly reduced the rate of NHE3 endocytosis. The effects of inhibiting phosphatidylinositol 3-kinase and of MbetaCD on NHE3 activity were not additive, indicating a common inhibitory mechanism. In contrast, 8-bromo-cAMP and MbetaCD inhibition of NHE3 was additive, indicating different mechanisms for inhibition of NHE3 activity. Approximately 50% of BB NHE3 and only approximately 11% of intracellular NHE3 in polarized OK cells were in LR. In summary, the BB pool of NHE3 in LR is functionally active because MbetaCD treatment decreased NHE3 basal activity. The LR pool is necessary for multiple kinetic aspects of normal NHE3 activity, including V(max) and K'(H+)(i), and also for multiple aspects of NHE3 trafficking, including at least basal endocytosis and phosphatidylinositol 3-kinase-dependent basal exocytosis. Because the C-terminal domain of NHE3 is necessary for its regulation and because the changes in NHE3 kinetics with MbetaCD resemble those with second messenger regulation of NHE3, these results suggest that the NHE3 C terminus may be involved in the MbetaCD sensitivity of NHE3.

Lysophosphatidic acid induces exocytic trafficking of Na +/H + exchanger 3 by E3KARP-dependent activation of phospholipase C

Lysophosphatidic acid (LPA) stimulates Na +/H + exchanger 3 (NHE3) activity in opossum kidney proximal tubule (OK) cells by increasing the apical membrane amount of NHE3. This occurs by stimulation of exocytic trafficking of NHE3 to the apical plasma membrane by an E3KARP-dependent mechanism. However, it is still unclear how E3KARP leads to the LPA-induced exocytosis of NHE3. In the current study, we demonstrate that stable expression of exogenous E3KARP increases LPA-induced phospholipase C (PLC) activation and subsequent elevation of intracellular Ca 2+ in opossum kidney proximal tubule (OK) cells. Pretreatment with U73122, a PLC inhibitor, prevented the LPA-induced NHE3 activation and the exocytic trafficking of NHE3. To understand how the elevation of intracellular Ca 2+ leads to the stimulation of NHE3, we pretreated OK cells with BAPTA-AM, an intracellular Ca 2+ chelator. BAPTA-AM completely blocked the LPA-induced increase of NHE3 activity and surface NHE3 amount by decreasing the LPA-induced exocytic trafficking of NHE3. Pretreatment with GF109203X, a PKC inhibitor, did not affect the percent of LPA-induced NHE3 activation and increase of surface NHE3 amount. From these results, we suggest that E3KARP plays a necessary role in LPA-induced PLC activation, and that PLC-dependent elevation of intracellular Ca 2+ but not PKC activation is necessary for the LPA-induced increase of NHE3 exocytosis.

Lysophosphatidic acid induces exocytic trafficking of Na+/H+ exchanger 3 by E3KARP-dependent activation of phospholipase C

Lysophosphatidic acid (LPA) stimulates Na+/H+ exchanger 3 (NHE3) activity in opossum kidney proximal tubule (OK) cells by increasing the apical membrane amount of NHE3. This occurs by stimulation of exocytic trafficking of NHE3 to the apical plasma membrane by an E3KARP-dependent mechanism. However, it is still unclear how E3KARP leads to the LPA-induced exocytosis of NHE3. In the current study, we demonstrate that stable expression of exogenous E3KARP increases LPA-induced phospholipase C (PLC) activation and subsequent elevation of intracellular Ca2+ in opossum kidney proximal tubule (OK) cells. Pretreatment with U73122, a PLC inhibitor, prevented the LPA-induced NHE3 activation and the exocytic trafficking of NHE3. To understand how the elevation of intracellular Ca2+ leads to the stimulation of NHE3, we pretreated OK cells with BAPTA-AM, an intracellular Ca2+ chelator. BAPTA-AM completely blocked the LPA-induced increase of NHE3 activity and surface NHE3 amount by decreasing the LPA-induced exocytic trafficking of NHE3. Pretreatment with GF109203X, a PKC inhibitor, did not affect the percent of LPA-induced NHE3 activation and increase of surface NHE3 amount. From these results, we suggest that E3KARP plays a necessary role in LPA-induced PLC activation, and that PLC-dependent elevation of intracellular Ca2+ but not PKC activation is necessary for the LPA-induced increase of NHE3 exocytosis.

Carbachol regulation of rabbit ileal brush border Na+–H+ exchanger 3 (NHE3) occurs through changes in NHE3 trafficking and complex formation and is Src dependent

The epithelial brush border membrane (BBM) Na(+)-H(+) exchanger 3 (NHE3) is the major transport protein responsible for ileal electroneutral Na(+) absorption. We have previously shown that ileal BBM NHE3 activity is rapidly inhibited by carbachol, an agonist that mimics cholinergic activation in digestion. In this study, we investigated the mechanisms involved in this NHE3 inhibition. Carbachol decreased the amount of ileal Na(+) absorptive cell BBM NHE3 within 10 min of exposure. Based on OptiPrep gradient centrifugation, carbachol increased the amount of NHE3 in early endosomes and decreased the amount of NHE3 in BBM, consistent with effects on NHE3 trafficking. The decrease in BBM NHE3 occurred in the detergent-soluble BBM fraction with no change in the amount of NHE3 in the BBM detergent-resistant membranes. The size of BBM NHE3 complexes increased in carbachol-exposed ileum, as studied with sucrose gradient centrifugation. The NHE3 complex size increased in the total BBM, but did not change in the detergent-soluble fraction. This suggests that carbachol treatment enhanced the association of proteins with NHE3 complexes specifically in the detergent-resistant fraction of ileal BBM. NHERF2, alpha-actinin-4 and protein kinase C were among those NHE3-associated proteins because they were more efficiently coimmunoprecipitated from total BBM after carbachol treatment. Moreover, Src was involved in the carbachol-mediated inhibition since: (1) c-Src was rapidly activated in the detergent-resistant membranes by carbachol; and (2) carbachol inhibition of ileal Na(+) absorption was completely abolished by the Src family inhibitor 4-amino-5-(4-chlorophenyl)-7-(t-butyl)pyrazolo[3,4-d]pyrimidine (PP2). Moreover, the carbachol-induced increase in the size of NHE3-containing complexes was reversed by PP2. These data demonstrate that regulation of NHE3 activity by carbachol can be achieved at several interrelated levels: (1) the subcellular level, at which NHE3 is rapidly endocytosed from BBM to endocytic vesicles upon treatment with carbachol; (2) multiple BBM pools, in which carbachol selectively decreases the amount of NHE3 in the BBM detergent-soluble fraction but not the detergent-resistant membrane; and (3) the molecular level, at which NHE3 complex-associated proteins can be changed upon carbachol treatment, with carbachol leading to larger BBM NHE3 complexes and increased co-IP of NHERF2 with alpha-actinin-4 and activated PKC. The study further describes NHE3 presence simultaneously in multiple dynamic BBM pools in which NHE3 distribution and associated proteins are altered as part of carbachol-induced and Src-mediated rapid signal transduction, which decreases the amount of BBM NHE3 and thus inhibits NHE3 activity.

Ca2+-dependent inhibition of NHE3 requires PKC alpha which binds to E3KARP to decrease surface NHE3 containing plasma membrane complexes.

The intestinal brush border (BB) Na+/H+ exchanger isoform 3 (NHE3) is acutely inhibited by elevation in the concentration of free intracellular Ca2+ ([Ca2+]i) by the cholinergic agonist carbachol and Ca2+ ionophores in a protein kinase C (PKC)-dependent manner. We previously showed that elevating [Ca2+]i with ionomycin rapidly inhibited NHE3 activity and decreased the amount of NHE3 on the plasma membrane in a manner that depended on the presence of the PDZ domain-containing protein E3KARP (NHE3 kinase A regulatory protein, also called NHERF2). The current studies were performed in PS120 fibroblasts (NHE-null cell line) stably transfected with NHE3 and E3KARP to probe the mechanism of PKC involvement in Ca2+ regulation of NHE3. Pretreatment with the general PKC inhibitor, GF109203X prevented ionomycin inhibition of NHE3 without altering basal NHE3 activity. Similarly, the Ca2+-mediated inhibition of NHE3 activity was blocked after pretreatment with the conventional PKC inhibitor Gö-6976 and a specific PKCalpha pseudosubstrate-derived inhibitor peptide. [Ca2+]i elevation caused translocation of PKCalpha from cytosol to membrane. PKCalpha bound to the PDZ1 domain of GST-E3KARP in vitro in a Ca2+-dependent manner. PKCalpha and E3KARP coimmunoprecipitated from cell lysates; this occurred to a lesser extent at basal [Ca2+]i and was increased with ionomycin exposure. Biotinylation studies demonstrated that [Ca2+]i elevation induced oligomerization of NHE3 in total lysates and decreased the amount of plasma membrane NHE3. Treatment with PKC inhibitors did not affect the oligomerization of NHE3 but did prevent the decrease in surface amount of NHE3. These results suggest that PKCalpha is not necessary for the Ca2+-dependent formation of the NHE3 plasma membrane complex, although it is necessary for decreasing the membrane amounts of NHE3, probably by stimulating NHE3 endocytosis.

Aldosterone stimulates surface expression of NHE3 in renal proximal brush borders.

The mineralocorticoid aldosterone is one of the major regulators of extracellular volume and blood pressure. It acts by enhancing Na(+) reabsorption across tight epithelia such as renal collecting ducts and colon. In addition, it has been shown that aldosterone stimulates NaCl and volume reabsorption in renal proximal tubules by an unknown mechanism. To test the hypothesis that the application of aldosterone results in greater activity of the apical Na(+)/H(+) exchanger-3 (NHE3), we investigated the effect of aldosterone on amiloride-sensitive, proximal tubular volume reabsorption and proximal tubular NHE3 abundance in adrenalectomized rats. Aldosterone at physiological concentrations (dosage 36 microg/100 g b.w. per day) increased NHE3-dependent proximal tubular volume reabsorption and the abundance of NHE3 in brush borders without changing the total amount of NHE3 in cortical homogenates. These results indicate that renal proximal tubular NHE3 is a target for aldosterone-mediated regulation resulting in increased Na(+) reabsorption and thus extracellular volume and blood pressure. Further studies are required to determine the precise mechanism of action, especially whether the action of aldosterone on proximal tubular function is direct or indirect.

Chronic renal injury-induced hypertension alters renal NHE3 distribution and abundance.

Renal cortical phenol injection provokes acute sympathetic nervous system-dependent hypertension and a shift of proximal tubule Na(+)/H(+) exchanger isoform 3 (NHE3) and Na(+)-P(i) cotransporter type 2 (NaPi2) to apical microvilli. This study aimed to determine whether proximal tubule (PT) Na(+) transporter redistribution persists chronically and whether the pool sizes of renal Na(+) transporters are altered. At 5 wk after a 50-microl 10% phenol injection, blood pressure is elevated: 154 +/- 8 vs. 113 +/- 11 mmHg after saline injection. Cortical membranes were fractionated into three "windows" enriched in apical brush border (WI), mixed apical and intermicrovillar cleft (WII), and intracellular membranes (WIII). NHE3 relative distribution in these windows, assessed by immunoblots and expressed as %total, remained shifted to apical from intracellular membranes (WI: 25.3 +/- 3 in phenol vs.12.7 +/- 3% in saline and WIII: 9.1 +/- 1.3 in phenol vs. 18.9 +/- 3% in saline). NaPi2 and dipeptidyl-peptidase IV also remained shifted to WI, and alkaline phosphatase activity increased 100.9 +/- 29.7 (WI) and 51.4 +/- 17.5% (WII) in phenol-injected membranes. Na(+) transporter total abundance [NHE3, NaPi2, thiazide-sensitive Na-Cl cotransporter, bumetanide-sensitive Na-K-2Cl cotransporter, Na-K-ATPase alpha(1)- and beta(1)-subunits, and epithelial Na(+) channel (ENaC) alpha- and beta-subunits] was profiled by immunoblotting. Only cortical NHE3 abundance was altered, decreasing to 0.56 +/- 0.06. The results demonstrate that phenol injury provokes a persistant shift of PT NHE3 and NaPi2 to the apical microvilli, along with a 44% decrease in total NHE3, evidence for an escape mechanism that would counteract the redistribution of a larger fraction of NHE3 to the apical surface by normalizing the total amount of NHE3 in apical membranes.

Na+-H+ exchanger 3 (NHE3) is present in lipid rafts in the rabbit ileal brush border: a role for rafts in trafficking and rapid stimulation of NHE3.

1. Rabbit ileal Na+-absorbing cell Na+-H+ exchanger 3 (NHE3) was shown to exist in three pools in the brush border (BB), including a population in lipid rafts. Approximately 50% of BB NHE3 was associated with Triton X-100-soluble fractions and the other approximately 50% with Triton X-100-insoluble fractions; approximately 33% of the detergent-insoluble NHE3 was present in cholesterol-enriched lipid microdomains (rafts). 2. The raft pool of NHE3 was involved in the stimulation of BB NHE3 activity with epidermal growth factor (EGF). Both EGF and clonidine treatments were associated with a rapid increase in the total amount of BB NHE3. This EGF- and clonidine-induced increase of BB NHE3 was associated with an increase in the raft pool of NHE3 and to a smaller extent with an increase in the total detergent-insoluble fraction, but there was no change in the detergent-soluble pool. In agreement with the rapid increase in the amount of NHE3 in the BB, EGF also caused a rapid stimulation of BB Na+-H+ exchange activity. 3. Disrupting rafts by removal of cholesterol with methyl-beta-cyclodextrin (MbetaCD) or destabilizing the actin cytoskeleton with cytochalasin D decreased the amount of NHE3 in early endosomes isolated by OptiPrep gradient fractionation. Specifically, NHE3 was shown to associate with endosomal vesicles immunoisolated by anti-EEA1 (early endosomal autoantigen 1) antibody-coated magnetic beads and the endosome-associated NHE3 was decreased by cytochalasin D and MbetaCD treatment. 4. We conclude that: (i) a pool of ileal BB NHE3 exists in lipid rafts; (ii) EGF and clonidine increase the amount of BB NHE3; (iii) lipid rafts and to a lesser extent, the cytoskeleton, but not the detergent-soluble NHE3 pool, are involved in the EGF- and clonidine-induced acute increase in amount of BB NHE3; (iv) lipid rafts and the actin cytoskeleton play important roles in the basal endocytosis of BB NHE3.

Short-term regulation of NHE3 by EGF and protein kinase C but not protein kinase A involves vesicle trafficking in epithelial cells and fibroblasts.

NHE3 is an intestinal epithelial isoform Na+/H+ exchanger that is present in the brush border of small intestinal, colonic, and gallbladder Na(+)-absorbing epithelial cells. NHE3 is acutely up- and downregulated in response to some G protein-linked receptors, tyrosine kinase receptors, and protein kinases when studied in intact ileum, when stably expressed in PS120 fibroblasts, and in the few studies reported in the human colon cancer cell line Caco-2. In most cases this is due to changes in Vmax of NHE3, although in response to cAMP and squalamine there are also changes in the K'(H+)i of the exchanger. The mechanism of the Vmax regulation as shown by cell surface biotinylation and confocal microscopy in Caco-2 cells and biotinylation in PS120 cells involves changes in the amount of NHE3 on the plasma membrane. In addition, in some cases there are also changes in turnover number of the exchanger. In some cases, the change in amount of NHE3 in the plasma membrane is associated with a change in the amount of plasma membrane. A combination of biochemical studies and transport/inhibitor studies in intact ileum and Caco-2 cells demonstrated that the increase in brush border Na+/H+ exchange caused by acute exposure to EGF was mediated by PI 3-kinase. PI 3-kinase was also involved in FGF stimulation of NHE3 expressed in fibroblasts. Thus, NHE3 is another example of a transport protein that is acutely regulated in part by changing the amount of the transporter on the plasma membrane by a process that appears to involve vesicle trafficking and also to involve changes in turnover number.