Protein histidine phosphatase 1 (PHPT-1) is an evolutionarily conserved 14-kDa protein that dephosphorylates phosphohistidine. PHPT-1−/− mice were generated to gain insight into the role of PHPT-1 and histidine phosphorylation/dephosphorylation in mammalian biology. PHPT-1−/− mice exhibited neonatal hyperinsulinemic hypoglycemia due to impaired trafficking of KATP channels to the plasma membrane in pancreatic β-cells in response to low glucose and leptin and resembled patients with congenital hyperinsulinism (CHI). The defect in KATP channel trafficking in PHPT-1−/− β-cells was due to the failure of PHPT-1 to directly activate transient receptor potential channel 4 (TRPC4), resulting in decreased Ca2+ influx and impaired downstream activation of AMPK. Thus, these studies demonstrate a critical role for PHPT-1 in normal pancreatic β-cell function and raise the possibility that mutations in PHPT-1 and/or TRPC4 may account for yet to be defined cases of CHI.

Although histidine phosphorylation has been proposed to account for >6% of the total phosphorylated proteins in mammals for >40 years, very little is known about the identity of the kinases or phosphatases that regulate histidine phosphorylation in mammals, their protein targets, or the biological consequences mediated by histidine phosphorylation. Two-component histidine kinases, whose identity and biological roles are well understood in bacteria, fungi, and plants, do not exist in mammals (1). To date, only two mammalian histidine kinases, nucleoside diphosphate kinase (NDPK)-A (NME1) and NDPK-B (NME2) (1), and two mammalian histidine phosphatases, protein histidine phosphatase 1 (PHPT-1) (2,3) and phosphoglycerate mutase family 5 (PGAM5) (4), have been identified.

NDPKs are encoded by the Nme (nonmetastatic cell) gene family and consist of 10 family members of between 16 and 20 kDa (5). Although early studies were mostly related to their ability to transfer the γ-phosphate of a nuclear triphosphate (NTP) to a NDP via a phosphohistidine intermediate (5), NDPK-A and NDPK-B are ubiquitously expressed and also function as histidine kinases. NDPK-A and -B bear no sequence similarity or structural resemblance to protein tyrosine or serine threonine kinases (1). PHPT-1 is an evolutionarily conserved 14-kDa protein that is encoded by a single gene and was first discovered based on its ability to dephosphorylate phosphohistidine (2,3,6). PHPT-1 does not resemble serine/threonine or tyrosine phosphatases and does not contain an invariant conserved cysteine motif (Cx5R) found in other phosphatases.

PGAM5 is a second mammalian histidine phosphatase that specifically dephosphorylates and inhibits NDPK-B (4). PGAM5 is 1 of 10 members of the phosphoglycerate mutase family that share a conserved PGAM motif (7). Many members of this family function as metabolic enzymes; however, PGAM5 does not exhibit mutase activity but rather has been shown to function as serine/threonine and, more recently, a histidine phosphatase that specifically dephosphorylates and inhibits NDPK-B (4,8,9). PGAM5 also does not contain a catalytic cysteine residue, but rather, like PHPT-1, uses a conserved histidine as a phosphoacceptor (3,10).

During the past several years, genetic and biochemical evidence has emerged demonstrating that NDPKs, PHPT-1, and PGAM5 regulate a variety of biological processes by reversible histidine phosphorylation, thus confirming the critical role for these molecules as well as histidine phosphorylation/dephosphorylation in mammals. NDPKs and PHPT-1 have been shown to regulate at least three distinct substrates by reversible histidine phosphorylation, which include the intermediate conductance K+ channel KCa3.1 (11,12), the β-subunit of heterotrimeric G proteins (Gβ) (1,13), and the Ca2+-conducting transient receptor potential (TRP) channel TRPV5 (14). NDPK-B phosphorylates H358 in the carboxy terminus of KCa3.1, and this phosphorylation is required for KCa3.1 channel activation, Ca2+ influx, and activation of CD4 T cells and mast cells (11,15,16). In contrast, PHPT-1 inhibits KCa3.1 and thereby T-cell and mast cell activation by dephosphorylating the same histidine residue (12,15,16). PGAM5 functions as a histidine phosphatase to specifically dephosphorylate H118 on NDPK-B, thereby inhibiting NDPK-B phosphorylation and activation of KCa3.1 and subsequent T-cell receptor (TCR)-stimulated Ca2+ influx and T-cell activation. TRPV5, which mediates Ca2+ reabsorption in the distal nephron of the kidney, is regulated in a similar manner to KCa3.1 (14). We have now generated PHPT-1−/− mice to gain further insight into the role for PHPT-1 in vivo. The studies reported here demonstrate that PHPT-1 plays a critical role in trafficking of KATP channels to the plasma membrane (PM) by directly activating TRPC4 in pancreatic β-cells and thereby regulates insulin release from pancreatic β-cells.

Pancreatic β-Cell Isolation and Rat Insulinoma Cells

Pancreatic islets were isolated from PHPT-1+/+ and PHPT-1−/− mice by collagenase digestion, and pancreatic β-cells were isolated after digestion with trypsin (17). To generate short hairpin (sh)PHPT-1 knockdown, rat insulinoma (INS-1) cells (clone 832/13) were infected with shRNA PHPT-1 (clone TRCN0000080981; Sigma-Aldrich) or shRNA vector control, and pools of cells were selected in puromycin.

Constructs and Cell Transfection

The cDNAs for TRPC4α and TRPC4β (18) were cloned into C1-green fluorescent protein (GFP) (Invitrogen) to generate GFP-TRPC4α and TRPC4β cDNAs. GFP-TRPC4α (H912N) was generated by overlapping PCR using the following oligos: forward 5′-GTGTTAGTAGACAACAGAGAAAGGA-3′, reverse 5′-TCCTTTCTCTGTTGTCTACTAACAC-3′. HIS–PHPT-1(wild type [WT]) or phosphatase-dead HIS–PHPT-1(H53A) were generated as previously described (19).

Immunoblotting and Antibodies

Anti–PHPT-1 antibodies were previously described (19). Anti-Kir6.2 H18 antibody was purchased from Santa Cruz Biotechnology, and anti-SUR1 antibodies were previously described (20).

Whole-Cell Patch Clamp

Whole-cell patch clamp was used to measure KATP or TRPC4 channel current. To measure KATP channel current, a voltage ramp of 4 s was applied, ranging from 0 to −100 mV from a holding potential of −45 mV (21). To pharmacologically inhibit TRPC4 and CaMKKβ, cells were pretreated respectively with 10 μmol/L ML-204 or TRPC4-069 (22,23).

Amphotericin was added in the pipette solution to measure membrane potential, and current clamp mode was used as previously described (24).

To measure TRPC4 channel current, a voltage ramp of 500 ms was applied, ranging from +100 to −100 mV from a holding potential of −60 mV, as previously described, with some modifications (25).

Surface Biotinylation

Biotinylation of surface proteins in INS-1 cells was performed by labeling cells with EZ-Link Sulfo-NHS-SS-Biotin (Thermo Scientific, Rockford, IL), as previously described (14). Biotinylated proteins were precipitated using high-capacity agarose resin, and cell-surface SUR1 was assessed by immunoblotting the avidin precipitates with anti-SUR1 antibodies, as previously described (20).

Expression and Detection of Cell Surface α-Bungarotoxin–Binding Sequence SUR1

WT and shRNA PHPT-1 INS-1 cells were transduced with α-bungarotoxin (BTX)–binding sequence (BTX-tag) SUR1 and Kir6.2 viruses, as previously described (20). Cells were then cultured in high or low glucose in the presence or absence of leptin (10 nmol/L) for the times as indicated, and cell surface expression of SUR1 was quantitated by FACS after staining with Alexa Fluor 555 α-BTX.

Perifused Islets and Insulin Secretion

Islets were isolated by collagenase digestion and cultured overnight in RPMI 1640 medium containing 10 mmol/L or 2 mmol/L glucose supplemented with 10% FBS, 2 mmol/L glutamine, and penicillin and streptomycin, as previously described (26). Separate perifusion was performed in 2 mmol/L glucose-cultured islets, and islets were perifused with a ramp of glucose (0–15 mmol/L with 0.5 mmol/L/min increment and then 15 mmol/L to 0 with 0.5 mmol/L/min decrements). Islets were finally exposed to 30 mmol/L KCl. Samples were collected every minute for insulin measurements, which were determined by HTRF Insulin Kit (Cisbio Bioassays, Bedford, MA).

Cytosolic Free Ca2+ Measurements in Islets

Measurements of cytosolic Ca2+ were described previously (27) using a dual wavelength fluorescence microscope. Fura-2 acetoxymethylester (Life Technologies) was used as calcium indicator (26).

Glucose Tolerance Test

All mice were housed in the New York University School of Medicine Central Animal Facility. Mice were maintained in accordance with the Animal Welfare Act, the U.S. Department of Agriculture Regulations (9 CFR, Parts 1, 2, and 3), and the Guide for the Care and Use of Laboratory Animals (National Academy Press, Revised 1996). Animals were fasted overnight and then injected intraperitoneally with 29% glucose (10 μL/g body weight), and blood glucose levels were assessed using a glucometer and test strips at 15, 30, 60, and 90 min.

PHPT-1 Is Associated With Perinatal Hypoglycemia

PHPT-1−/− mice derived from gene-targeted albino C57Bl/6 ES cells were generated (Fig. 1A and B). PHPT-1−/− mice were born at the expected Mendelian ratio, indicating that PHPT-1 is not essential for embryonic development. However, some PHPT-1−/− mice exhibited increased perinatal mortality. Further evaluation demonstrated a statistically significant decrease in blood glucose levels in PHPT-1−/− newborn mice (day 1) compared with PHPT-1+/+ mice (in mg/dL, PHPT-1+/+ 77.8 [SEM 2.2] and PHPT-1−/− 51.6 [SEM 2.8], n = 10 mice in each group; P < 0.01 PHPT-1−/− compared with PHPT-1+/+ mice), which was associated with increased serum insulin (ng/mL)/blood glucose (mg/dL) levels (PHPT-1+/+ 0.0017 [SEM 2E−4], PHPT-1−/− 0.055 [SEM 7E−4], n = 10 mice in each group; P < 0.01 PHPT-1−/− compared with PHPT-1+/+ mice). Blood glucose levels normalized in PHPT-1−/− mice 48–72 h after birth, and differences in blood glucose levels between PHPT-1−/− and PHPT-1+/+ mice were no longer present.

KATP Channel Activity Is Decreased in β-Cells From PHPT-1−/− Mice Leading to Decreased PM Potential

Congenital hyperinsulinism (CHI) in newborns is characterized by neonatal hypoglycemia due to inappropriate release of insulin from pancreatic β-cells (28,29). A common cause for CHI in newborns is loss-of-function mutations in the Kir6.2 and SUR1 subunits of KATP channels (KATP CHI) (28,30). Similar to patients with KATP CHI who do not respond to diazoxide therapy, basal and maximal KATP channel activity after diazoxide treatment was markedly decreased in PHPT-1−/− β-cells compared with PHPT-1+/+ β-cells, as assessed by whole-cell patch clamp (Fig. 2A). These results were confirmed in rat INS-1 cells in which PHPT-1 was knocked down with a PHPT-1 shRNA (Fig. 2B), which was rescued by overexpressing an shRNA-resistant PHPT-1 mutant (GFP-PHPT) (Fig. 2Biii). The decreased KATP channel current was not due to decreased amounts of KATP channels because PHPT-1−/− and PHPT-1+/+ β-cells and INS-1 cells expressed similar amounts of Kir6.2 and SUR1 protein (Figs. 2Aiv and Biv, and 3B). The decrease in the whole-cell KATP channel current was confirmed in excised membrane patches PHPT-1−/− β-cells. (see Supplementary Fig. 1).

KATP channels are critical in setting the membrane potential in pancreatic β-cells (31). Decreased KATP channel activity leads to membrane depolarization, activation of voltage-dependent Ca2+ channels (VDCCs), Ca2+ influx, and ultimately, exocytosis of insulin (32). Consistent with decreased KATP current, PHPT-1−/− β-cells were significantly depolarized compared with PHPT-1+/+ cells (PHPT-1+/+ = −72 ± 1.5 mV, PHPT-1−/− = −42 ± 6 mV; P = 0.001 by one- way ANOVA) (Fig. 2Ci–iii), and, unlike WT cells, exhibited rapid fluctuations in 1 mmol/L glucose, which is consistent with a basal elevated membrane potential being at the threshold of stimulating electrical activity, as previously described for SUR1−/− β-cells (33) (Fig. 2Ci and ii).

PHPT-1−/− β-Cells Have Decreased Trafficking of KATP Channels to the PM in Response to Low Glucose and Leptin Stimulations

Trafficking of KATP channels to and from the PM has emerged as an important regulator of β-cell function and insulin release (20,34). AMPK has been shown to play a critical role in inducing the trafficking of KATP channels from an intracellular compartment to the plasma membrane (20,34,35). In low glucose, the AMP-to-ATP ratio increases in β-cells and, via activation of AMPK, induces the translocation of KATP channels to the PM. However, when the extracellular glucose concentration is high, the AMP-to-ATP ratio is low, which results in most of the KATP channels being localized to an intracellular compartment due to decreased AMPK activation. Consistent with previous reports, the KATP channel current was much higher under low glucose (1 mmol/L) compared with high glucose (10 mmol/L) conditions in PHPT-1+/+ β-cells (Fig. 3Ai). In contrast, KATP currents were low under both high and low glucose conditions in PHPT-1−/− β-cells (Fig. 3Ai). Treatment of PHPT-1−/− β-cells with the AMPK activator AICAR restored KATP channel currents to similar levels as PHPT-1+/+ β-cells, suggesting that defective activation of AMPK could account for the impaired trafficking of KATP channels in PHPT-1−/− cells (Fig. 3Ai). The effect of AICAR was mediated by AMPK activation because treatment with the AMPK inhibitor compound C blocked AICAR-induced KATP channel currents (Fig. 3Aiib). Similar findings were also found in shRNA PHPT-1 knockdown INS-1 cells (Fig. 3Aii).

Leptin also stimulates the translocation of KATP channels to the PM in pancreatic β-cells via activation of AMPK (20,34,36). Leptin stimulation increased KATP currents in PHPT-1+/+ β-cells and WT INS-1 cells under high glucose conditions but failed to stimulate KATP currents in PHPT-1−/− β-cells or PHPT-1 shRNA INS-1 cells (Fig. 3Aia and iia).

Consistent with the patch clamp data, surface labeling of WT and shRNA PHPT-1 INS-1 cells with biotin showed decreased low glucose (Fig. 3Bi) and leptin (Fig. 3Bii) stimulated increase in PM SUR1 in PHPT-1 shRNA INS-1 cells. These findings were also confirmed in WT and PHPT-1 shRNA INS-1 cells that were transduced with BTX-tag SUR1 and Kir6.2 viruses and incubated under similar conditions (20); PM KATP channels were assessed by FACS after labeling with Alexa Fluor 355 α-BTX (Fig. 3C) (20).

Impaired Glucose-Stimulated Insulin Release in Isolated Islets From PHPT-1−/− Mice

Glucose-stimulated insulin release was next assessed in isolated islets. Consistent with the electrophysiology results, we did not detect differences in basal or amino acid and glucose-stimulated intracellular Ca2+ levels or insulin release between PHPT-1−/− and PHPT-1+/+ islets when islets were cultured under high glucose (10 mmol/L) conditions before performing the experiments (Fig. 4Ai and ii). In contrast, basal intracellular Ca2+ levels were significantly increased in PHPT-1−/− islets compared with PHPT-1+/+ islets when cultured under low glucose (2 mmol/L) conditions (Fig. 4Bi). In addition, whereas glucose-stimulated insulin release was significantly decreased in PHPT-1+/+ islets cultured under low glucose conditions compared with PHPT-1+/+ islets cultured under high glucose conditions (Fig. 4Aii and Bii), glucose-stimulated insulin release was further impaired in PHPT-1−/− islets compared with PHPT-1+/+ islets (Fig. 4Biii and iv). The difference between PHPT-1−/− and PHPT-1+/+ islets was not due to decreased intracellular insulin levels or the inability of PHPT-1−/− islets to release insulin because KCl-stimulated insulin release was increased in PHPT-1−/− islets, presumably due to decreased glucose-stimulated insulin release before exposure to KCl. The impaired glucose-stimulated insulin response of PHPT-1−/− islets cultured under low glucose conditions is consistent with findings in mouse Kir6.2−/− and SUR1−/− islets (3741) and is also consistent with impaired glucose-stimulated insulin release or “glucose blindness” in humans with loss-of-function KATP channel mutations (27).

Leptin Activation of TRPC4 Is Defective in PHPT-1−/− β-Cells

A signaling pathway coupling leptin signaling to AMPK has recently been mapped. This signaling pathway includes leptin activation of the Ca2+ permeable TRPC4 channel leading to an influx in extracellular Ca2+ (34). Increased Ca2+ then activates CaMKKβ, which ultimately activates AMPK by phosphorylating threonine 172 (34). To identify where PHPT-1 functions on this signaling pathway, we assessed whether impaired activation of endogenous TRPC4α could account for the defect in PHPT-1−/− β-cells. Cesium (Cs)- and leptin-induced activation of TRPC4 Ca2+ currents was impaired in PHPT-1−/− β-cells compared with PHPT-1+/+ β-cells (Fig. 5A). The TRPC4 current measured was specific because it was induced by Cs and inhibited with the selective TRPC4 inhibitor ML-204 (42). Leptin- and Cs-stimulated TRPC4α current was also reduced in shRNA PHPT-1 INS-1 cells transfected with GFP-TRPC4α (WT) (Fig. 5B). Similar levels of GFP-TRPC4α localized to the plasma membrane in WT and PHPT-1 shRNA INS-1 cells, indicating that decreased amounts of TRPC4 protein at the PM did not account for the decreased TRPC4 current (Fig. 5C). Leptin also activated TRPC4β, a spliced isoform of TRPC4α lacking 84 amino acids in the carboxy terminus of TRPC4α (Fig. 5D).

TRPC5 is the closest homolog of TRPC4 and shares 64% sequence identify (43). Leptin failed to activate TRPC5 in INS-1 cells, and PHPT-1 was not necessary for Cs-stimulated activation of TRPC5 current (Fig. 5E), indicating that PHPT-1 is specifically required for TRPC4 activation. In addition, TRPC4 and PHPT-1, coimmunoprecipitated with TRPC4 (Fig. 5F).

H912 in the C Terminus of TRPC4 Mediates the Inhibition of TRPC4 in PHPT-1−/− β-Cells

The decreased TRPC4 current in PHPT-1−/− β-cells suggested that increased phosphorylation of a histidine residue in the C terminus of TRPC4, which is normally dephosphorylated by PHPT-1, accounted for the inhibition of TRPC4 channel activity. To assess this possibility, individual histidine residues in the C terminus of TRPC4 were mutated to asparagine, transfected into shRNA PHPT-1 INS-1 cells, and leptin-induced TRPC4 and KATP channel current were assessed. These studies demonstrated that mutation of only histidine 912 (TRPC4[H912N]) restored TRPC4 channel activation in response to leptin stimulation in shRNA PHPT-1 INS-1 cells (Fig. 6Ai). TRPC4(H912N) also rescued leptin-induced KATP currents in shRNA PHPT-1 INS-1 cells to similar levels as control WT INS-1 cells (Fig. 6Aii) and KATP currents in shRNA PHPT-1 INS-1 cells in response to low glucose (1 mmol/L) (Fig. 6Aii).

PHPT-1 Activates TRPC4 in Inside/Out Patch Experiments

To directly assess the effect of PHPT-1 on TRPC4 channel activity, inside/out (I/O) patch clamp experiments were performed on INS-1 cells transfected with TRPC4(WT) or TRPC4(H912N). Application of PHPT-1(WT), but not the phosphatase-dead PHPT-1(H53A), to TRPC4(WT) I/O patches led to an increase in TRPC4 current in the presence of 100 nmol/L free Ca2+ (Fig. 6Bi), indicating that PHPT-1 directly activates TRPC4 channel activity and that this activation requires the catalytic activity of PHPT-1. Basal TRPC4(H912N) channel activity was similar to TRPC4(WT) at substimulatory free Ca2+ concentrations (100 nmol/L), but TRPC4(H912N) channel activity was markedly increased with increased free Ca2+ concentrations (750 nmol/L) compared with TRPC4(WT) (Fig. 6Bi–iii), and PHPT-1 did not further activate TRPC4(H912N) under any of the conditions tested (Fig. 6Bii and iii). Thus, these data are consistent with phospho-H912 functioning to negatively regulate TRPC4 and suggest that this inhibition can be mitigated by PHPT-1–stimulated dephosphorylation or by mutation of H912 to asparagine.

Adult PHPT-1−/− Mice Exhibit Impaired Glucose Tolerance and Insulin Release

Mouse knockouts of KATP channel subunits Kir6.2 or SUR1 maintain normal blood glucose levels but exhibit glucose intolerance as they age due to impaired insulin release in response to glucose stimulation from pancreatic β-cells (3739). PHPT-1−/− mice also exhibited impaired glucose tolerance in response to a glucose tolerance test, which was associated with decreased serum insulin levels (Fig. 7A and B). The defect in insulin release was not due to abnormalities in the pancreatic islets because PHPT-1+/+ and PHPT-1−/− islets looked phenotypically normal and stained similarly with anti-insulin (β-cells) and anti-glucagon (α-cells) antibodies (Fig. 7C).

It is now evident that histidine phosphorylation/dephosphorylation is not only relevant to prokaryotes and fungi but also plays important roles in normal mammalian biology. The most striking phenotype in PHPT-1−/− mice was neonatal hyperinsulinemic hypoglycemia due to impaired trafficking of KATP channels to the PM, which resulted in a phenotype that mimicked mouse knockouts of the KATP channel subunits Kir6.2 and SUR1 (3739).

KATP channel activity is central to coupling β-cells to glucose-stimulated insulin secretion (32). In the setting of high plasma glucose concentrations, the resultant metabolism of glucose leads to ATP generation and the direct inhibition of KATP channels in the PM. Closure of KATP channels subsequently leads to membrane depolarization, Ca2+ influx, and insulin release. CHI in people generally occurs due to loss-of-function mutations in Kir6.2 (KCNJ11) or SUR1 (ABCC8) subunits resulting in membrane depolarization, which then results in persistent and unregulated insulin secretion and, ultimately, low plasma glucose levels. Kir6.2−/− and SUR1−/− mice have surprisingly mild phenotypes compared with humans with KATP CHI, who exhibit persistent hyperinsulinemic hypoglycemia that frequently requires nearly total pancreatectomy (29,30,44). In contrast, Kir6.2−/− or SUR1−/− mice exhibit neonatal hyperinsulinemic hypoglycemia for the first 2 days after birth, which then normalizes shortly after birth, and Kir6.2 and SUR1 knockouts both develop glucose intolerance later in life (3739). These phenotypes were recapitulated in PHPT-1−/− mice, where decreased KATP channel currents was not due to gene inactivation but rather to intracellular retention of KATP channels due to their failure to traffic to the PM. The importance of PHPT-1 in regulating KATP trafficking is further reinforced by our finding that KATP trafficking is also impaired in PHPT-1−/− cardiomyocytes (Supplementary Fig. 2).

In addition to direct regulation of KATP channel currents by AMP-to-ADP-to-ATP ratios, recent studies have highlighted the importance for KATP channel trafficking to and from the PM to KATP channel current and membrane potential to normal β-cell physiology (36). Under low glucose conditions, KATP channel currents are maximal, leading to hyperpolarization of the membrane potential, which is mediated both by a direct effect of decreased ATP-to-ADP-to-AMP ratio levels on the activity of KATP channels in the PM and AMPK-stimulated redistribution of KATP channels to the PM (20,34,36). Maximal KATP channel current ensures that the β-cell is hyperpolarized, thereby preventing inappropriate insulin release that could lead to hypoglycemia. Although activation of AMPK by low glucose has been known to promote trafficking of KATP channels to the PM (20,34,36), the need for other upstream signals has been unappreciated. We now find that PHPT-1 is also required for low glucose–stimulated translocation of KATP channels to the PM. This is supported by our finding of decreased KATP channels at the PM and decreased KATP channel current in PHPT-1−/− β-cells cultured under low glucose conditions but not under high glucose conditions. The physiological effects of low glucose conditions on KATP channel trafficking to the PM were also associated with membrane depolarization leading to increased basal intracellular Ca2+ levels and impaired glucose-stimulated insulin release in PHPT-1−/− perifused islets compared with PHPT-1+/+ islets in low glucose media, which was not evident when PHPT-1−/− islets were cultured under high glucose conditions. The phenotype of PHPT-1−/− islets cultured under low glucose conditions recapitulates previous studies of Kir6.2−/− and SUR1−/− islets (3741,44).

Leptin has also been shown to play an important role in KATP trafficking (45,46), and studying the pathways regulated by leptin has provided important insight into the signaling molecules downstream of the leptin receptor that regulate KATP trafficking (20,34). In addition to acting centrally, leptin has direct effects on a wide range of peripheral organs, including the pancreatic β-cell, where it facilitates the redistribution of KATP channels to the PM leading the hyperpolarization of β-cells and decreased insulin release (34,45,46). Leptin signaling in β-cells and its role in inhibiting insulin secretion is thought to have important physiological effects under normal and disease states. Leptin has been proposed to function in an adipoinsular axis where it provides feedback inhibition from adipocytes to limit insulin release from β-cells, thereby preventing an increase in adipocyte mass from the proadipogenic effects of insulin (46). Leptin has also been shown to be essential for the relocalization of KATP channels to the PM in fasting animals in vivo (34). On the flip side, increased release of leptin from adipose tissue in obese individuals may contribute to the impaired glucose intolerance in type 2 diabetes (46). The signaling pathway activated by leptin includes the activation of Janus kinase 2 and phosphatidyl inositol 3-kinase, which function to activate the Ca2+-permeable TRPC4 channel leading to an influx in extracellular Ca2+ (20,34). Increased Ca2+ then activates CaMKKβ, which by phosphorylating and activating AMPK stimulates the translocation of KATP channels to the PM (20,34,36). We now show that PHPT-1 plays a critical role in TRPC4 activation by dephosphorylating H912 in the C terminus of TRPC4 (schematic Fig. 8). This is supported by our findings that TRPC4 channel activity is impaired in PHPT-1−/− β-cells and activation of AMPK, which functions downstream of TRPC4 with AICAR, rescues KATP current in PHPT-1−/− β-cells. In addition, expression of a TRPC4 mutant, TRPC4(H912N), rescues TRPC4 channel activity and KATP channel current in PHPT-1 shRNA INS-1 cells in response to leptin signaling and low glucose, and PHPT-1(WT), but not a mutant PHPT-1 devoid of phosphatase activity, activates TRPC4 in I/O patches. These findings strongly suggest that phosphorylation of TRPC4 at H912 inhibits channel activity and that PHPT-1 directly activates TRPC4 by dephosphorylating H912. So far we have been unable to directly demonstrate phosphorylation of H912 on TRPC4 or changes in phosphorylation in response to leptin signaling in vivo with the recently described anti–1- or anti–3-phosphohistidine antibodies (47), which is likely due to the instability of phosphohistidine and the finding that these antibodies only detect a subset of phosphohistidine proteins (47,48).

TRPC4 is a Ca2+-activated monovalent cation channel (49). TRPC4 contains six transmembrane domains and functions as a hexamer. Interestingly, TRPC5, which is the TRP channel most closely related to TRPC4, does not contain a conserved histidine that is similar to H912 on TRPC4 and is not regulated by PHPT-1. The mechanism whereby PHPT-1 is regulated by leptin or how dephosphorylation of histidine 912 activates TRPC4 as the regulation of TRPC4 is complex and remains poorly understood (49). PHPT-1 does not regulate the trafficking or number of TRPC channels in the PM because we detected the same amount of transfected TRPC4 at the PM in WT and shRNA PHPT-1 INS-1 cells. Future studies will address the mechanism for PHPT-1 activation, whether phosphorylation of H912 directly affects gating of TRPC4, and whether PHPT-1 is also required for TRPC4 activation by other signaling pathways that have been shown to affect TRPC4 activity. In addition, ongoing studies will address whether mutations in PHPT-1 and/or TRPC4 may account for yet-to-be defined cases of CHI hypoglycemia.

Acknowledgments. The authors thank Show-Ling Shyng (Oregon Health & Science University) for the generous gift of BTX-tag SUR1 and Kir6.2 virus.

Funding. C.L. is supported by National Institutes of Health grant 1R01-DK-098517-01A1. E.Y.S. is supported by National Institutes of Health grants R01-GM-099873 and R01-AI-080583.

Duality of Interest. No potential conflicts of interest relevant to this article were reported.

Author Contributions. S.S. designed and performed experiments, analyzed and interpreted the data, and drafted and revised the manuscript. Z.L. and I.S. performed experiments and interpreted the data. Y.S. and J.W. performed experiments. L.B. and C.L. performed experiments and analyzed and interpreted the data. W.A.C. analyzed and interpreted the data. C.A.S. drafted and revised the manuscript. E.Y.S. conceptualized and supervised the project, analyzed data, and wrote and edited the manuscript. E.Y.S. is the guarantor of this work and, as such, had full access to all the data in the study and takes responsibility for the integrity of the data and the accuracy of the data analysis.

1.
Attwood
PV
,
Wieland
T
.
Nucleoside diphosphate kinase as protein histidine kinase
.
Naunyn Schmiedebergs Arch Pharmacol
2015
;
388
:
153
160
[PubMed]
2.
Ek
P
,
Pettersson
G
,
Ek
B
,
Gong
F
,
Li
JP
,
Zetterqvist
O
.
Identification and characterization of a mammalian 14-kDa phosphohistidine phosphatase
.
Eur J Biochem
2002
;
269
:
5016
5023
[PubMed]
3.
Klumpp
S
,
Krieglstein
J
.
Reversible phosphorylation of histidine residues in proteins from vertebrates
.
Sci Signal
2009
;
2
:
pe13
[PubMed]
4.
Panda
S
,
Srivastava
S
,
Li
Z
, et al
.
Identification of PGAM5 as a mammalian protein histidine phosphatase that plays a central role to negatively regulate CD4(+) T cells
.
Mol Cell
2016
;
63
:
457
469
[PubMed]
5.
Boissan
M
,
Dabernat
S
,
Peuchant
E
,
Schlattner
U
,
Lascu
I
,
Lacombe
ML
.
The mammalian Nm23/NDPK family: from metastasis control to cilia movement
.
Mol Cell Biochem
2009
;
329
:
51
62
[PubMed]
6.
Klumpp
S
,
Hermesmeier
J
,
Selke
D
,
Baumeister
R
,
Kellner
R
,
Krieglstein
J
.
Protein histidine phosphatase: a novel enzyme with potency for neuronal signaling
.
J Cereb Blood Flow Metab
2002
;
22
:
1420
1424
[PubMed]
7.
Jedrzejas
MJ
.
Structure, function, and evolution of phosphoglycerate mutases: comparison with fructose-2,6-bisphosphatase, acid phosphatase, and alkaline phosphatase
.
Prog Biophys Mol Biol
2000
;
73
:
263
287
[PubMed]
8.
Chen
G
,
Han
Z
,
Feng
D
, et al
.
A regulatory signaling loop comprising the PGAM5 phosphatase and CK2 controls receptor-mediated mitophagy
.
Mol Cell
2014
;
54
:
362
377
[PubMed]
9.
Wang
Z
,
Jiang
H
,
Chen
S
,
Du
F
,
Wang
X
.
The mitochondrial phosphatase PGAM5 functions at the convergence point of multiple necrotic death pathways
.
Cell
2012
;
148
:
228
243
[PubMed]
10.
Busam
RD
,
Thorsell
AG
,
Flores
A
,
Hammarström
M
,
Persson
C
,
Hallberg
BM
.
First structure of a eukaryotic phosphohistidine phosphatase
.
J Biol Chem
2006
;
281
:
33830
33834
[PubMed]
11.
Srivastava
S
,
Li
Z
,
Ko
K
, et al
.
Histidine phosphorylation of the potassium channel KCa3.1 by nucleoside diphosphate kinase B is required for activation of KCa3.1 and CD4 T cells
.
Mol Cell
2006
;
24
:
665
675
[PubMed]
12.
Di
L
,
Srivastava
S
,
Zhdanova
O
, et al
.
Inhibition of the K+ channel KCa3.1 ameliorates T cell-mediated colitis
.
Proc Natl Acad Sci U S A
2010
;
107
:
1541
1546
[PubMed]
13.
Hippe
HJ
,
Lutz
S
,
Cuello
F
, et al
.
Activation of heterotrimeric G proteins by a high energy phosphate transfer via nucleoside diphosphate kinase (NDPK) B and Gbeta subunits. Specific activation of Gsalpha by an NDPK B.Gbetagamma complex in H10 cells
.
J Biol Chem
2003
;
278
:
7227
7233
[PubMed]
14.
Cai
X
,
Srivastava
S
,
Surindran
S
,
Li
Z
,
Skolnik
EY
.
Regulation of the epithelial Ca2+ channel TRPV5 by reversible histidine phosphorylation mediated by NDPK-B and PHPT1
.
Mol Biol Cell
2014
;
25
:
1244
1250
[PubMed]
15.
Srivastava
S
,
Cai
X
,
Li
Z
,
Sun
Y
,
Skolnik
EY
.
Phosphatidylinositol-3-kinase C2β and TRIM27 function to positively and negatively regulate IgE receptor activation of mast cells
.
Mol Cell Biol
2012
;
32
:
3132
3139
[PubMed]
16.
Feske
S
,
Wulff
H
,
Skolnik
EY
.
Ion channels in innate and adaptive immunity
.
Annu Rev Immunol
2015
;
33
:
291
353
[PubMed]
17.
Kim-Muller
JY
,
Zhao
S
,
Srivastava
S
, et al
.
Metabolic inflexibility impairs insulin secretion and results in MODY-like diabetes in triple FoxO-deficient mice
.
Cell Metab
2014
;
20
:
593
602
[PubMed]
18.
McKay
RR
,
Szymeczek-Seay
CL
,
Lievremont
JP
, et al
.
Cloning and expression of the human transient receptor potential 4 (TRP4) gene: localization and functional expression of human TRP4 and TRP3
.
Biochem J
2000
;
351
:
735
746
[PubMed]
19.
Srivastava
S
,
Zhdanova
O
,
Di
L
, et al
.
Protein histidine phosphatase 1 negatively regulates CD4 T cells by inhibiting the K+ channel KCa3.1
.
Proc Natl Acad Sci U S A
2008
;
105
:
14442
14446
[PubMed]
20.
Chen
PC
,
Kryukova
YN
,
Shyng
SL
.
Leptin regulates KATP channel trafficking in pancreatic β-cells by a signaling mechanism involving AMP-activated protein kinase (AMPK) and cAMP-dependent protein kinase (PKA)
.
J Biol Chem
2013
;
288
:
34098
34109
[PubMed]
21.
Bao
L
,
Taskin
E
,
Foster
M
, et al
.
Alterations in ventricular K(ATP) channel properties during aging
.
Aging Cell
2013
;
12
:
167
176
[PubMed]
22.
Miller
MR
,
Shi
J
,
Wu
M
, et al
.
Novel chemical inhibitor of TRPC4 channels.
In
Probe Reports from the NIH Molecular Libraries Program
.
Bethesda
,
National Center for Biotechnology Information
,
2010
23.
Abbott
MJ
,
Edelman
AM
,
Turcotte
LP
.
CaMKK is an upstream signal of AMP-activated protein kinase in regulation of substrate metabolism in contracting skeletal muscle
.
Am J Physiol Regul Integr Comp Physiol
2009
;
297
:
R1724
R1732
[PubMed]
24.
Lippiat
JD
.
Whole-cell recording using the perforated patch clamp technique
.
Methods Mol Biol
2008
;
491
:
141
149
[PubMed]
25.
Kim
H
,
Jeon
JP
,
Hong
C
, et al
.
An essential role of PI(4,5)P2 for maintaining the activity of the transient receptor potential canonical (TRPC)4β
.
Pflugers Arch
2013
;
465
:
1011
1021
[PubMed]
26.
Li
C
,
Matter
A
,
Kelly
A
, et al
.
Effects of a GTP-insensitive mutation of glutamate dehydrogenase on insulin secretion in transgenic mice
.
J Biol Chem
2006
;
281
:
15064
15072
[PubMed]
27.
Li
C
,
Ackermann
AM
,
Boodhansingh
KE
, et al
.
Functional and metabolomic consequences of KATP channel inactivation in human islets
.
Diabetes
2017
;
66
:
1901
1913
[PubMed]
28.
Flanagan
SE
,
Clauin
S
,
Bellanné-Chantelot
C
, et al
.
Update of mutations in the genes encoding the pancreatic beta-cell K(ATP) channel subunits Kir6.2 (KCNJ11) and sulfonylurea receptor 1 (ABCC8) in diabetes mellitus and hyperinsulinism
.
Hum Mutat
2009
;
30
:
170
180
[PubMed]
29.
Stanley
CA
.
Hypoglycemia in the neonate
.
Pediatr Endocrinol Rev
2006
;
4
(
Suppl. 1
):
76
81
[PubMed]
30.
Dunne
MJ
,
Cosgrove
KE
,
Shepherd
RM
,
Aynsley-Green
A
,
Lindley
KJ
.
Hyperinsulinism in infancy: from basic science to clinical disease
.
Physiol Rev
2004
;
84
:
239
275
[PubMed]
31.
Ashcroft
FM
,
Rorsman
P
.
K(ATP) channels and islet hormone secretion: new insights and controversies
.
Nat Rev Endocrinol
2013
;
9
:
660
669
[PubMed]
32.
Rorsman
P
,
Braun
M
.
Regulation of insulin secretion in human pancreatic islets
.
Annu Rev Physiol
2013
;
75
:
155
179
[PubMed]
33.
Düfer
M
,
Haspel
D
,
Krippeit-Drews
P
,
Aguilar-Bryan
L
,
Bryan
J
,
Drews
G
.
Oscillations of membrane potential and cytosolic Ca(2+) concentration in SUR1(-/-) beta cells
.
Diabetologia
2004
;
47
:
488
498
[PubMed]
34.
Park
SH
,
Ryu
SY
,
Yu
WJ
, et al
.
Leptin promotes K(ATP) channel trafficking by AMPK signaling in pancreatic β-cells
.
Proc Natl Acad Sci U S A
2013
;
110
:
12673
12678
[PubMed]
35.
Lim
A
,
Park
SH
,
Sohn
JW
, et al
.
Glucose deprivation regulates KATP channel trafficking via AMP-activated protein kinase in pancreatic beta-cells
.
Diabetes
2009
;
58
:
2813
2819
[PubMed]
36.
Holz
GG
,
Chepurny
OG
,
Leech
CA
.
Leptin-stimulated KATP channel trafficking: a new paradigm for β-cell stimulus-secretion coupling
?
Islets
2013
;
5
:
229
232
[PubMed]
37.
Miki
T
,
Nagashima
K
,
Tashiro
F
, et al
.
Defective insulin secretion and enhanced insulin action in KATP channel-deficient mice
.
Proc Natl Acad Sci U S A
1998
;
95
:
10402
10406
[PubMed]
38.
Seghers
V
,
Nakazaki
M
,
DeMayo
F
,
Aguilar-Bryan
L
,
Bryan
J
.
Sur1 knockout mice. A model for K(ATP) channel-independent regulation of insulin secretion
.
J Biol Chem
2000
;
275
:
9270
9277
[PubMed]
39.
Shiota
C
,
Larsson
O
,
Shelton
KD
, et al
.
Sulfonylurea receptor type 1 knock-out mice have intact feeding-stimulated insulin secretion despite marked impairment in their response to glucose
.
J Biol Chem
2002
;
277
:
37176
37183
[PubMed]
40.
Szollosi
A
,
Nenquin
M
,
Aguilar-Bryan
L
,
Bryan
J
,
Henquin
JC
.
Glucose stimulates Ca2+ influx and insulin secretion in 2-week-old beta-cells lacking ATP-sensitive K+ channels
.
J Biol Chem
2007
;
282
:
1747
1756
[PubMed]
41.
Nakazaki
M
,
Crane
A
,
Hu
M
, et al
.
cAMP-activated protein kinase-independent potentiation of insulin secretion by cAMP is impaired in SUR1 null islets
.
Diabetes
2002
;
51
:
3440
3449
[PubMed]
42.
Miller
M
,
Shi
J
,
Zhu
Y
, et al
.
Identification of ML204, a novel potent antagonist that selectively modulates native TRPC4/C5 ion channels
.
J Biol Chem
2011
;
286
:
33436
33446
[PubMed]
43.
Wu
LJ
,
Sweet
TB
,
Clapham
DE
.
International Union of Basic and Clinical Pharmacology. LXXVI. Current progress in the mammalian TRP ion channel family
.
Pharmacol Rev
2010
;
62
:
381
404
[PubMed]
44.
Düfer
M
,
Gier
B
,
Wolpers
D
,
Krippeit-Drews
P
,
Ruth
P
,
Drews
G
.
Enhanced glucose tolerance by SK4 channel inhibition in pancreatic beta-cells
.
Diabetes
2009
;
58
:
1835
1843
[PubMed]
45.
Roujeau
C
,
Jockers
R
,
Dam
J
.
New pharmacological perspectives for the leptin receptor in the treatment of obesity
.
Front Endocrinol (Lausanne)
2014
;
5
:
167
[PubMed]
46.
Kieffer
TJ
,
Habener
JF
.
The adipoinsular axis: effects of leptin on pancreatic beta-cells
.
Am J Physiol Endocrinol Metab
2000
;
278
:
E1
E14
[PubMed]
47.
Fuhs
SR
,
Meisenhelder
J
,
Aslanian
A
, et al
.
Monoclonal 1- and 3-phosphohistidine antibodies: new tools to study histidine phosphorylation
.
Cell
2015
;
162
:
198
210
[PubMed]
48.
Attwood
PV
,
Piggott
MJ
,
Zu
XL
,
Besant
PG
.
Focus on phosphohistidine
.
Amino Acids
2007
;
32
:
145
156
[PubMed]
49.
Freichel
M
,
Tsvilovskyy
V
,
Camacho-Londoño
JE
.
TRPC4- and TRPC4-containing channels
.
Handb Exp Pharmacol
2014
;
222
:
85
128
[PubMed]
Readers may use this article as long as the work is properly cited, the use is educational and not for profit, and the work is not altered. More information is available at http://www.diabetesjournals.org/content/license.

Supplementary data