Regulation of K_ATP Channel Trafficking in Pancreatic β-Cells by Protein Histidine Phosphorylation

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 K_ATP channels to the plasma membrane (PM) by directly activating TRPC4 in pancreatic β-cells and thereby regulates insulin release from pancreatic β-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.

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).

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 was used to measure K_ATP or TRPC4 channel current. To measure K_ATP 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).

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).

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.

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).

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).

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^−/− 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.

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 K_ATP channels (K_ATP CHI) (28,30). Similar to patients with K_ATP CHI who do not respond to diazoxide therapy, basal and maximal K_ATP 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 K_ATP channel current was not due to decreased amounts of K_ATP 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 K_ATP channel current was confirmed in excised membrane patches PHPT-1^−/− β-cells. (see Supplementary Fig. 1).

K_ATP channels are critical in setting the membrane potential in pancreatic β-cells (31). Decreased K_ATP channel activity leads to membrane depolarization, activation of voltage-dependent Ca²⁺ channels (VDCCs), Ca²⁺ influx, and ultimately, exocytosis of insulin (32). Consistent with decreased K_ATP 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).

Trafficking of K_ATP 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 K_ATP 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 K_ATP 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 K_ATP channels being localized to an intracellular compartment due to decreased AMPK activation. Consistent with previous reports, the K_ATP 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, K_ATP 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 K_ATP channel currents to similar levels as PHPT-1^+/+ β-cells, suggesting that defective activation of AMPK could account for the impaired trafficking of K_ATP 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 K_ATP 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 K_ATP channels to the PM in pancreatic β-cells via activation of AMPK (20,34,36). Leptin stimulation increased K_ATP currents in PHPT-1^+/+ β-cells and WT INS-1 cells under high glucose conditions but failed to stimulate K_ATP 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 K_ATP channels were assessed by FACS after labeling with Alexa Fluor 355 α-BTX (Fig. 3C) (20).

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 (37–41) and is also consistent with impaired glucose-stimulated insulin release or “glucose blindness” in humans with loss-of-function K_ATP channel mutations (27).

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).

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 K_ATP 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 K_ATP currents in shRNA PHPT-1 INS-1 cells to similar levels as control WT INS-1 cells (Fig. 6Aii) and K_ATP currents in shRNA PHPT-1 INS-1 cells in response to low glucose (1 mmol/L) (Fig. 6Aii).

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.

Mouse knockouts of K_ATP 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 (37–39). 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 K_ATP channels to the PM, which resulted in a phenotype that mimicked mouse knockouts of the K_ATP channel subunits Kir6.2 and SUR1 (37–39).

K_ATP 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 K_ATP channels in the PM. Closure of K_ATP 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 K_ATP 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 (37–39). These phenotypes were recapitulated in PHPT-1^−/− mice, where decreased K_ATP channel currents was not due to gene inactivation but rather to intracellular retention of K_ATP channels due to their failure to traffic to the PM. The importance of PHPT-1 in regulating K_ATP trafficking is further reinforced by our finding that K_ATP trafficking is also impaired in PHPT-1^−/− cardiomyocytes (Supplementary Fig. 2).

In addition to direct regulation of K_ATP channel currents by AMP-to-ADP-to-ATP ratios, recent studies have highlighted the importance for K_ATP channel trafficking to and from the PM to K_ATP channel current and membrane potential to normal β-cell physiology (36). Under low glucose conditions, K_ATP 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 K_ATP channels in the PM and AMPK-stimulated redistribution of K_ATP channels to the PM (20,34,36). Maximal K_ATP 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 K_ATP 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 K_ATP channels to the PM. This is supported by our finding of decreased K_ATP channels at the PM and decreased K_ATP 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 K_ATP 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 (37–41,44).

Leptin has also been shown to play an important role in K_ATP 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 K_ATP 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 K_ATP 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 K_ATP 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 K_ATP 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 K_ATP current in PHPT-1^−/− β-cells. In addition, expression of a TRPC4 mutant, TRPC4(H912N), rescues TRPC4 channel activity and K_ATP 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.