Na⁺-dependent transporters mediate HCO₃^– salvage across the luminal membrane of the main pancreatic duct

To study the roles of Na⁺-dependent H⁺ transporters, we characterized H⁺ efflux mechanisms in the pancreatic duct in wild-type, NHE2^–/–, and NHE3^–/– mice. The pancreatic duct expresses NHE1 in the basolateral membrane, and NHE2 and NHE3 in the luminal membrane, but does not contain NHE4 or NHE5. Basolateral Na⁺-dependent H⁺ efflux in the microperfused duct was inhibited by 1.5 μM of the amiloride analogue HOE 694, consistent with expression of NHE1, whereas the luminal activity required 50 μM HOE 694 for effective inhibition, suggesting that the efflux might be mediated by NHE2. However, disruption of NHE2 had no effect on luminal transport, while disruption of the NHE3 gene reduced luminal Na⁺-dependent H⁺ efflux by ∼45%. Notably, the remaining luminal Na⁺-dependent H⁺ efflux in ducts from NHE3^–/– mice was inhibited by 50 μM HOE 694. Hence, ∼55% of luminal H⁺ efflux (or HCO₃^– influx) in the pancreatic duct is mediated by a novel, HOE 694–sensitive, Na⁺-dependent mechanism. H⁺ transport by NHE3 and the novel transporter is inhibited by cAMP, albeit to different extents. We propose that multiple Na⁺-dependent mechanisms in the luminal membrane of the pancreatic duct absorb Na⁺ and HCO₃^– to produce a pancreatic juice that is poor in HCO₃^– and rich in Cl^– during basal secretion. Inhibition of the transporters during stimulated secretion aids in producing the HCO₃^–-rich pancreatic juice.

Pancreatic ducts serve a secretory function to produce a fluid rich in Na⁺ and HCO₃^– in response to secretin stimulation, and also function as a conduit to carry pancreatic enzyme and fluid secretions to the intestine (1). Secretin-stimulated HCO₃^– secretion across the luminal membrane (LM) requires H⁺ secretion or HCO₃^– absorption across the basolateral membrane (BLM) to maintain constant cytosolic pH (pH_i) and replenish the secreted HCO₃^–. Current evidence suggests that a Na⁺ and HCO₃^– cotransporter mediates most HCO₃^– influx across the BLM (2, 3). A Na⁺/H⁺ exchanger (NHE) in the BLM is assumed to help HCO₃^– secretion by controlling pH_i to prevent intracellular acidification during HCO₃^– secretion (4). Because the pancreatic duct secretes HCO₃^– and Na⁺, NHEs are not expected to be present in the LM. However, using perfused ducts, we and others have reported NHE activity in the LM of pancreatic ducts of different species (4, 5). The proteins mediating this activity, their regulation, and their physiological significance are unknown.

NHEs are a family of membrane proteins involved in regulation of pH_i, cell volume, and electrolyte transport. Since the initial cloning of NHE1 (6), at least six NHE isoforms have been found in mammalian cells; five of these were identified in rodent tissue (7). The physiological role of only a few of the isoforms has been established with any certainty. NHE1 is ubiquitously expressed, and has a housekeeping role (8). NHE1 is always expressed in the BLM of epithelial cells (9). In many epithelia, such as salivary glands, small intestine, colon, and kidney, NHE2 and NHE3 are the major isoforms expressed in the LM (7, 9–11). In the kidney, NHE3 in the proximal tubule mediates 60% of Na⁺ and HCO₃^– absorption (12, 13). NHE2 is believed to mediate a large portion of Na⁺ absorption in the small intestine (9). The phenotype of a targeted NHE3 knockout mouse supports a major role for NHE3 in renal Na⁺ and HCO₃^– absorption (14). However, based on the phenotype of the NHE2^–/– mouse, so far the function of NHE2 appears to be essential only in gastric parietal cells (15). The role of NHE2 and NHE3 in other tissues that express these proteins is not known.

The availability of NHE2^–/– and NHE3^–/– animals provided an opportunity to clarify the role of these NHE isoforms and other H⁺ efflux mechanisms in the HCO₃^–-secreting pancreatic duct. We find that NHE1 is expressed in the BLM, whereas NHE2 and NHE3 are expressed in the LM. Furthermore, we found a novel amiloride-sensitive, Na⁺-dependent H⁺ efflux (or HCO₃^– influx) mechanism that does not correspond to any known NHE isoform. The use of ducts from wild-type (WT), NHE2^–/–, and NHE3^–/– mice showed that NHE3 was responsible for about 45% of the luminal H⁺ efflux, and the novel mechanism for about 55%. Despite the expression of NHE2 in the LM, no functional role for this isoform could be established. Luminal Na⁺-dependent H⁺ efflux mechanisms were decreased by cAMP. Based on these findings, we proposed a role for luminal Na⁺-dependent H⁺ efflux mechanisms in controlling electrolyte composition of the pancreatic juice during basal pancreatic fluid secretion.

Identification of NHE isoforms in the pancreatic duct. The identities of the NHE isoforms expressed in the pancreatic duct were first analyzed by RT-PCR. Although secretory mechanisms may be slightly different between the mouse and the rat pancreatic ducts, we performed RT-PCR on rat mRNA because sequence data specific for all NHE isoforms are available only for the rat. To circumvent contamination by other cell types, acinar cells and vessel structures were removed from the main and interlobular ducts under microscopic inspection, and the ducts were washed twice with fresh pancreatic solution A. Figure 1 shows that the pancreatic duct expresses mRNA for NHE1, NHE2, and NHE3, but not for NHE4 and NHE5. The ability of the primers for NHE4 and NHE5 to detect the respective isoforms was verified using mRNA prepared from kidney and brain, respectively (data not shown). The identity of each of the PCR-amplified products was confirmed by nucleotide sequencing.

Figure 1

RT-PCR analysis of NHE isoforms in pancreatic ducts. The rat pancreatic duct expresses mRNA for β-actin (211 bp), NHE1 (160 bp), NHE2 (196 bp), and NHE3 (243 bp), but not for NHE4 and NHE5. The identity of each of the PCR-amplified products was confirmed by sequencing.

Next, we used immunostaining in an attempt to determine localization of the NHE isoforms. The results obtained with the mouse pancreas are shown in Figure 2. The staining with anti-NHE1 antibody in Figure 2a shows clear basolateral localization and an absence of luminal staining. However, because more than 95% of the plasma membrane is BLM, in many ducts staining with NHE1 appeared to encircle the cells, with occasional absence of staining in luminal borders. The polyclonal antibody recognizing NHE2 stained the LM of the duct (Figure 2b); the specificity of staining was verified by the absence of a significant signal in pancreatic sections obtained from NHE2^–/– mice. Expression of NHE3 was localized to the LM of duct cells (Figure 2c), and the specificity of the staining with this antibody was verified using the pancreas from NHE3^–/– mice. Sections from NHE2^–/– mice showed clear luminal NHE3 staining; those from NHE3^–/– mice showed clear luminal NHE2 staining (data not shown). However, the signal/noise was not sufficient to determine whether there was any compensatory increment of NHE expression in the pancreatic ducts of either mutant mouse.

Figure 2

Immunolocalization of NHE isoforms in murine pancreas. Pancreatic sections were prepared from the indicated mouse lines and then stained with antibodies specific for NHE1 (a), NHE2 (b), and NHE3 (c). In each image pair, the first image is the phase contrast and the second image is the corresponding fluorescent image. Because it is difficult to visualize the sparse ducts in the pancreas that are embedded in the acinar structures without a counterstain, and because counterstaining is not compatible with immunofluorescence, the luminal (LM) and basal (BLM) borders are highlighted by black lines and marked by arrows in all phase-contrast images.

Previously, it was shown that both the LM and BLM of the rat pancreatic duct express functional Na⁺-dependent H⁺ efflux activity, which was inhibited by 0.5 mM amiloride (4). Similar results were found in the mouse pancreatic duct (Figure 3). Resting pH_i of WT pancreatic duct cells was 7.38 ± 0.04 (n = 5) in the absence of HCO₃^–. Perfusing the bath with a Na⁺-free medium caused a small decrease in pH_i, whereas perfusing both the bath and the lumen with Na⁺-free medium caused rapid and intensive acidification. The cells were further acidified by a luminal application of 20 mM NH₄Cl for 1–3 minutes. After removal of NH₄⁺, pH_i stabilized at 6.4–6.5. Application of Na⁺-containing solution to the bath caused rapid pH_i recovery. In five experiments, HOE 694 at 1.5 μM inhibited the Na⁺-dependent pH_i recovery by an average of 87% (see Table 1). Among the known NHE isoforms, only the K_i value for inhibition of NHE1 by HOE 694 is below 1.5 μM (20). Hence, the functional data substantiates the immunolocalization of NHE1 in the BLM. Subsequent application of a medium containing Na⁺ and 1.5 μM HOE 694 to the lumen showed that the luminal transporters have a lower affinity for HOE 694 than for the BLM NHE (Figure 3). Multiple experiments summarized in Table 1 showed that HOE 694 inhibited luminal NHE activity by 78% at 15 μM, and by 86% at 50 μM.

Figure 3

Na⁺-dependent H⁺ efflux in the BLM and LM of the pancreatic duct. NHE activity was measured in the main pancreatic duct. After NH₄⁺-induced acidification, the BLM was perfused with a Na⁺-containing solution with or without 1.5 μM HOE 694, as indicated. Subsequently, the LM was perfused with a Na⁺-containing solution supplemented with the indicated concentration of HOE 694.

Table 1

Inhibition of Na⁺-dependent pH_i recovery by HOE 694

Identification of the NHE isoform mediating luminal Na⁺/H⁺ exchange activity. RT-PCR analysis and immunolocalization studies suggested the expression of NHE2 and NHE3 in the LM of the mouse pancreatic duct. Because inhibition of NHE3 in model systems required a high concentration of HOE 694 (K_i of 0.65 mM), and 50 μM HOE 694 had no effect on luminal NHE activity in the mouse proximal tubule (21), we expected NHE2 to be the active isoform in the LM of the pancreatic duct (9, 20). To obtain unequivocal evidence for the role of each NHE isoform, we compared NHE activity in pancreatic ducts from WT, NHE2^–/–, and NHE3^–/– mice. Figure 4a shows representative traces from each mouse line; Figure 4b shows the summary of at least four experiments under each condition. The rate of luminal Na⁺-dependent pH_i recovery at an initial pH_i of 6.4–6.5 in ducts from WT mice was 0.93 ± 0.08 pH units/min (n = 5). Surprisingly, the rate of luminal Na⁺-dependent H⁺ efflux in pancreatic ducts from NHE2^–/– mice was 0.86 ± 0.09 pH units/min (n = 4), which was not significantly different from that in WT mice. By contrast, the rate of luminal Na⁺-dependent pH_i recovery was significantly reduced in ducts from NHE3^–/– mice (0.51 ± 0.02 pH units/min; n = 6). When interpreting these results, it is important to consider the buffer capacity of duct cells in all mouse lines. An increase in buffer capacity in ducts from NHE3^–/– mice could explain the reduced rate of Na⁺-dependent pH_i recovery. To exclude this possibility, the buffer capacity of duct cells from WT and NHE knockout mice was measured. Table 2 shows that there were no significant differences between the buffer capacity of duct cells from any of these mouse lines.

Figure 4

Luminal Na⁺-dependent H⁺ efflux in pancreatic ducts of WT, NHE2^–/–, and NHE3^–/– mice. Luminal Na⁺-dependent H⁺ efflux was measured in ducts acidified to the same level using the indicated protocol. (a) Examples of individual traces from each mouse line. (b) Summary of five, four, and six experiments performed with ducts from WT, NHE2^–/–, and NHE3^–/– mice, respectively. AP < 0.05 compared eith WT.

Table 2

Buffer capacity of mouse pancreatic ducts at pH_i 7.0

Of note is the remaining 55% of Na⁺-dependent H⁺ efflux in the LM of ducts from NHE3^–/– mice, and the normal activity in ducts from NHE2^–/– mice. Therefore, it was of interest to determine whether these activities could still be inhibited by HOE 694. Figure 5 shows that 50 μM HOE 694 inhibited the luminal Na⁺-dependent H⁺ efflux by more than 85% in ducts from NHE2^–/– and NHE3^–/– mice. Similar results were obtained with three ducts from each knockout line. Hence, it appears that a novel Na⁺-dependent and HOE 694–sensitive mechanism mediates 55% of luminal H⁺ efflux (or HCO₃^– influx) in the mouse pancreatic duct.

Figure 5

The luminal Na⁺-dependent H⁺ efflux is inhibited by HOE 694. The protocol outlined in the legend to Figure 3 was used to measure inhibition of luminal Na⁺-dependent H⁺ efflux in pancreatic ducts from NHE2^–/– (upper trace) and NHE3^–/– (lower trace) mice.

Regulation of luminal H⁺ efflux by cAMP. The main function of pancreatic duct cells is secretion of an HCO₃^–-rich fluid in response to feeding stimuli such as secretin (22). Having found at least two luminal Na⁺-dependent H⁺ efflux mechanisms, we proceeded to determine their possible regulation by the secretin-generated, second-messenger cAMP. After measuring luminal NHE activity under basal conditions, duct cells were treated with 5 μM forskolin and 100 μM 3-isobutyl-1-methylxanthine (IBMX) for 5 minutes (Figure 6a). Stimulation with forskolin and IBMX reduced luminal Na⁺-dependent H⁺ efflux in ducts from WT mice by 43 ± 5% (n = 5) (Figure 6b). This protocol was also performed with ducts from NHE2^–/– and NHE3^–/– mice. Inhibition by forskolin and IBMX in NHE2^–/– mice (45 ± 2%; n = 4) was not different from that measured in WT mice. The residual activity in ducts from NHE3^–/– mice was also inhibited by cAMP stimulation, although the inhibition was only 23 ± 6% (n = 6). Table 2 shows that inhibition of luminal Na⁺-dependent H⁺ efflux by cAMP was not due to a change in buffer capacity, because stimulation with forskolin and IBMX did not change cytoplasmic buffer capacity of pancreatic ducts from any of the lines. The effect of cAMP stimulation on BLM NHE was also investigated. In five experiments, the BLM NHE activity was not affected by forskolin and IBMX treatment (data not shown).

Figure 6

Regulation of luminal NHE activity by cAMP. After the control period, ducts were treated with 5 μM forskolin and 100 μM IBMX for 5 minutes. (a) A representative experiment using the pancreatic duct of a WT mouse. (b) Summary of the multiple experiments. Forskolin treatment reduced luminal NHE activity by 43 ± 5% (n = 5) in WT mice, 45 ± 2% (n = 6) in NHE2^–/– mice, and 23 ± 6% (n = 6) in NHE3^–/– mice. AP < 0.05 compared with WT.

The major aim of this study was to identify the Na⁺-dependent H⁺ secretory mechanisms in the LM of the pancreatic duct, and their possible roles in ductal physiology. Immunohistochemistry, backed by RT-PCR analysis, suggests expression of NHE2 and NHE3 in the LM of pancreatic duct cells. This is confirmed by the elimination of staining from the ducts of the respective knockout mice. The duct also expressed NHE1, which could be localized by a functional assay (sensitivity to HOE 694) and immunostaining to the BLM. Therefore, we can conclude that pancreatic ducts express NHE2 and NHE3 in the LM, and NHE1 in the BLM.

Functional analysis of Na⁺-dependent H⁺ transport in the LM of ducts from WT, NHE2^–/–, and NHE3^–/– mice revealed several surprises. The first unexpected finding was normal luminal Na⁺-dependent H⁺ efflux in ducts from NHE2^–/– mice. From the sensitivity of the activity to HOE 694 (Table 1) and the reported K_i of the different NHE isoforms for inhibition by HOE 694 (20), we expected that most luminal H⁺ efflux would be mediated by NHE2. Several findings confirmed disruption of the NHE2 gene in the knockout mice. The mice lacked mRNA coding for WT NHE2, and displayed severe histopathology of the gastric mucosa and a lack of acid secretory function (15). Furthermore, the NHE2 protein was absent from the parotid gland (23), the kidney (21), and the pancreas (this study). Yet, deletion of NHE2 protein had no effect on luminal Na⁺-dependent H⁺ efflux in pancreatic duct. Several explanations can account for this finding. Upregulation of other Na⁺-dependent transporters might have offset the effect of deletion of NHE2. A prediction of this scenario is that the residual activity in the LM of ducts from NHE2^–/– mice will show different HOE 694 sensitivity than that in LM from ducts from WT mice. This possibility was excluded by the finding that 50 μM HOE 694 inhibited more than 85% of luminal Na⁺-dependent H⁺ efflux in ducts from NHE2^–/– mice. These findings also suggest that sensitivity to HOE 694 cannot be used in all cases to discern the NHE isoform expressed in a given tissue or cell type.

Another alternative explanation for the lack of effect of deletion of NHE2 is that the NHE2 expressed in the LM of the pancreatic duct is not active or may not function as a Na⁺/H⁺ exchanger. Some support for this unconventional interpretation comes from the absence of any apparent abnormalities in renal, intestinal, or pancreatic function in the NHE2^–/– animals (ref. 15 and this study). In addition, deletion of NHE2 had no effect on Na⁺-dependent H⁺ efflux in the proximal tubule (21). Hence, the role of luminal NHE2 in epithelial physiology remains to be found.

In contrast to the results with NHE2, deletion of the NHE3 gene reduced luminal pancreatic ductal Na⁺-dependent H⁺ efflux by 45%. The unexpected finding in this case was the 85–90% inhibition of overall luminal H⁺ transport by 50 μM HOE 694. This indicates that most of the NHE3-mediated activity in the pancreatic duct is inhibited by 50 μM HOE 694. Yet, 50 μM HOE 694 had no effect on Na⁺-dependent H⁺ transport in the LM of the mouse proximal tubule (21). It is therefore conceivable that the pancreatic duct expresses an NHE3 splice variant different from that expressed in the kidney. Further cloning efforts are needed to examine this possibility. Alternatively, posttranslational modification of NHE3 may confer tissue-specific sensitivity to HOE 694 (and possibly other amiloride analogues). This raises further questions regarding the general use of sensitivity to amiloride and its analogues as diagnostic tools of NHE isoforms.

An additional unexpected finding was the novel HOE 694–inhibitable; Na⁺-dependent H⁺ efflux mechanism in the LM of pancreatic duct. Recently, we reported analogous activity in the proximal tubule, with the difference that the renal activity required 100 μM EIPA for maximal inhibition (21). The particular significance of this activity is that it mediates as much as 55% of luminal Na⁺-dependent H⁺ efflux. Hence, it may have an important physiological role in HCO₃^– absorption in various tissues.

At present, we do not know what protein may mediate this activity. An obvious possibility is an as-yet unknown NHE isoform, different from NHE1–5. Additional possibilities are the newly described Na⁺ and HCO₃^– cotransporter NBC3 (24) or a similar isoform. Unique features of NBC3, when compared with other NBC isoforms, are that it can transport HCO₃^– and OH^–, it is inhibitable by amiloride analogues, and it is insensitive to the stilbene derivative DIDS (24). These features are reminiscent of those found for the novel luminal Na⁺-dependent H⁺ (or HCO₃^–) transport in the pancreatic duct (this study) and proximal tubule (21). We are in the process of evaluating the contribution of this or a similar transporter to luminal Na⁺-dependent H⁺ efflux.

The pancreatic duct secretes the bulk of HCO₃^– found in secretin-stimulated pancreatic juice. Secretin effects in duct cells are mediated by stimulation of adenylyl cyclase. It is believed that the increase in cAMP activates the cystic fibrosis transmembrane conductance regulator (CFTR) to facilitate HCO₃^– secretion by a Cl^–/ HCO₃^– exchange mechanism. Na⁺-dependent H⁺ secretion was not expected to be present in the LM of pancreatic ducts, because it can reduce pancreatic HCO₃^– and fluid secretion by secreting H⁺ and absorbing Na⁺ (and consequently water) from the luminal fluid. Therefore, it was of particular interest to determine possible regulation of the luminal NHE transporters by cAMP. Pancreatic NHE3 and the luminal Na⁺-dependent H⁺ efflux transporter are inhibited by a cAMP-dependent mechanism. Thus, in pancreatic ducts from both WT and NHE2^–/– animals, an increase in cAMP reduced NHE activity by an average of about 45%. Stimulation with cAMP reduced the novel Na⁺-dependent H⁺ efflux by about 25%, which amounts to about 12.5% of total luminal Na⁺-dependent H⁺ efflux. Thus, cAMP stimulation inhibited NHE3 activity by about 37.5%. Regulatory inhibition of NHE3 by cAMP in the kidney proximal tubule is well understood (25). Protein kinase–mediated(PKA-mediated) phosphorylation may regulate NHE3 activity directly or through changes in the availability of NHERF (a PDZ motif containing protein) for interaction with NHE3 (26). In this respect, it is interesting that NHERF can also downregulate the activity of NBC transporters (27). Such a mechanism may be related to the inhibition of the novel Na⁺-dependent H⁺ efflux by cAMP stimulation.

What can the physiological relevance of luminal Na⁺-dependent H⁺ efflux in the HCO₃^– secreting pancreatic duct be? A plausible scenario is illustrated in the models shown in Figure 7. The main pancreatic duct maintains a resting transepithelial potential difference of –5 mV, lumen negative (28), and has Na⁺-leaky tight junctions (1). The duct also expresses CFTR and has Cl^–/HCO₃^– exchange activity that is modulated by the PKA-phosphorylated CFTR (18). The basal Cl^–/HCO₃^– exchange activity of the pancreatic duct is about 50% of the cAMP-stimulated activity (18). Basal Cl^–/HCO₃^– exchange and Na⁺ leaking through the tight junction will result in a basal secretion of a fluid rich in Na⁺ and HCO₃^–. To prevent loss of Na⁺ and HCO₃^– under the basal state, these ions must be reabsorbed from the luminal fluid. The Na⁺-dependent H⁺ efflux mechanisms described here can perform this function in a mode analogous to that used by the proximal tubule to absorb Na⁺ and HCO₃^– (Figure 7a). Na⁺ and HCO₃^– absorption will also increase absorbtion of fluid from the duct lumen. Low pancreatic secretion in the interdigestive basal state is of particular importance in intermittent feeders such as humans (22). There is evidence for Na⁺ and HCO₃^– absorption by the duct in the basal state. In fact, basal secretion of digestive enzymes by acinar cells is about 10% of maximal. By contrast, basal secretion of HCO₃^– by the duct is only 1–2% of maximal. Equally important, the basal secretory fluid contains high levels of Cl^– (22). This suggests that indeed, the pancreatic duct absorbs HCO₃^– rather than Cl^– under basal conditions.

Figure 7

Roles of luminal Na⁺-dependent H⁺ efflux mechanisms in pancreatic duct. (a) The resting pancreatic duct maintains a transepithelial voltage of –5 mV, lumen negative, and has a leaky tight junction. Hence, Na⁺ flows from the interstitial space to the luminal space. The luminal Na⁺-dependent H⁺ efflux mechanisms absorb the Na⁺ in exchange for H⁺. H⁺ efflux is equivalent to HCO₃^– reabsorption, which is secreted by the basal Cl^–/HCO₃^– exchange activity. (b) When feeding, the gastrointestinal hormone secretin is released from intestinal cells to stimulate pancreatic secretion by increasing intracellular cAMP in duct cells. The activity of the luminal Na⁺-dependent H⁺ efflux mechanisms are downregulated by a cAMP-dependent mechanism to reduce Na⁺-HCO₃^– reabsorption.

When feeding, the gastrointestinal hormone secretin is released from intestinal cells to stimulate pancreatic ductal fluid and HCO₃^– secretion by increasing cellular cAMP (1). cAMP activates CFTR (29), which in turn results in activation of Cl^–/HCO₃^– exchange (18, 30). Consequently, a large amount of HCO₃^– is secreted into the lumen, which is followed by Na⁺ and water transport through the leaky tight junctions. The results presented here show that in parallel with stimulation of HCO₃^– secretion, cAMP inhibits Na⁺-dependent H⁺ secretion by NHE3 and the novel luminal efflux mechanism. This will reduce Na⁺ and HCO₃^– absorption to allow Cl^–/HCO₃^– exchange and CFTR to produce HCO₃^–-rich pancreatic juice.

In summary, we believe that this study highlights an underappreciated but important function of the pancreatic duct: the control of pancreatic secretion and the composition of the pancreatic juice under basal conditions. It appears that a luminal NHE3 mechanism and a novel Na⁺-dependent, amiloride-sensitive H⁺ efflux mechanism reabsorb Na⁺, HCO₃^–, and fluid, probably to produce an acidic, Cl^–-rich pancreatic juice under resting conditions. Upon cell stimulation by agonists that act through cAMP, these mechanisms are inhibited to allow production of an HCO₃^–-rich pancreatic juice.

This work was supported by research grants from The Korea Research Foundation (1997–1998, to K.H. Kim) and National Institutes of Health grants DE-12309 and DK-38938 (to S. Muallem). W. Ahn is a PhD student supported by the Brain Korea 21 Project for Medical Sciences from the Ministry of Education, Korea.

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