Introduction
Epithelial Na
+ channel (ENaC)
3The abbreviations used are:
ENaC
epithelial Na+ channel
WT
wildtype
ECD
extracellular domain
ASIC
acid-sensing ion channel
MTSET
2-(trimethylammonium)ethyl methanethiosulfonate, bromide
FRT
Fisher rat thyroid
MBS
modified Barth's solution
HA
hemagglutinin
Glc-6-P
glucose-6-phosphate dehydrogenase
ANOVA
analysis of variance
cRNA
complementary RNA.
-mediated Na
+ transport regulates extracellular fluid volume and K
+ homeostasis, as well as blood pressure (
1- Warnock D.G.
- Kusche-Vihrog K.
- Tarjus A.
- Sheng S.
- Oberleithner H.
- Kleyman T.R.
- Jaisser F.
Blood pressure and amiloride-sensitive sodium channels in vascular and renal cells.
). Three homologous subunits typically constitute a functional trimeric ENaC complex (
1- Warnock D.G.
- Kusche-Vihrog K.
- Tarjus A.
- Sheng S.
- Oberleithner H.
- Kleyman T.R.
- Jaisser F.
Blood pressure and amiloride-sensitive sodium channels in vascular and renal cells.
2- Jasti J.
- Furukawa H.
- Gonzales E.B.
- Gouaux E.
Structure of acid-sensing ion channel 1 at 1.9-Å resolution and low pH.
,
3- Kashlan O.B.
- Kleyman T.R.
ENaC structure and function in the wake of a resolved structure of a family member.
4- Kleyman T.R.
- Kashlan O.B.
- Hughey R.P.
Epithelial Na+ channel regulation by extracellular and intracellular factors.
). Distinct from most ion channels composed of subunits with two transmembrane domains, ENaC/Degenerin family members possess a large extracellular domain (ECD) composed of over 400 residues per subunit. The resolved structure of an acid-sensing ion channel (ASIC1), a member of the ENaC/Degenerin family, revealed a highly organized ECD with five distinct subdomains. These include a central core formed by β strand-structured palm and β-ball domains, and peripheral α-helical thumb, finger, and knuckle domains. Sequence similarities suggest that the ECDs of other members of the ENaC/Degenerin family have a similar structure (
2- Jasti J.
- Furukawa H.
- Gonzales E.B.
- Gouaux E.
Structure of acid-sensing ion channel 1 at 1.9-Å resolution and low pH.
3- Kashlan O.B.
- Kleyman T.R.
ENaC structure and function in the wake of a resolved structure of a family member.
,
4- Kleyman T.R.
- Kashlan O.B.
- Hughey R.P.
Epithelial Na+ channel regulation by extracellular and intracellular factors.
5- Kashlan O.B.
- Adelman J.L.
- Okumura S.
- Blobner B.M.
- Zuzek Z.
- Hughey R.P.
- Kleyman T.R.
- Grabe M.
Constraint-based, homology model of the extracellular domain of the epithelial Na+ channel α subunit reveals a mechanism of channel activation by proteases.
). Previous studies also suggest that the well-organized and tightly packed ECDs sense a variety of external cues that modulate channel open probability (
6- Sheng S.
- Perry C.J.
- Kleyman T.R.
External nickel inhibits epithelial sodium channel by binding to histidine residues within the extracellular domains of α and γ subunits and reducing channel open probability.
7- Chraïbi A.
- Horisberger J.D.
Na self-inhibition of human epithelial Na channel: temperature dependence and effect of extracellular proteases.
,
8- Sheng S.
- Bruns J.B.
- Kleyman T.R.
Extracellular histidine residues crucial for Na+ self-inhibition of epithelial Na+ channels.
,
9- Sheng S.
- Perry C.J.
- Kleyman T.R.
Extracellular Zn2+ activates epithelial Na+ channels by eliminating Na+ self-inhibition.
,
10Extracellular protons regulate human ENaC by modulating Na+ self-inhibition.
,
11Extracellular chloride regulates the epithelial sodium channel.
,
12- Winarski K.L.
- Sheng N.
- Chen J.
- Kleyman T.R.
- Sheng S.
Extracellular allosteric regulatory subdomain within the γ subunit of the epithelial Na+ channel.
,
13- Chen J.
- Myerburg M.M.
- Passero C.J.
- Winarski K.L.
- Sheng S.
External Cu2+ inhibits human epithelial Na+ channels by binding at a subunit interface of extracellular domains.
,
14- Chen J.
- Winarski K.L.
- Myerburg M.M.
- Pitt B.R.
- Sheng S.
Probing the structural basis of Zn2+ regulation of the epithelial Na+ channel.
,
15- Shi S.
- Ghosh D.D.
- Okumura S.
- Carattino M.D.
- Kashlan O.B.
- Sheng S.
- Kleyman T.R.
Base of the thumb domain modulates epithelial sodium channel gating.
,
16- Shi S.
- Carattino M.D.
- Kleyman T.R.
Role of the wrist domain in the response of the epithelial sodium channel to external stimuli.
,
17- Shi S.
- Blobner B.M.
- Kashlan O.B.
- Kleyman T.R.
Extracellular finger domain modulates the response of the epithelial sodium channel to shear stress.
18- Kashlan O.B.
- Blobner B.M.
- Zuzek Z.
- Tolino M.
- Kleyman T.R.
Na+ inhibits the epithelial Na+ channel by binding to a site in an extracellular acidic cleft.
).
ENaCs in both native tissues and heterologous expression systems are constitutively open, albeit with considerably variable open probability. The latter can be attributed to factors that regulate ENaC gating, including ions, pH, proteases, temperature, mechanic forces, acidic phospholipids, and palmitoylation (
4- Kleyman T.R.
- Kashlan O.B.
- Hughey R.P.
Epithelial Na+ channel regulation by extracellular and intracellular factors.
). ENaC has been suggested to function as a ligand-regulated ion channel (
9- Sheng S.
- Perry C.J.
- Kleyman T.R.
Extracellular Zn2+ activates epithelial Na+ channels by eliminating Na+ self-inhibition.
,
19- Horisberger J.D.
- Chraïbi A.
Epithelial sodium channel: a ligand-gated channel?.
). ENaC open probability is reduced by extracellular Na
+, a process referred to as Na
+ self-inhibition (
7- Chraïbi A.
- Horisberger J.D.
Na self-inhibition of human epithelial Na channel: temperature dependence and effect of extracellular proteases.
,
8- Sheng S.
- Bruns J.B.
- Kleyman T.R.
Extracellular histidine residues crucial for Na+ self-inhibition of epithelial Na+ channels.
,
18- Kashlan O.B.
- Blobner B.M.
- Zuzek Z.
- Tolino M.
- Kleyman T.R.
Na+ inhibits the epithelial Na+ channel by binding to a site in an extracellular acidic cleft.
,
20- Fuchs W.
- Larsen E.H.
- Lindemann B.
Current-voltage curve of sodium channels and concentration dependence of sodium permeability in frog skin.
). Many of the identified residues where substitutions either enhance or suppress Na
+ self-inhibition are located within the ECDs, including a region in the α subunit containing a putative Na
+-binding site that mediates the inhibitory effect of external Na
+ (
18- Kashlan O.B.
- Blobner B.M.
- Zuzek Z.
- Tolino M.
- Kleyman T.R.
Na+ inhibits the epithelial Na+ channel by binding to a site in an extracellular acidic cleft.
). We hypothesized that the independently folded helical domains (thumb, finger, and knuckle) serve as functional modules to regulate gating and mediate the interactions of ENaC with various factors, including extracellular Na
+. In support of this hypothesis, we reported that deletion of the α subunit knuckle domain leads to hyperactive channels with a loss of the Na
+ self-inhibition response, whereas deletion of either the β or γ subunit knuckle domain diminished channel expression and activity (
21- Chen J.
- Kleyman T.R.
- Sheng S.
Deletion of alpha-subunit exon 11 of the epithelial Na+ channel reveals a regulatory module.
,
22- Chen J.
- Ray E.C.
- Yates M.E.
- Buck T.M.
- Brodsky J.L.
- Kinlough C.L.
- Winarski K.L.
- Hughey R.P.
- Kleyman T.R.
- Sheng S.
Functional roles of clusters of hydrophobic and polar residues in the epithelial Na+ channel Knuckle domain.
). In this study, we sought to determine specific functional roles of the helical thumb domains within ENaC subunits. Our data indicate that the thumb domain supports various ENaC-specific activities. Surprisingly the thumb domain in each subunit exhibits distinct functional roles. These results emphasize the complex inter- and intra-protein interactions to which this regulated channel is exposed.
Discussion
In this study we sought to dissect the functional roles of the extracellular thumb domains within each of the ENaC subunits. Our results suggest that thumb domains in each subunit are required for optimal ENaC surface expression in Xenopus oocytes. Channels with an α or β subunit thumb domain deletion had a very low level of surface expression, whereas the absence of the γ subunit thumb domain resulted in only a moderate reduction in channel surface expression. Furthermore, deletions of the α or γ subunit thumb domain were associated with a loss of the Na+ self-inhibition response, whereas channels with a β subunit thumb domain deletion had a Na+ self-inhibition response that was similar to the WT channel. These results indicate that the thumb domains within the three ENaC subunits have distinct and nonequivalent roles for ENaC activity and surface expression. Although deletion of the γ subunit thumb domain did not significantly change macroscopic Na+ currents in oocytes, we found that γΔT channels had reduced surface expression as well as a loss of the Na+ self-inhibition response that should increase channel open probability.
Previous studies analyzing channels with specific point mutations in the α or γ subunit thumb domain suggested that these domains are involved in the Na
+ self-inhibition response. A reduction in the Na
+ self-inhibition response was seen with channels bearing individual mutations in six of the 10 Cys residues in the γ subunit thumb domain (
42- Sheng S.
- Maarouf A.B.
- Bruns J.B.
- Hughey R.P.
- Kleyman T.R.
Functional role of extracellular loop cysteine residues of the epithelial Na+ channel in Na+ self-inhibition.
). Mutation of a Met residue (γMet-438) within α5 helix was also associated with a loss of the Na
+ self-inhibition response (
24- Maarouf A.B.
- Sheng N.
- Chen J.
- Winarski K.L.
- Okumura S.
- Carattino M.D.
- Boyd C.R.
- Kleyman T.R.
- Sheng S.
Novel determinants of epithelial sodium channel gating within extracellular thumb domains.
). Although oocytes expressing αΔTβγ channels had very low currents in oocytes, reflecting markedly reduced ENaC surface expression, we observed that these channels lost Na
+ self-inhibition. Previous findings have also suggested that the α subunit thumb domain has a role in this response. Specifically, channels with an individual mutation in five of the 10 α subunit thumb domain Cys residues exhibited altered responses to extracellular Na
+ (
42- Sheng S.
- Maarouf A.B.
- Bruns J.B.
- Hughey R.P.
- Kleyman T.R.
Functional role of extracellular loop cysteine residues of the epithelial Na+ channel in Na+ self-inhibition.
). Interestingly, mutations at four sites led to a reduction of the Na
+ self-inhibition response, whereas one mutation enhanced the response (
42- Sheng S.
- Maarouf A.B.
- Bruns J.B.
- Hughey R.P.
- Kleyman T.R.
Functional role of extracellular loop cysteine residues of the epithelial Na+ channel in Na+ self-inhibition.
). Other work indicated that mutation of a Cys residue (αC479R) in the α subunit thumb domain of human ENaC was associated with Liddle syndrome in a sibling pair (
43- Salih M.
- Gautschi I.
- van Bemmelen M.X.
- Di Benedetto M.
- Brooks A.S.
- Lugtenberg D.
- Schild L.
- Hoorn E.J.
A missense mutation in the extracellular domain of αENaC causes Liddle syndrome.
). As a mutation of the homologous residue in mouse αENaC (αC506A) eliminated Na
+ self-inhibition (
42- Sheng S.
- Maarouf A.B.
- Bruns J.B.
- Hughey R.P.
- Kleyman T.R.
Functional role of extracellular loop cysteine residues of the epithelial Na+ channel in Na+ self-inhibition.
), the phenotype seen with the human αC579R mutant activity likely reflects suppressed Na
+ self-inhibition and an increase in channel open probability. Interestingly, most mutations in the region linking α helices in the thumb domain of the α subunit dramatically enhanced Na
+ self-inhibition and reduced ENaC activity (
42- Sheng S.
- Maarouf A.B.
- Bruns J.B.
- Hughey R.P.
- Kleyman T.R.
Functional role of extracellular loop cysteine residues of the epithelial Na+ channel in Na+ self-inhibition.
,
44- Kashlan O.B.
- Boyd C.R.
- Argyropoulos C.
- Okumura S.
- Hughey R.P.
- Grabe M.
- Kleyman T.R.
Allosteric inhibition of the epithelial Na+ channel through peptide binding at peripheral finger and thumb domains.
). In contract to channels with an αΔT or γΔT subunit, channels with a βΔT subunit had a Na
+ self-inhibition response that was similar to WT channels. This result is also in agreement with several previous studies where β subunit mutations did not affect Na
+ self-inhibition (
8- Sheng S.
- Bruns J.B.
- Kleyman T.R.
Extracellular histidine residues crucial for Na+ self-inhibition of epithelial Na+ channels.
,
15- Shi S.
- Ghosh D.D.
- Okumura S.
- Carattino M.D.
- Kashlan O.B.
- Sheng S.
- Kleyman T.R.
Base of the thumb domain modulates epithelial sodium channel gating.
,
16- Shi S.
- Carattino M.D.
- Kleyman T.R.
Role of the wrist domain in the response of the epithelial sodium channel to external stimuli.
,
22- Chen J.
- Ray E.C.
- Yates M.E.
- Buck T.M.
- Brodsky J.L.
- Kinlough C.L.
- Winarski K.L.
- Hughey R.P.
- Kleyman T.R.
- Sheng S.
Functional roles of clusters of hydrophobic and polar residues in the epithelial Na+ channel Knuckle domain.
,
24- Maarouf A.B.
- Sheng N.
- Chen J.
- Winarski K.L.
- Okumura S.
- Carattino M.D.
- Boyd C.R.
- Kleyman T.R.
- Sheng S.
Novel determinants of epithelial sodium channel gating within extracellular thumb domains.
,
42- Sheng S.
- Maarouf A.B.
- Bruns J.B.
- Hughey R.P.
- Kleyman T.R.
Functional role of extracellular loop cysteine residues of the epithelial Na+ channel in Na+ self-inhibition.
).
ENaCs are cleaved and activated by proteases within the biosynthetic pathway and at the cell surface (
4- Kleyman T.R.
- Kashlan O.B.
- Hughey R.P.
Epithelial Na+ channel regulation by extracellular and intracellular factors.
,
45- Kleyman T.R.
- Carattino M.D.
- Hughey R.P.
ENaC at the cutting edge: regulation of epithelial sodium channels by proteases.
). ENaC proteolysis appears to have a role in activating the channel under certain physiologic conditions, including volume depletion and hyperkalemia (
46- Yang L.
- Xu S.
- Guo X.
- Uchida S.
- Weinstein A.M.
- Wang T.
- Palmer L.G.
Regulation of renal Na transporters in response to dietary K.
,
47- Frindt G.
- Yang L.
- Bamberg K.
- Palmer L.G.
Na restriction activates epithelial Na channels in rat kidney through two mechanisms and decreases distal Na+ delivery.
). Studies of ENaC activation by proteases have provided clues regarding a mechanism by which the α and γ thumb domains influence the Na
+ self-inhibition response. Channel activation by proteases involves cleavage at sites flanking imbedded inhibitory tracts in the α and γ subunit finger domains (
4- Kleyman T.R.
- Kashlan O.B.
- Hughey R.P.
Epithelial Na+ channel regulation by extracellular and intracellular factors.
,
45- Kleyman T.R.
- Carattino M.D.
- Hughey R.P.
ENaC at the cutting edge: regulation of epithelial sodium channels by proteases.
,
48- Sheng S.
- Carattino M.D.
- Bruns J.B.
- Hughey R.P.
- Kleyman T.R.
Furin cleavage activates the epithelial Na+ channel by relieving Na+ self-inhibition.
,
49- Bruns J.B.
- Carattino M.D.
- Sheng S.
- Maarouf A.B.
- Weisz O.A.
- Pilewski J.M.
- Hughey R.P.
- Kleyman T.R.
Epithelial Na+ channels are fully activated by furin- and prostasin-dependent release of an inhibitory peptide from the γ-subunit.
50- Carattino M.D.
- Sheng S.
- Bruns J.B.
- Pilewski J.M.
- Hughey R.P.
- Kleyman T.R.
The epithelial Na+ channel is inhibited by a peptide derived from proteolytic processing of its α subunit.
). Release of these inhibitory tracts transitions channels to a higher open probability state with an associated loss of Na
+ self-inhibition (
48- Sheng S.
- Carattino M.D.
- Bruns J.B.
- Hughey R.P.
- Kleyman T.R.
Furin cleavage activates the epithelial Na+ channel by relieving Na+ self-inhibition.
,
49- Bruns J.B.
- Carattino M.D.
- Sheng S.
- Maarouf A.B.
- Weisz O.A.
- Pilewski J.M.
- Hughey R.P.
- Kleyman T.R.
Epithelial Na+ channels are fully activated by furin- and prostasin-dependent release of an inhibitory peptide from the γ-subunit.
50- Carattino M.D.
- Sheng S.
- Bruns J.B.
- Pilewski J.M.
- Hughey R.P.
- Kleyman T.R.
The epithelial Na+ channel is inhibited by a peptide derived from proteolytic processing of its α subunit.
). Peptides corresponding to the released tracts are reversible channel inhibitors, and a putative inhibitory binding region for the α subunit-based peptide mapped to sites in α helices (α1 and α2) in the finger domain, and the loop connecting the thumb domain α helices in the α subunit (
5- Kashlan O.B.
- Adelman J.L.
- Okumura S.
- Blobner B.M.
- Zuzek Z.
- Hughey R.P.
- Kleyman T.R.
- Grabe M.
Constraint-based, homology model of the extracellular domain of the epithelial Na+ channel α subunit reveals a mechanism of channel activation by proteases.
,
51- Kashlan O.B.
- Blobner B.M.
- Zuzek Z.
- Carattino M.D.
- Kleyman T.R.
Inhibitory tract traps the epithelial Na+ channel in a low activity conformation.
). It was proposed that the α subunit thumb-finger domain interface is dynamic, and that the α subunit-imbedded inhibitory tract stabilizes the thumb-finger domain interface, conferring a low activity channel state (
5- Kashlan O.B.
- Adelman J.L.
- Okumura S.
- Blobner B.M.
- Zuzek Z.
- Hughey R.P.
- Kleyman T.R.
- Grabe M.
Constraint-based, homology model of the extracellular domain of the epithelial Na+ channel α subunit reveals a mechanism of channel activation by proteases.
,
44- Kashlan O.B.
- Boyd C.R.
- Argyropoulos C.
- Okumura S.
- Hughey R.P.
- Grabe M.
- Kleyman T.R.
Allosteric inhibition of the epithelial Na+ channel through peptide binding at peripheral finger and thumb domains.
,
51- Kashlan O.B.
- Blobner B.M.
- Zuzek Z.
- Carattino M.D.
- Kleyman T.R.
Inhibitory tract traps the epithelial Na+ channel in a low activity conformation.
). In support of this model, cross-linking the finger domain α1 helix and the loop connecting the thumb domain in the α subunit stabilized ENaC in a low activity state (
51- Kashlan O.B.
- Blobner B.M.
- Zuzek Z.
- Carattino M.D.
- Kleyman T.R.
Inhibitory tract traps the epithelial Na+ channel in a low activity conformation.
). As this model was based on functional studies, a resolved ENaC structure including the finger and thumb domains is needed to confirm key components of the model. We predict that the γ subunit imbedded inhibitory tract stabilizes the channel in a lower activity channel state by a similar mechanism.
Our model suggests that the α and γ subunit thumb domains help restrain the activity of noncleaved channels. As ENaC activation by proteases occurs in association with reductions in (or a loss of) the Na
+ self-inhibition response (
7- Chraïbi A.
- Horisberger J.D.
Na self-inhibition of human epithelial Na channel: temperature dependence and effect of extracellular proteases.
,
48- Sheng S.
- Carattino M.D.
- Bruns J.B.
- Hughey R.P.
- Kleyman T.R.
Furin cleavage activates the epithelial Na+ channel by relieving Na+ self-inhibition.
), the model also predicts that channels lacking an α and γ subunit thumb domain will exhibit a reduced Na
+ self-inhibition response. This is exactly what was observed (
Fig. 4). We also found that the Na
+ self-inhibition response of βΔT channels was similar to WT channels. This is notable as β subunits are not processed by proteases (
38- Hughey R.P.
- Mueller G.M.
- Bruns J.B.
- Kinlough C.L.
- Poland P.A.
- Harkleroad K.L.
- Carattino M.D.
- Kleyman T.R.
Maturation of the epithelial Na+ channel involves proteolytic processing of the α- and γ-subunits.
,
39- Hughey R.P.
- Bruns J.B.
- Kinlough C.L.
- Kleyman T.R.
Distinct pools of epithelial sodium channels are expressed at the plasma membrane.
40- Hughey R.P.
- Bruns J.B.
- Kinlough C.L.
- Harkleroad K.L.
- Tong Q.
- Carattino M.D.
- Johnson J.P.
- Stockand J.D.
- Kleyman T.R.
Epithelial sodium channels are activated by furin-dependent proteolysis.
), and there is no evidence that β subunits have imbedded inhibitory tracts.
Thumb domain interactions with other extracellular components likely impact channel gating. For example, intersubunit cross-linking between thumb and palm domains alters ENaC activity in a length-dependent manner (
52- Collier D.M.
- Tomkovicz V.R.
- Peterson Z.J.
- Benson C.J.
- Snyder P.M.
Intersubunit conformational changes mediate epithelial sodium channel gating.
). Furthermore, there is evidence that ASIC1 thumb domain interactions with the β-ball and palm domains influence channel gating (
53- Krauson A.J.
- Carattino M.D.
The thumb domain mediates acid-sensing ion channel desensitization.
,
54- Vullo S.
- Bonifacio G.
- Roy S.
- Johner N.
- Bernèche S.
- Kellenberger S.
Conformational dynamics and role of the acidic pocket in ASIC pH-dependent gating.
). The recently published closed state structure of ASIC1 highlights a prominent role of the thumb domains in channel gating (
55- Yoder N.
- Yoshioka C.
- Gouaux E.
Gating mechanisms of acid-sensing ion channels.
).
Although the reduced Na
+ self-inhibition response in channels with an αΔT or γΔT subunit should result in an increase in channel activity, thumb domain deletions were also associated with significant reductions in ENaC surface expression. Expression of WT or mutant channels in FRT cells suggested that the α and β subunit thumb domains are required for subunit maturation. Cycloheximide-chase studies suggested that the β subunit thumb domain has a role in stabilizing the immature β subunit (
Fig. 7). In addition, the γ thumb domain appears to have a role in processing leading to the mature forms of the α and β subunits, or stabilizing the mature form of the γ subunit (
Figure 8,
Figure 9). These findings are in agreement with previous studies of ENaCs with deletions of specific extracellular domains, where reductions in subunit maturation and channel surface expression were seen. For example, deletion of either the β or γ subunit knuckle domain, but not the α subunit knuckle domain, dramatically reduced ENaC surface expression and prevented subunit maturation (
22- Chen J.
- Ray E.C.
- Yates M.E.
- Buck T.M.
- Brodsky J.L.
- Kinlough C.L.
- Winarski K.L.
- Hughey R.P.
- Kleyman T.R.
- Sheng S.
Functional roles of clusters of hydrophobic and polar residues in the epithelial Na+ channel Knuckle domain.
).
In summary, we found that ENaC extracellular thumb domains are important regulators of ENaC gating by extracellular Na
+, steady state levels of channels at the cell surface, and channel subunit maturation. Our observations support the notion that specific extracellular domain structures serve as key functional modules (
3- Kashlan O.B.
- Kleyman T.R.
ENaC structure and function in the wake of a resolved structure of a family member.
,
21- Chen J.
- Kleyman T.R.
- Sheng S.
Deletion of alpha-subunit exon 11 of the epithelial Na+ channel reveals a regulatory module.
,
22- Chen J.
- Ray E.C.
- Yates M.E.
- Buck T.M.
- Brodsky J.L.
- Kinlough C.L.
- Winarski K.L.
- Hughey R.P.
- Kleyman T.R.
- Sheng S.
Functional roles of clusters of hydrophobic and polar residues in the epithelial Na+ channel Knuckle domain.
).
Author contributions
S. S. and T. R. K. conceptualization; S. S., J. C., and T. M. B. data curation; S. S., J. L. B., and T. R. K. formal analysis; S. S., J. L. B., and T. R. K. supervision; S. S. validation; S. S., J. C., A. M., M. E. Y., T. M. B., M. A. T., and R. P. H. investigation; S. S., J. C., T. M. B., R. P. H., and T. R. K. methodology; S. S., R. P. H., and T. R. K. writing-original draft; S. S. and T. R. K. project administration; S. S., J. C., A. M., M. E. Y., T. M. B., J. L. B., M. A. T., R. P. H., and T. R. K. writing-review and editing; J. L. B. and T. R. K. funding acquisition.
Article info
Publication history
Published online: September 18, 2018
Received in revised form:
September 13,
2018
Received:
April 20,
2018
Edited by Mike Shipston
Footnotes
This work was supported by National Institutes of Health Grants P30 DK079307, K01 DK90195, R03 DK109024, R37 DK051391, and R01 GM075061. The authors declare that they have no conflicts of interest with the contents of this article. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health.
Copyright
© 2018 Sheng et al.