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A Long Isoform of the Epithelial Sodium Channel Alpha Subunit Forms a Highly Active Channel a
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Jonathan M. Berman , Cristin Brand & Mouhamed S. Awayda a
Department of Physiology and Biophysics, State University of New York at Buffalo, Buffalo New York Accepted author version posted online: 17 Dec 2014.
Click for updates To cite this article: Jonathan M. Berman, Cristin Brand & Mouhamed S. Awayda (2014): A Long Isoform of the Epithelial Sodium Channel Alpha Subunit Forms a Highly Active Channel, Channels, DOI: 10.4161/19336950.2014.985478 To link to this article: http://dx.doi.org/10.4161/19336950.2014.985478
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Extended N-Terminus Alters Alpha ENaC Activity
A Long Isoform of the Epithelial Sodium Channel Alpha Subunit Forms a Highly Active Channel Jonathan M. Berman, Cristin Brand, and Mouhamed S. Awayda 1
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From: 1Department of Physiology and Biophysics, State University of New York at
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Address correspondence to: Mouhamed S. Awayda, Department of Physiology and
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Biophysics, SUNY at Buffalo, Buffalo NY, E-Mail:
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Buffalo, Buffalo New York
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Extended N-Terminus Alters Alpha ENaC Activity
Abstract: A long isoform of the human Epithelial Sodium Channel (ENaC) α subunit has been identified, but little data exist regarding the properties or regulation of channels formed by α728. The baseline whole cell conductance of oocytes expressing trimeric α728βγ
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channels was 898.1 ± 277.2 and 49.59 ± 13.2 µS in low and high sodium solutions,
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respectively, and was 11 and 2 fold higher than the conductances of α 669βγ in same
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proteases subtilisin and trypsin than α669βγ in low and high Na+ conditions. The long isoform exhibited lower levels of full length and cleaved protein at the plasma
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membrane and a rightward shifted sensitivity to inhibition by increases of [Na+]i. Both
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channels displayed similar single channel conductances of 4 pS, and both were activated to a similar extent by reducing temperature, altogether indicating that
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activation of baseline conductance of α728βγ was likely mediated by enhanced channel activity or open probability. Expression of α728 in native kidneys was validated in human
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urinary exosomes. These data demonstrate that the long isoform of αENaC forms the
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structural basis of a channel with different activity and regulation, which may not be easily distinguishable in native tissue, but may underlie sodium hyperabsorption and salt sensitive differences in humans.
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solutions. α728βγ channels were also 2 to 5 fold less sensitive to activation by the serine
Keywords:
Electrophysiology, Ion Channels, Protease, Epithelial Sodium Channel (ENaC), Alternative splicing, hypertension.
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Extended N-Terminus Alters Alpha ENaC Activity
Introduction: Background Hypertension is a risk factor for myocardial infarction and stroke. One of the primary
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determinants of an individual’s blood pressure is blood volume, which is chronically regulated by the kidneys. Reabsorption of water from the nephron lumen to the plasma
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is tied to the osmotic gradient created by the movement of sodium. The epithelial
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the nephron (CD) and the rate-limiting step in reabsorption in these epithelia.
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High activity ENaC is a heteromultimer consisting of a structural subunit, typically alpha, as well as beta and gamma subunits with a membrane stoichiometry which
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electrophysiology indicates consists of two alpha subunits, one beta and one gamma 1, while structural homology to the crystalized acid sensing ion channel (ASIC)2,3,
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suggests a trimer of each subunit.
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Modularity and Alternate Splicing
Alpha ENaC is critical for channel formation.1,4 Alpha-like subunits such as Xenopus
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epsilon5, ASIC16, or human delta, can exhibit different properties and may substitute for alpha in some tissues yielding channels with different activity and/or regulation. In
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sodium channel (ENaC) is the final step in sodium reabsorption in the collecting duct of
humans, a longer alpha isoform has been detected in the kidney but it remains incompletely characterized.7 We examined the properties of this 728 a.a isoform because little is known about its physiological function and regulation despite being first identified by Thomas et al. in 1998 where it was shown to have single channel conductance and macroscopic currents similar to those of the 669 a.a. isoform. It was
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Extended N-Terminus Alters Alpha ENaC Activity
also shown to be expressed in multiple tissues including colon, lung, and kidney,7 however, there are no detailed comparisons between channels formed with α 728 and α669. A significant part of ENaC regulation occurs through membrane trafficking/recycling
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leading to differences of membrane protein expression.8 Another major aspect of
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regulation occurs through changes to open probability mainly through proteolytic
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extracellular Na+ leading to downregulation in response to increases of [Na+].10,11 The
the current work.
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Regulation by [Na+] and Proteolysis
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response and regulation of α728 channel by [Na+] is undetermined and is examined in
Two types of inhibition by sodium have been described and differentiated based on
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time course: 12–14 a slow effect due to high [Na+], termed “feedback inhibition,” and mediated by PKC15 and a fast effect termed “self-inhibition” likely mediated by the Na+
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ions interaction with the channel, 11 and this represents an intrinsic channel property
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that does not require further second messenger.14 ENaC in native epithelia, and especially in the kidney, is exposed to different [Na+] making both regulatory processes physiologically relevant. Further, there is variation in the degree to which blood pressure
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cleavage of the channel.9 The channel also responds to changes of intra and
is sensitive an individual’s salt intake, and it is unknown if such differences could be, at least in part, mediated by differences in the structural isoform expressed (α669 vs α728). Another regulator of channel activity is cleavage by internal and external proteases.9,16,17 This occurs on two ENaC subunits, with α being one of these subunits.9 Cleavage markedly increases open probability (Po) either by removal of an inhibitory 4
Extended N-Terminus Alters Alpha ENaC Activity
tract,18 or loss of the first transmembrane domain.19 The baseline intracellular and exogenous extracellular cleavage of channels formed with α728 by proteases is unknown. Given the chronic exposure of ENaC to urinary proteases, differences in proteolytic activation of these subunits is of potential significance to renal sodium
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handling in the CD.
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Effects of Temperature
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by cooling.20,21 This effect increases channel Po possibly by increased membrane order
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and rigidity and interaction with the lipid bilayer.20 This activation of Po is likely separate from that caused by cleavage, as it is immediate and reversible, and, it is unknown if the
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two human alpha isoforms exhibit similar responses to cooling. We report that α728βγ forms a high activity channel despite low plasma membrane
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density of the full length and cleaved forms. This indicates that ENaC may be highly active in the absence of cleavage. Regulation by Na+ was also different with α728
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channels exhibiting larger inhibition by chronic and acute high [Na+]i with sensitivity
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shifted to higher [Na+]. Effects of temperature were similar indicating that the interaction with the lipid bilayer was not likely modified. Altogether, these data indicate that α728 can form a high activity channel in vivo that is less dependent on proteolysis for its activity
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In addition to the above processes, ENaC is stimulated by membrane rigidification
and is further stimulated in low [Na+]i, and that the existence of channels with this isoform may predispose collecting duct epithelia to Na+ retention.
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Extended N-Terminus Alters Alpha ENaC Activity
Results Structure and Function The structure and function of the α ENaC N-terminus remains largely unstudied
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despite being 1st identified by Thomas and colleagues in 1998.7 We examined the amino acid sequence of the extended α728 N-terminus with reference to the known α669
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sequence and homology to rat. Figure 1A, represents the alternative splice sites which
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transcript produces upstream an additional 59 amino acids from a longer first exon of
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α669 which also contains a unique 5’ UTR. Because the ASIC structure solved by Jasti et al.2 did not include the N-terminus there is no homologous structure to which this
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sequence can be compared to estimate a secondary structure. We compared the human α728 sequence to that of rat α699 (Figure 1B) because the
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rat N-terminus is longer than that of human α669 and because there are notable electrophysiological differences between rat and human ENaC especially in terms of
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voltage activation and sensitivity to Na+.22 The first 24 a.a. of the rat sequence are
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homologous to human α728 a.a 13-37, and these are absent from the human α669 sequence.
Rectification and Voltage Activation
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give rise to the differences in the mRNA sequence between α669 and α728. The α728
Two of the characteristic electrophysiological properties of α669βγ are its current-
voltage relationship which shows inward rectification (Figure 2), and a voltage dependent activation possibly by voltage induced changes to [Na+] at the inner mouth of the channel.22 Rectification and voltage activation are in addition to Na+-self inhibition
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Extended N-Terminus Alters Alpha ENaC Activity
intrinsic channel properties and changes in them reflect differences in channel properties and function. We tested rectification and voltage activation of α728βγ to determine if the longer N-terminus led to differences in these properties. Rectification of the α728βγ and α669βγ channels was assessed from the ratio of the
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slope conductances at -100 mV and +40 mV in ND94 recording solution. Conductance
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Voltage dependent activation at -100 mV in α728βγ was apparently less than that
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observed in α669βγ. The observed activation in α728βγ was longer than the 500ms measurement and for this reason it was not possible to determine a finite time constant
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or magnitude of activation (Figure 2C). Therefore, we cannot rule out that the reduced activation is actually just due to a much longer time constant of activation of α728βγ.
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Given complications with Na+ loading in prolonged clamping to -100 mV, it was not possible to extend the time course of the measurement to accurately obtain a time
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constant. Nonetheless, these data indicate marked differences in the way these two
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channels respond to acute changes of voltage. These differences of voltage activation and rectification led to a different characteristic I/V relationship for the two isoforms. Consistent with effects of Na + and
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hyperpolarizing voltages (Figure 2D, p