K Pump Mutations Associated with Primary Hyperaldosteronism

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Na/K pump mutations associated with primary hyperaldosteronism cause loss of function. Dylan J Meyer, Craig Gatto, and Pablo Artigas Biochemistry, Just Accepted Manuscript • DOI: 10.1021/acs.biochem.9b00051 • Publication Date (Web): 27 Feb 2019 Downloaded from http://pubs.acs.org on March 13, 2019

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Biochemistry

Na/K pump mutations associated with primary hyperaldosteronism cause loss of function.

Dylan J. Meyer1, Craig Gatto2 and Pablo Artigas1* 1

Department of Cell Physiology and Molecular Biophysics, Center for Membrane Protein

Research, Texas Tech University Health Sciences Center, Lubbock, TX. 2

School of Biological Sciences, Illinois State University, Normal, IL

*To whom correspondence should be addressed [email protected] Keywords:

Na/K

pump,

Conn’s

syndrome,

electrophysiology,

primary

hyperaldosteronism, aldosterone-producing adenoma, ATP1A1, ATP1B3. Abbreviations & Acronyms: Primary hyperaldosteronism (PHA), Aldosteroneproducing adenoma (APA), micro aldosterone-producing adenoma (mAPA), Na+-Ca2+ exchanger (NCX).

ACS Paragon Plus Environment

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Abstract Primary hyperaldosteronism (Conn’s syndrome), a common cause of secondary hypertension, is frequently produced by unilateral aldosterone-producing adenomas that carry mutations in ion-transporting genes, including ATP1A1, encoding the Na/K pump’s α1-subunit. Whether Na/K-pump-mutant-mediated inward currents are required to depolarize the cell and increase aldosterone production remains unclear, as such currents were observed in four out of five mutants described so far. Here, we use electrophysiology and uptake of the K+-congener 86Rb+, to characterize the effects of eight additional Na/K pump mutations in transmembrane segments TM1 (delM102-L103, delL103-L104, and delM102-I106), TM4 (delI322-I325 and I327S), and TM9 (delF956E961, delF959-E961, and delE960-L964), expressed in Xenopus oocytes. All deletion mutants induced abnormal inward currents of different amplitudes at physiological voltages, while I327S lacked such currents. A detailed functional characterization revealed that I327S significantly reduces intracellular Na+ affinity without altering affinity for external K+.

86Rb+-uptake

experiments show that I327S dramatically impairs function

under physiological concentrations of Na+ and K+. Since Na/K pumps in the adrenal cortex may be formed by association of α1 with β3 instead of β1 subunits, we evaluated whether G99R (another mutant without inward currents when associated with β1) would show inward currents when associated with β3. We found that the kinetic characteristics of either mutant, or wild-type α1β3 pumps expressed in Xenopus oocytes to be indistinguishable from those of α1β1 pumps. The observed functional consequences of each hyperaldosteronism mutant points to loss of Na/K pump function as the common feature of all mutants, which is sufficient to induce hyperaldosteronism.


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Biochemistry

Introduction Aldosterone is a hormone that raises blood pressure by increasing Na+ reabsorption and K+ secretion in the kidney. It is produced by the zona glomerulosa of the adrenal cortex, where oscillations in membrane potential (regulated by angiotensin II and serum K+ 1) control intracellular Ca2+ concentration 1, which in turn regulates aldosterone synthesis (for reviews, see

2-4).

Depolarization of the resting potential leads to higher

voltage-oscillation frequency 1, augmented intracellular [Ca2+], and increased aldosterone production. In primary hyperaldosteronism (PHA), aldosterone is produced independently of regulation by the renin-angiotensin system, causing severe hypertension with hypokalemia. Roughly 40% of PHA cases are due to a unilateral aldosterone-producing adenoma

5, 6

or micro-aldosterone-producing-adenoma

7,

that we will collectively

abbreviate as APA, within the adrenal cortex. Genomic sequencing of such APAs has identified recurrent somatic mutations of several genes involved in ion homeostasis. Thus far, mutations to ion-channel genes lead to increased currents through inward rectifier K+ channels (KCNJ5) 8-11, voltage-dependent Ca2+ channels (CACNA1D and CACNA1H) 1214

or Cl- channels (CLCN2)

15, 16.

Because they result in larger-than-normal depolarizing

inward currents through the mutated channels, these are called “gain-of-function” mutations

8, 10, 14.

Mutations were also found in two P-type II ATPases, including the

Na+,K+-ATPase 12, 17-19. The Na+,K+-ATPase, or Na/K pump, builds and maintains the electrochemical gradients for Na+ and K+ across the plasma membrane of human cells. This pump is formed by association of a catalytic α subunit and an auxiliary β subunit, but is frequently found as a trimer as the αβ dimer associates with a regulatory FXYD protein (Fig. 1). Mutations in ATP1A1, the gene encoding the Na/K pump α1 subunit, have been associated with PHA

7, 12, 17-19.

The mutants L104R, delF100-L104, V332G, and

EETA963S were shown to transport abnormal inward currents at negative voltages 12, 20, suggesting that, like ion-channel mutations, Na/K-pump mutations induce a cell depolarization leading to elevated aldosterone production

12.

Although intriguing, this

inward-current hypothesis was challenged by recent experiments showing that inward currents were absent from oocytes expressing the PHA-mutant G99R, and that the inward


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currents through L104R and EETA963S were likely too small to induce significant depolarization under physiological conditions 20.

Figure 1. Na/K pump structure with 3 Na+ ions bound. A) Lateral view with the extracellular side on the top. The α subunit is comprised of 10 transmembrane segments (TM, gray), and the intracellular domains: actuator (A, magenta), nucleotide-binding (N, forest green), and phosphorylation (P, orange). The β subunit (yellow) has a globular extracellular domain and one TM. The α subunit is comprised of 10 transmembrane segments (TM, gray), and the intracellular domains: actuator (A, magenta), nucleotide-binding (N, forest green), and phosphorylation (P, orange). The β subunit (yellow) has a globular extracellular domain and one TM. The membrane spans the length of the γ subunit (FXYD2, lime green), one of 7 members of the FXYD family or Na/K pump regulators. B) Close-up view of the TM region showing the ion binding sites (I – III) indicating several ioncoordinating side chains (gray carbons), and the TM1, TM4, and TM9 residues that were mutated in this study. Note that some TM1 and TM9 deletion mutations comprise only specific portions of the highlighted residues. The purple spheres represent the three bound Na+ ions.

The functional characteristics of several ATP1A1 mutations recently associated to PHA have yet to be described

7, 18.

Like previously described mutations, these are located in

the vicinity of the ion transport sites within the α-subunit (Fig. 1B, PDB 3WGV,

21),

in

transmembrane segments TM1 (deletions delM102-L103, delL103-L104, and delM102I106), TM4 (deletion delI322-I325 and missense mutant I327S), and TM9 (deletions delF956-E961, delF959-E961, and delE960-L964). Here, we report the characterization of these α1 mutants expressed with β1 in Xenopus oocytes. Using two-electrode voltage clamp (TEVC), we demonstrate that seven of the eight novel mutants have abnormalinward currents (also known as “leak” currents) carried by Na+ while one mutant, I327S in TM4, does not. Additional inside-out patch clamp and


86Rb-uptake

experiments show

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Biochemistry

that I327S induces a loss of function due to a reduced apparent affinity for intracellular Na+, resembling the effect of G99R

20.

Since only two (G99R and I327S) of thirteen

hyperaldosteronism mutants characterized lack passive inward currents in the presence of external Na+, we searched for other scenarios that might impede our ability to observe an inward current in these two variants. In particular, because zona glomerulosa cells express β3 subunits, we evaluated if mutants without inward currents when associated with β1 may produce passive inward currents when associated with β3. However, we show that G99R-α1β3 pumps also lack inward current and have identical functional characteristics as G99R-α1β1 pumps when esxpressed in Xenopus oocytes. Our data demonstrate that the non-leaking mutants must induce PHA due to their loss of function and not to the presence of an abnormal inward current. Thus, we discuss how loss of function is the common feature of all PHA-mutants, which is sufficient to induce PHA, irrespective of the presence or absence of abnormal “leak” currents.


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Materials and Methods Oocyte Isolation and Molecular Biology. Oocytes were isolated, injected with in vitro transcribed cRNA, and cultured in SOS media as described

20, 22.

Animals were used in

accordance with approved TTUHSC IACUC protocols. Female Xenopus laevis frogs were anesthetized with tricaine, oocytes were surgically removed, and incubated with collagenase type I (2 mg/ml, Sigma) for 2 hours in Ca2+-free OR2 (in mM: 82.5 NaCl, 1 MgCl2, 2 KCl, 5 HEPES, pH to 7.5 with NaOH). After washing the collagenase away with Ca2+-free OR2, oocytes were rinsed three times for 30 minutes in OR2 + 2 mM Ca2+ and subsequently transferred to SOS media (in mM: 100 NaCl, 1 MgCl2, 2 KCl, 1.8 CaCl2, 5 HEPES, 2.5 Pyruvic Acid [Sigma], 1X Antibiotic-Antimycotic [Gibco], and 5% Horse Serum [Gibco], pH to 7.5 with NaOH). Mutations were introduced into cDNA encoding the human Na/K pump α1 subunit (in the pSD5 plasmid) by PCR-based mutagenesis and confirmed by sequencing. Plasmids were linearized with NdeI (for α1) or BglII (for β1 and β3) and cRNA was in vitro transcribed using the SP6 mMessage machine kit (Ambion). An equimolar mixture of cRNA for α1 (75 ng) and β1 or β3 (25 ng) was injected into oocytes. Oocytes were kept in SOS media at 16 °C until experimentation. Of note, we chose to use the wild-type human α1 as the background for mutagenesis, because introducing ouabain-sensitive conferring mutations, to allow for pharmacologic separation of endogenous and heterologous pumps, would alter ion-affinities in a physiologic-relevant manner. Signals from endogenous pumps are negligible (less than 10% of total in most experiments due to heterologous overexpression) and do not affect neither quantitative nor qualitative conclusions. This issue was discussed at length in 20. Electrophysiology. TEVC was performed at room temperature (21-23°C) as previously described

20, 22,

utilizing an OC-725C amplifier (Warner Instruments) or CA-1B amplifier

(Dagan) with a digidata A/D converter (Molecular Devices) controlled by pClamp software (Molecular Devices). Data were acquired at 10 kHz and at 1 kHz (using a minidigi 1A, Molecular Devices). Resistance of the glass pipettes used was 0.2 – 1.0 MΩ (filled with 3 M KCl). Before recording, oocytes were incubated (for 30 min in most experiments, 1 hr in Fig. 5) in Na+-loading solution (in mM: 90 NaOH, 20 TEA-OH, 40 HEPES, 0.2 EGTA,


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Biochemistry

pH to 7.2 with sulfamic acid) and kept in 125 mM Na+ external solution (pH 7.6) until recording. External solutions of two osmolalities were used for TEVC, as indicated. The 125 mM Na+ or 125 mM NMG+ external solutions (~ 260 mOsm/kg) contained (in mM) 133 methane sulfonic acid (MS), 5 Ba(OH)2, 1 Mg(OH)2, 0.5 Ca(OH)2, 10 HEPES, and 125 NaOH or 125 NMG, pH to 7.6 with MS. The 150 mM external Na+ solution (~ 300 mOsm/kg) contained (in mM) 135 MS, 5 BaCl2, 1 MgCl2, 0.5 CaCl2, 5 HEPES, and 150 NaOH, pH to 7.4 with MS. Ouabain was directly dissolved in external solutions. External K+ was added to 125 mM solutions from a 450 mM potassium-MS stock and to 150 mM Na+ solutions by mixing with a 150 mM external K+ solution (~ 300 mOsm/kg, where 150 mM KOH replaced 150 mM NaOH). Inside-out patch clamp was performed on the animal pole, at room temperature, as previously described 20. Currents were acquired with pClamp software at 100 kHz (using a Dagan 3900A amplifier with a digidata 1550A A/D), and continuously at 1 kHz (with a minidigi 1A). Pipettes were fire polished (~ 20 µm diameter) and coated with Sylgard. The osmolality of bath and pipette solutions was ~ 275 mOsm/kg. The extracellular (pipette) solution contained (in mM) 140 NMG, 5 KCl, 5 BaCl2, 1 MgCl2, 0.5 CaCl2, and 5 HEPES, titrated to pH 7.4 with HCl. The intracellular (bath) solution contained (in mM) 1 MgCl2, 10 TEA-Cl, 5 EGTA, 5 HEPES, and 140 NaOH (internal Na+ solution) or 140 KOH (internal K+ solution), titrated to pH 7.4 with L-glutamic acid. Intermediate intracellular Na+ concentrations were made by mixing the Na+ and K+ solutions. MgATP (Sigma) was added from a frozen 200 mM stock, just prior to recording. Radioactive

86Rb+

Uptake. Na/K pump have similar affinity for Rb+ and K+

experiments were performed as in

20

23, 24.Uptake

with slight changes to the incubation times and

solutions. Oocytes were either Na+-loaded for 30 minutes and kept in external 125 mM Na+ solution until the experiment, or taken from SOS culture media and briefly washed in external 125 mM Na+ solution before incubation. Oocytes were then transferred to 125 mM Na+ solution with 4.5 mM RbCl, containing radioactive

86Rb+,

for 5 minutes.

Throughout washes and incubation Na/K pump-independent uptake was measured in parallel using oocytes from the same frog and injection groups with 100 µM ouabain in


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the extracellular solutions. Uptake was ended by five washes in external Na+ with 100 µM ouabain before the oocytes were transferred to individual scintillation vials to determine total Rb+ uptake during the 5 minute period. Data Analysis. Electrophysiological data were analyzed using pClamp and Origin (Originlab). During each recording, a voltage pulse protocol was executed in each experimental condition to measure the currents between +40 and -140 mV in 20 mV increments. The steady-state currents at each voltage were determined by averaging current over the last 5 ms of the pulse. Ouabain-sensitive current-voltage (I-V) plots were calculated by subtracting the steady-state currents after ouabain application from the steady-state currents prior to ouabain application in the same experimental condition. Charge-voltage (Q-V) curves were obtained by integrating the ouabain-sensitive transient currents following the return to holding voltage (-50 mV) after a 100 ms-long voltage pulse. They were fitted with a Boltzmann distribution 25: 𝑄 = 𝑄ℎ𝑦𝑝 ―

𝑄𝑡𝑜𝑡 𝑧𝑞𝑒(𝑉 ― 𝑉1/2) 1 + 𝑒𝑥𝑝 𝑘𝑇

(

)

where Qhyp is the charge moved by hyperpolarizing voltage changes, Qtot is the total charge moved, V1/2 is the voltage at the center of the distribution, zq is the apparent valence of a charge crossing the whole electric field, e is the elementary charge, k is the Boltzmann constant, and T is temperature (in Kelvin). The slope factor is kT/ezq. To account for variable expression levels amongst oocytes, the Q-V curves were normalized: Qhyp was subtracted from charge measured at each voltage and the result divided by Qtot, so that all Q-V curves are displayed between 0 and 1. Ion-concentration dependencies of pump current were fitted with a Hill equation:

(

𝐼 = 𝐼𝑚𝑎𝑥

[𝑖𝑜𝑛]𝑛𝐻

)

𝑛𝐻 𝐾𝑛𝐻 0.5 + [𝑖𝑜𝑛]

where maximal pump current (Imax) is the current stimulated by saturating ion concentrations, and K0.5 is the [ion] at which I = Imax/2. For fits to the external K+dependence of outward current (TEVC recordings), the Hill coefficient (nH) was fixed at 1.6 for experiments with external Na+ solution (Fig. 5) according to previous reports 26, 27.


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Biochemistry

For fits to the intracellular Na+-dependence of outward current (patch clamp recordings), nH and K0.5 were shared globally for all experiments with each pump.


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Results We expressed each α1-subunit mutant with the β1 subunit in Xenopus oocytes. Figure 2 depicts TEVC experiments using oocytes expressing wild-type pumps (Fig. 2A) or the TM1 deletions (delM102-L103, Fig. 2B, delL103-L104, Fig. 2C, and delM102-I106, Fig.

Figure 2. Effect of TM1 mutations. TEVC recording from oocytes expressing Wild type – WT (A) delM102-L103 (B), delL103-L104 (C), and delM102-I106 (D). Left panels illustrate the effects of applying external K+ (in mM, indicated by numbers above the recording) and 125 mM Na+ on the current of oocytes held at −50 mV. The gray dashed line is the zero current level. Ouabain (ouab, 0.5 mM) was applied for 2 min to inhibit pump-mediated currents. The vertical deflections throughout each recording represent 100-ms voltage pulses between +40 and −140 mV, in 20 mV increments. Right panels are the mean, ouabain-sensitive, steady-state current vs. voltage (I-V) plots in 125 mM NMG+ (filled squares), 125 mM NMG+ + 3 mM K+ (open squares), 125 mM Na+ (filled circles), and 125 mM Na+ + 4.5 mM K+ (open circles) measured for each pump. Wild type I-V curves are from reference 20. n = 3-9 oocytes each; error bars represent SEM.

2D). The left panels show representative current at -50 mV. Each recording begins with the oocyte in NMG+ solution, where the net current is zero for wild-type-expressing oocytes and outward for oocytes expressing the three mutants. Application of 3 mM K+ in NMG+ activates outward Na/K-pump current in wild-type-expressing oocytes, and partially inhibits the outward current in mutant-expressing oocytes. Replacement of all


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Biochemistry

NMG+ by Na+ has no effect on wild-type-expressing oocytes, but activates inward current in oocytes expressing each TM1 mutant, resembling reported observations with most PHA-associated mutants 12, 20. Application of K+ in Na+ solution activates outward current for wild-type pumps, and inhibits each mutant’s inward current to a variable extent. All changes in holding currents are inhibited by application of 0.5 mM ouabain (irreversibly within the duration of the experiment). Vertical deflections along the continuous traces correspond to 100-ms voltage pulses

Figure 3. Effects of TM9 mutations. TEVC recordings from oocytes expressing delF956-E961 (A), delF959-E961 (B), and delE960-L964 (C). Left panels, current at −50 mV from representative oocytes. Application of external K+ (in mM) is indicated by the numbers above the recordings. The gray dashed line indicates the zero current level. Application of 0.5 mM ouabain (ouab) inhibited inward currents to different extent for all mutants. Vertical deflections along the recording represent a series of 100 ms-long pulses voltages from +40 to −140 mV, in 20 mV increments. Right panels, mean, ouabain-sensitive, steady-state currents in 125 mM NMG+ (closed squares), 125 mM NMG+ + 3 mM K+ (open squares), 125 mM Na+ (filled circles), and 125 mM Na+ + 4.5 mM K+ (open circles). Data from 5-9 oocytes each; error bars represent SEM. Some symbols have reduced size for clarity.

to obtain steady-state current-voltage (I-V) curves. The right-side panels in figure 2 show the ouabain-sensitive I-V curves (current before ouabain minus current after ouabain). The I-V plots for wild-type pumps show that ouabain-sensitive current is outward when K+ is present, and zero when Na+ is the only monovalent cation in the external solution; in NMG+, there is a small inward current activated at voltages more negative than -60 mV due to passive import of H+ 28-32. The reversal potential of the I-V curves for the mutants


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is left-shifted by more than 70 mV in NMG+ solution compared to Na+ solution, indicating that these passive currents are mainly carried by Na+ 20. Note that in oocytes expressing delM102-L103 and delL103-L104 (Fig. 2B and 2C) 4.5 mM K+ completely inhibits the inward current in Na+ solution, a result similar to reported observations with EETA963S 12, 20.

Passive currents were also observed in oocytes expressing the TM9 mutants (delF956E961, delF959-E961, and delE960-L964, Figs. 3A-C, respectively). The slow-time-base recordings (Fig. 3, left-side panels) illustrate the distinct characteristics of these mutants at -50 mV. There was a small outward current in NMG+ where application of 3 mM K+ induced 213 ± 16 nA (n = 8) in delF956-E961 oocytes, 315 ± 25 nA (n = 11) in delF959E961 oocytes and 57 ± 18 nA (n = 11) in delE960-L964; significantly more than in control oocytes injected with β1 alone ~ 25 nA,

20, 22.

All three mutants presented large Na+-

activated currents. However, only the current through delF956-E961 was completely inhibited by ouabain, indicated by the return of the holding current to zero (gray dashed line) during ouabain application (Fig 3A, left). The Na+-activated currents through delF959-E961 and delE960-L964 were only partially inhibited by 0.5 mM ouabain (Fig 3B and 3C, left) and not further inhibited by 10 mM ouabain (n = 2, not illustrated). Note that there is no outward current upon reapplication of K+ in NMG+ solution at the end of the experiment (i.e. ouabain remained bound to most mutant pumps). This indicates that Na+ permeates through the ouabain-bound delF959-E961 and delE960-L964 mutants. The reversal potential of ouabain-sensitive I-V curves (Fig. 3, right panels) was shifted to negative voltages in NMG+ compared to Na+ solution (by ~ −70 mV for delF956-E961, ~ −50 mV for delF959-E961, and ~ −70 mV for delE960-L964), indicating that Na+ is the main carrier of this current. The behavior of TM4 mutants (delI322-I325 and I327S, Figs. 4 and 5, respectively) is much different from that of TM1 and TM9 mutants. DelI322-I325 has exceptionally reduced ouabain sensitivity (described below) likely arising from deletion of residues directly interacting with the inhibitor

33, 34.

This strong ouabain resistance allowed us to

use pretreatment with 10 μM ouabain to inhibit endogenous pumps and measure currents produced exclusively by mutant pumps.


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Biochemistry

A representative trace from an oocyte expressing delI322-I325 pretreated with 10 µM ouabain is shown in figure 4A. The current in NMG+ at -50 mV was outward. Application of extracellular K+ further activated outward current, demonstrating that this mutant mediates electrogenic Na+/K+ exchange. This K+-induced outward current is insensitive to 10 mM ouabain due to a combination of the mutant’s strong ouabain resistance exacerbated by the well-known antagonistic effect of K+ on ouabain binding

33, 35.

Replacement of NMG+ with Na+ activated an inward current (albeit of smaller amplitude than in oocytes expressing TM1 and TM9 mutants). Subsequent application of 4.5 mM K+ or 10 mM ouabain in Na+-only external solution, partially inhibited the inward current. Upon ouabain withdrawal, the inward current quickly returned to its original amplitude, indicating a substantial increase of the ouabain unbinding rate; the mean time constant for ouabain unbinding in Na+ solution was estimated from the recovery of inward current in Na+ solution following ouabain removal 18.5 ± 0.6 s (n = 3), contrasting with the nearly irreversible inhibition of wild-type pumps 20.

Figure 4. Effect of delI322-I325. A) Current at −50 mV from a Na+-loaded oocyte expressing the delI322I325, pre-treated with 10 µM ouabain for 30 min. The dashed line indicates the zero-current level. Numbers above the trace indicate the concentration of K+ applied (in mM). Substitution of NMG+ with Na+ induced inward current that was only partially inhibited by application of 10 mM ouabain (ouab). The effect of ouabain rapidly reverses upon ouabain withdrawal. The red line represents a monoexponential fit to quantify the ouabain unbinding rate; the mean unbinding time constant was = 18.5 ± 0.6 s. B) Mean, steady-state currents inhibited by 10 mM ouabain in NMG+ (filled squares) and Na+ (filled circles) solutions. Data from 4 oocytes; error bars represent SEM.

The I-V curves for the small current fraction inhibited by 10 mM ouabain (Fig. 4B) show a negative shift of the reversal potential when Na+ is replaced with NMG+ (~ −70 mV), indicating that Na+ is the main current carrier through this mutant. Although quantification of the exact current amplitudes in this mutant is hindered by its strong ouabain resistance,


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it is safe to conclude that in the presence of normal external Na+ and K+, the net current through delI322-I325 is inward at voltages below −50 mV.

Figure 5. Effect of I327S on interaction with external K+ and Na+. A) Current from an oocyte expressing I327S bathed in Na+ solution, held at −50 mV. Application of K+ (in mM, indicated by the numbers) evoked outward currents. Ouabain (ouab, 0.5 mM) application for 2 min inhibited subsequent response to K+. To obtain stable K+-induced currents these oocytes had to be Na+-loaded for 1 hr due to the reduction in intracellular Na+ apparent affinity described in Fig. 6. B) Voltage dependence of ouabainsensitive currents at various [K+] (calculated by subtracting the current at the times indicated by smallcaps letters in A. C) Voltage dependence of mean K0.5 for K+, estimated by fitting a Hill equation to the [K+]-dependence (n = 4). The K0.5 of WT is also shown. D) Q-V curves plotting the integral of the ouabain-sensitive currents for I327S (shown in the inset) elicited by 100 ms-long deviations from the holding potential compared to WT. Q-Vs from individual cells were normalized to the total charge (29 ± 3 nC for I327S, n =7) from Boltzmann fits. The solid line is the Boltzmann distribution fitted to the normalized I327S data, with parameters V1/2 = −39.8 ± 2.9 mV and slope factor 42.18 ± 1.0 mV (from 7 individual determinations). Dashed line is the Boltzmann fit for WT. Error bars represent SEM (smaller than the symbol size in C and D). Wild type data from reference 20.

The functional effects of I327S in TM4 (Fig. 5) deviate from the other seven mutants described here. A representative recording from an oocyte expressing I327S held at −50 mV (Fig. 5A) illustrates that application of K+ activates large outward currents. Stepwise changes of the K+ concentration in Na+-induced outward currents in a concentrationdependent manner. Ouabain inhibited K+-induced currents, irreversibly, as these did not return when K+ was applied after ouabain withdrawal.


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Biochemistry

The ouabain-sensitive I-V curves demonstrate that I327S produces outward current at physiological concentrations of extracellular Na+ and K+, at all voltages (Fig. 5B). Surprisingly, when K+ was absent from the bathing solution, the ouabain-sensitive current at −50 mV was outward in both Na+ and NMG+ (~ 15% of maximal K+-induced current in both

cases)

suggesting

that

I327S

increases

the

rate

of

K+-independent

dephosphorylation, probably leading to uncoupled Na+ efflux 36, 37. The K0.5-V curve (Fig. 5C) illustrates a slight increase in the apparent affinity for external K+ in I327S compared to wild type, which significant (p < 0.01, two-tailed, two-sample t-test) at voltages deviating from the holding voltage. Ouabain-sensitive currents produced by voltage-dependent Na+-binding reactions elicited by square voltage pulses from −50 mV to voltages ranging from −160 to +80 mV in 20 mV increments (Fig. 5D, inset) represent the movement of Na+ ions between the external milieu and their binding sites in the protein, rate limited by the pump’s conformational change

38.

The charge (integral of the transient currents) was plotted vs.

voltage (Q-V) curve (Fig. 5D) whose position along the voltage axis relates to external Na+ affinity. The solid line is the fit of a Boltzmann distribution (Methods) to the mean data and the dotted line represents the fit to the Q-V curve of wild type pumps from

20.

The

center (V1/2) of the Q-V curve of I327S (-39.8 ± 2.9 mV) is identical to wild type (-38.2 ± 0.9 mV), suggesting that the overall apparent affinity for external Na+ is mostly unaffected. The effect of I327S on extracellular ion binding seems paradoxical because aldosterone production is stimulated by Na/K pump inhibition 39, 40, yet I327S appears to slightly enhance K+ apparent and thus increase Na+/K+ exchange in Na+-loaded oocytes (i.e. when intracellular Na+ binding is saturating). We measured Na/K pump current in inside-out patches (Fig. 6) to evaluate if I327S alters interaction with intracellular Na+.


Biochemistry 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Current recorded at 0 mV from representative patches expressing wild-type (Fig. 6A) or I327S mutant pumps (Fig. 6B) illustrate activation of pump current by Mg-ATP pulses

Figure 6. Effect of I327S on Na/K pump activation by intracellular Na+. A and B) ATP-activated currents at 0 mV recorded from a giant, inside-out patch excised from an oocyte expressing the wildtype human α1β1 pump (A) or the I327S mutant pump (B). Intracellular ATP application is indicated by the black bars. The intracellular [Na+] is indicated above each ATP-activated current. Vertical deflections along each recording indicate 25 ms-long voltage pulses between +40 and −100 mV in 20 mV increments. C) The intracellular Na+-dependence of ATP-activated currents (normalized) recorded from wild-type pumps (black squares) and I327S pumps (gray squares). Error bars represent SEM (smaller than the symbol size in most cases). The number of patches is indicated in parentheses.

in the in the presence of variable intracellular [Na+]. The normalized ATP-activated outward current plotted as a function of [Na+] (Fig. 6C) illustrates the profound reduction in apparent affinity for intracellular Na+. Hill fits to the data yielded best fit parameters K0.5,Na+ = 14.0 ± 0.4 mM with nH = 2.75 ± 0.17 for wild type (n = 18), and 33.0 ± 2.1 mM with nH = 1.83 ± 0.09 for I327S (n = 3). Although our measurements of intracellular Na+ interaction demonstrate that I327S reduces the pump’s apparent intracellular Na+ affinity (Fig. 6), it is also clear that the I327S mutant pump produces large outward currents under maximal transport conditions (Fig. 5). To test whether I327S performs meaningful Na+/K+ exchange under physiological Na+ and K+ concentrations, we measured the uptake of 86Rb+ (Fig. 7), a K+ congener with similar affinity as the physiologic cation. Figure 7A illustrates that when intracellular Na+ was increased by incubation in Na+-loading solution (like in the TEVC experiments), oocytes expressing I327S pumps show similar Rb+ uptake to oocytes expressing wildtype pumps. Uninjected oocytes from the same batch (with only endogenous pumps) show less Rb+ uptake. When intracellular Na+ was not experimentally increased (Fig. 7B), oocytes expressing wild type have much larger Rb+ uptake than oocytes expressing ACS Paragon Plus Environment

Page 16 of 33

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Biochemistry

I327S (which have similar uptake to uninjected oocytes). These results indicate that the reduced intracellular Na+ binding affinity caused by I327S dramatically impairs ion transport under physiological conditions. Finally, considering that all ion channel-mutations are thought to induce hyperaldosteronism by “gain-of-function” inward currents, we have remained puzzled by the lack of inward currents in two (G99R and I327S) out of 13 ATP1A1-mutants evaluated so far. We have previously demonstrated that the lack of inward currents in G99R is unrelated to FXYD1 interaction or to the reduced temperature in most oocyte experiments 20.

These characterizations were performed by expressing G99R-α1 with the β1-subunit.

Recently, we became aware that the adrenal cortex expresses significant levels of both RNA and protein for α1, β1, and β3 (www.proteinatlas.org). Therefore, in search for the elusive inward currents, which are touted as the cause of APA, we also examined the function of α1 and G99R-α1 when associated with β3 (Fig. 8).

Figure 7. Radioactive 86Rb+ uptake by wild-type and I327S pumps. A) Uptake in Na+-loaded oocytes (see Methods) B) Uptake in oocytes that were not exposed to Na+-loading solution. All measurements were made on the same day in oocytes from the same batch in the absence (solid bars) or presence (striped bars) of ouabain. WT – α1β1, I327S – I327Sα1β1, UI – uninjected. Error bars represent SEM from the number of oocytes indicated in parentheses.

Representative current recordings at −50 mV from Na+-loaded oocytes expressing wild-type α1β3 (Fig. 8A) or G99R-α1β3 (Fig. 8B) start in NMG+ solution, where extracellular K+ stimulates large outward currents in both constructs. There was no significant inward current when NMG+ was replaced by 150 mM external Na+. Subsequent application of 4.5 mM K+ (normokalemic conditions) in Na+ induced robust outward currents in both constructs (albeit smaller than in NMG+ due to competition between Na+ ACS Paragon Plus Environment

Biochemistry 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

and K+ for the externally-accessible binding sites). The ouabain-sensitive I-V curves (Fig. 8C) show that the net current produced by both pumps in external Na+ and K+ is always outward. The ratio of 4.5 mM K+-induced current in Na+ solution to the 3 mM K+-induced current in NMG+ solution was 0.79 ± 0.02 for wild-type α1β3 pumps (n = 4), and 0.57 ±

Figure 8. Effect of G99R on α1β3 pumps. A and B) TEVC recording from oocytes expressing α1β3 (A) and G99R-α1β3 (B) at −50 mV. In both cases, application of K+ in 150 mM Na+ or in NMG+ solutions provoked outward currents which did not return after a 2-min application of 0.5 mM ouabain (ouab). Vertical deflections along each current trace indicate time points where a protocol was executed to sample the currents at voltages between +60 mV and −140 mV, in 20-mV increments, for 100 ms each. Shown below, are the transient currents calculated by subtracting currents in ouabain from the currents in 150 mM Na+ alone. For G99R, the voltage pulses were extended to −200 mV to capture a larger range of charge movement. (C) Mean ouabain-sensitive I-V plots in 150 mM Na+ (squares) with 4.5 mM K+ (triangles) or 2 mM K+ (circles) for α1β3 (n = 4, closed symbols) and G99R-α1β3 (n = 5, open symbols) pumps. Note that G99R lacks ouabain-sensitive inward current in all cases. (D) Q-V curves in 150 mM Na+ for wild-type α1β3 (closed circles) and G99R-α1β3 (open circles). Solid line plots are Boltzmann distribution fits to the mean normalized data, with parameters V1/2 = −23.6 ± 0.6 mV and kT/ezq = 47.9 ± 1.2 mV for wild-type α1β3 (n = 9) and V1/2 = −111.0 ± 8.7 mV and kT/ezq = 75.6 ± 8.4 mV for G99Rα1β3 (n = 6). Error bars represent SEM (not visible when smaller than the symbol size).

0.01 (n = 5) for G99R-α1β3 pumps. This indicates that the apparent K+ affinity of G99Rα1β3 pumps is reduced at physiological external [Na+] and [K+], as previously shown for G99R-α1β1 20.


Page 18 of 33

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Biochemistry

The insets in figures 8A and 8B show, at a faster time-scale, each pump’s ouabainsensitive transient currents when the oocytes were bathed in 150 mM Na+ without K+. The G99R mutation shifts the Q-V curve V1/2 by ~ −90mV compared to α1β3. This indicates that G99R decreases α1β3’s apparent external Na+ affinity by 8- to 16-fold (2-fold per 25 mV shift 41), nearly identical to its effect on α1β1 pumps 20. Thus, G99R-α1 lacks inward “leak” currents and has nearly identical function whether expressed with β1 or β3. For thoroughness, we compared the kinetic parameters of α1β1 and α1β3 pumps (Fig. S1). At all voltages, α1β3 pumps have slightly lower apparent affinity for extracellular K+ than α1β1 pumps (Fig. S1-C), a reduction which may reflect an ~ 1.5-fold increase in apparent affinity for the competing Na+ (the V1/2 of the Q-V curve in the absence of Na+ is shifted by +11 mV, Fig. S1-E). Overall, the voltage-dependence of the pump current of both isozymes (Fig. S1-D) and their apparent affinities for intracellular Na+ (Fig. S1-F and S1-G) are indistinguishable.


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Page 20 of 33

Discussion We used electrophysiology and

86Rb-uptake

to characterize the functional effects of

several PHA-associated Na/K pump α1-subunit mutations. Our results further knowledge of the functional effects of individual Na/K pump mutants and provide additional evidence demonstrating that loss of function is sufficient to induce hyperaldosteronism. We show that: i) seven of the eight newly described mutations transport passive inward currents carried by Na+, ii) all these “leaky” mutants show a clear loss of function, iii) there are two pathways for inward currents by TM9 mutations, iv) the mutation I327S lacks inward currents, but causes a loss of function by reducing the pump’s ability to bind intracellular Na+, and v) association with the β3 subunit does not produce inward currents in mutants that lack them when associated with β1. The first report describing the functional effects of four PHA-associated ATP1A1 mutants showed that passive inward currents are carried by the Na/K pumps formed by association of the α1-mutants with β1 12. The analogy of these passive inward currents to the “gain-of-function” currents in PHA-associated ion-channel mutations led to the attractive hypothesis that all Na/K pump mutants may induce hyperaldosteronism by depolarizing the membrane. However, subsequent demonstration that inward current was absent from the PHA-associated mutant G99R, and was of relatively small amplitude for two other mutants, challenged this hypothesis

20.

The results presented here, reinforce

the conclusion the loss of Na/K pump function suffices to induce hyperaldosteronism and show that impaired active ion transport is a common feature of all PHA-associated Na/K pump mutants. The I327S mutant While seven of the eight newly characterized mutants have abnormal inward current, the I327S mutant does not (Fig. 5). Instead, the effect of I327S on Na/K pump function resembles the effect of G99R

20:

it reduces intracellular Na+ binding (Fig. 6) and inhibits

Na+/K+ exchange at physiological ion concentrations (Fig. 7). I327S is the second PHAassociated Na/K pump mutation identified that causes loss of function without introducing inward passive transport of Na+. As the I327S mutant cannot possibly depolarize the membrane via an inward current. Intracellular Ca2+ is the second messenger for


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Biochemistry

aldosterone production, and its regulation in zona glomerulosa is highly dependent on NCX 42 and consequently on a steep electrochemical gradient for Na+. Therefore, the loss of function of I327S pumps must elevate Ca2+ signaling and aldosterone production from APAs due to haploinsufficiency leading to increased intracellular Na+ and reduced driving force for Ca2+ extrusion via NCX. Structurally speaking, I327S is located to the extracellular side of the bound ions on TM4 where its side chain extends towards TM3 (in the Na+-bound E1 state, Fig. 1B) and towards the space between TM4 and TM5 in the K+-bound E2 state (not illustrated, cf. 43).

The alterations in apparent ion affinities resulting from substitution of this hydrophobic

isoleucine with a hydrophilic serine likely reflects a disruption of the hydrophobic interactions of TM4 with other TMs, which lead to changes at the ion-coordinating residues located 1.5 - 2 helix turns towards the cytoplasm. Hydrophobic residues on the extracellular portion of TM4 are thought to comprise a “gating latch” that stabilizes the position of the TM1-TM2 helices in the E2P open state” of P-type II ATPases 44, 45. Binding of the imported ion induces movement of the TM1-TM2 bundle that activates the pump’s intrinsic phosphatase in the A-domain (i.e. the TGES motif). Mutation of Met334 to Ala on TM4 in the gastric H/K pump, equivalent to I325 in the Na/K pump uncouples its ATPase activity from K+ binding, likely by destabilizing the interaction between TM4 and TM1-TM2 44.

Our observation that I327S generates outward current in the absence of extracellular

K+ (Fig. 5A and 5B) probably reflects a similar uncoupling mechanism, increasing the rate of K+-independent dephosphorylation. Na+ permeability in leaky mutants ─ plausible pathophysiological relevance. Can the effect of all leaky mutants induce significant depolarization of the cells that carry these mutations? We contend that in most cases the answer is no. It is well known that in most cells the normal hyperpolarizing outward current of the Na/K pump contributes little to the resting membrane potential and experimental evidence suggest that wild-type Na/K pumps contribute minimally to the resting potential of adrenocortical cells

46, 47.

It appears that half of the Na/K pumps in an APA are wild type and half are

mutants, because mRNA expression of ATP1A1 is unchanged

17.

Therefore, the

amplitude of the inward currents through mutant pumps has to be larger than the outward


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Page 22 of 33

current produced by the wild-type pumps in order for them to profoundly affect membrane voltage. The rates of inward “leak” current through the PHA mutants L104R and EETA963S are similar to or smaller than the rate of outward current produced by wildtype pumps, making it unlikely that they induce significant depolarization 20. While we thought it unnecessary to measure the rates of all the mutants described here, it can be seen that in the presence of normal external K+, the inward current through many mutants is either negligible (Figs. 2B and 2C) or smaller (Figs. 3A, and 4) than outward currents observed with wild-type pumps (−0.1 μA to −0.5 µA

20, 22).

Thus, they

are unlikely to depolarize the cell. Two mutants, delM102-I106 and delF956-E961, with current amplitudes comparable to delF100-L104 (with a turnover rate ~ 15-fold faster than wild type pump-mediated outward current 20) are expected to significantly depolarize the membrane of the cells where they are expressed. Overall, the current through five out of thirteen mutants (G99R, I327S, EETA963S, delM102-L103, and delL103-L104) is either zero or negligible under physiological conditions, making it impossible for depolarization to be the cause of hyperaldosteronism. In addition, hypokalemia will make the equilibrium potential for K+ more negative, and the normal resting voltage of zona glomerulosa cells is ~ −80 mV because of the predominant K2P and inward rectifier K+ conductances 2, 48. Although the charge influx through abnormal "leak” currents may not be relevant to induce hyperaldosteronism, the passive Na+ import occurring through all “leaky” mutants (Figs. 2, 3, and 4,

20)

may still contribute to pathophysiology. This is because the

increased workload on wild-type Na/K pumps caused by the loss of Na+ extrusion by mutant pumps would be aggravated by the dissipation of the Na+ gradient when the leak pathway is present. Nevertheless, I327S and G99R demonstrate that neither charge, nor Na+ entry are required to induce hyperaldosteronism. This is consistent with the observation that simple Na/K pump inhibition induces NCX-mediated increase in intracellular Ca2+ in zona glomerulosa cells 42. Ouabain-insensitive ion pathway through some TM9 mutants “Leak” currents through delF959-E961 and delE960-L964 were only partially inhibited by ouabain (Figs. 3B and 3C), which blocks the external ion access pathway by which extracellular Na+ or K+ reach ion-binding binding sites I & II


49, 50.

Because under the

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Biochemistry

chosen experimental conditions (10 mM ouabain) the mutant pumps are fully occupied by ouabain, this observation suggests that external access of the permeating ions may occur through two pathways, with only one being blocked by ouabain. Similar dual external pathways have been proposed to explain the effect of quaternary amines and other ions on the passive inward current carried by protons in wild-type Na/K pumps (observed when Na+ and K+ are absent from the extracellular milieu)

51.

Both, the Na+

ions in TM9 mutants 20, 52 and the protons in wild-type pumps 30, 31 are thought to traverse through the Na+-exclusive binding site (site III). Wild-type inward currents are only partially inhibited (~ 70%) by tetrapropylammonium+

51,

which competitively blocks the access

pathway for K+ 51, 53, 54, suggesting that extracellular protons may simultaneously access site-III by two pathways: 1) the normal ion-access pathway used by external K+ to reach the binding sites, and/or 2) a proton-wire composed of E961 on TM9, Y778 on TM5, and D933 on TM8 31. We propose that two out of four TM9 deletions transform the proton wire region into a Na+-permissive pathway continuously accessible in the presence of ouabain. This can be envisioned as an inward movement of the external-most part of TM9 due to

Figure 9. Na/K pump in the ouabain-bound conformation. View from the membrane similar to Fig. 1. Side chains of most ion-coordinating residues (gray carbons) are shown. Ouabain (ouab, violet carbons, dot cloud) blocks the pathway to site II between TMs 4, 5 & 6. Roman numerals (I-III) indicate the approximate locations of the binding sites as in Figure 1. Blue carbons illustrate TM9. The side chains of residues affected by various TM9 deletions are shown. The γ-subunit is shown in green. Mg2+ in the cation-binding pocket and the β-subunit are not shown for clarity.


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Page 24 of 33

deletion of the residues in the center of TM9 (Fig. 9, PDB 4HYT, 34), although the precise structural effect of each deletion would be hard to model accurately. Impaired Na/K pump function in ATP1A1 mutants All PHA-associated Na/K pump mutations cause loss of Na/K pump function; the five characterized previously 17, 19, 20, and the eight we describe in this report. Under conditions that maximally stimulate wild-type pumps (i.e. Na+-loaded oocytes bathed with extracellular NMG+ and 3 mM K+) delM102-L103, delL103-L104, delM102-I106, delF956E961, and delE960-L964 appear severely impaired (Figs. 2 and 3). Although delF959E961 and delI322-I325 produce outward current under maximal transport conditions (Figs. 3 and 4), loss of function is evident for both mutants in the presence of external Na+, where the current stimulated by 2 mM K+ is about half of that simulated by 4.5 mM K+ (Figs. 3B and 4A), an indication of reduced apparent affinity for extracellular K+. I327S causes loss of function by severely reducing apparent affinity for intracellular Na+ activation in the presence of physiological K+ concentration (Figs. 6 and 7). It is important to note that the normal function of the Na/K pump is to build the plasma membrane Na+ gradient. Therefore, even if some mutants perform active Na/K exchange in the presence of external K+ (delL103-L104, Fig.2C, delF959-E961 Fig. 3B, delI322-I325 Fig. 4A, and EETA963S), the dissipation of the Na+ gradient through the leak pathway of the same mutants is, de facto, a loss of Na/K-pump active-transport function. Characteristics of the pumps formed by association of wild-type or mutant α1 with β3 Human adrenal cortex expresses Na/K pump β3 subunits. However, G99R-α1β3 mutant pumps also lack inward currents at physiological concentrations of extracellular Na+ and K+, and the mutation induces identical effects on ion apparent affinities whether it is associated with β1 20 or β3 (Fig. 8). Comparison of the wild-type pumps shows that α1β3 has only slightly lower apparent extracellular K+ affinity than α1β1 (Fig. S1-C), in agreement with previous reports showing similar directional trends with K0.5 values of ~ 1-2 mM for both pumps

55, 56.

This minor

difference is unlikely to have physiological significance because the normal extracellular K+ concentration is ~ 4-fold larger than the K0.5. In addition, α1β1 and α1β3 isozymes


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Biochemistry

have identical Na+ affinities (Fig. S1-G), a result that contrasts with a report indicating that α1β3 affinity is ~ 3-fold higher than α1β1

56.

We suspect that the difference reflects the

use of distinct experimental systems for α1β3 (Sf-9 insect cells) and α1β1 (HeLa cells) in 56.

The similar ion affinities we observe for α1β1 and α1β3 pumps also contrast with observations with α1β2 pumps

22, 57.

Compared to α1β1, the α1β2 isozyme has a lower

apparent extracellular K+ affinity at voltages more negative than -60 mV and a higher apparent affinity for external Na+ (indicated by the strong voltage dependent inhibition of pump current in external Na+ cf. Supplementary Fig. 2 in shift of its Q-V curve V1/2

57).

22

and a ~ +60 mV rightward

That α1β1 and α1β3 pumps are nearly indistinguishable in

our experiments raises an important question for future studies: is there a larger role for the β3 subunit? The β3 subunit is expressed in several tissues (reviewed in 58), but always appears to be present with β1 (and sometimes β2). It is possible that β3 pumps are sensitive to different regulatory cues than pumps formed by β1 or β2 pumps. For instance, Cys45 in β1 has been linked to glutathionylation regulation

59,

a process that could not

occur in β3 pumps which lack this cysteine. Perspectives Loss of function is common to all PHA-associated Na/K pump α1 mutants characterized to date (17, 19, 20, Fig. 2-7). As half of the Na/K pumps in APA cells are wild type and half are mutants, there is a reduced transmembrane Na+ gradient. There is clear evidence for Na/K pump haploinsuficency-induced hyperaldosteronism in the literature 39, 40,

where ouabain concentrations that inhibit half the Na/K pumps in the glomerulosa cells,

increased aldosterone production. The mechanism of this must be by cellular Ca2+ dysregulation, as 200 μM ouabain (slightly above half-maximal for rat Na/K pumps) activates Ca2+ entry via NCX in rat glomerulosa cells. These experiments demonstrate that loss of function in half the pumps suffices to induce hyperaldosteronism. This loss of function hypothesis fits well with results describing Na/K-pump loss of function as the main mechanism for other illnesses. Loss of function mutations to α2 (ATP1A2) cause familial hemiplegic migraine (reviewed by

60),

to α3 (ATP1A3) cause

rapid-onset-dystonia parkinsonism and alternating hemiplegia of childhood (reviewed by


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61),

and to α1 cause Charcot-Marie-Tooth Type 2 (CMT) syndrome

Page 26 of 33

62.

Less clear is the

involvement of an abnormal inward current in the induction of hypomagnesemia, seizures, and intellectual disability in patients with α1 mutations 63. Convincing evidence in favor of a depolarizing inward current mechanism underlying a Na/K pump mutant-mediated illness has only been presented for one case of hypokalemic periodic paralysis by the α2 mutation S779N (equivalent to S783 in Fig. 1), in site I 64. Somewhat surprisingly, patients with CMT are not reported to have high blood pressure 62,

a finding that perhaps is due to compensatory mechanisms that take place when

ATP1A1 haploinsufficiency appears early in life. Consistent with this interpretation, heterozygous α1 knockout mice have increased aldosterone production without increased blood pressure

65.

Given the importance of Na/K pump function for normal

membrane physiology, and the ubiquitous presence of α1 in the body, it is likely that Na/K pump-α1 dysfunction will be identified as an underlying cause of other pathologies in the future. Acknowledgements This work was supported by the National Science Foundation (MCB-1515434 to PA) and National Institutes of Health (GM061583 to CG). DJM is supported by an American Heart Association predoctoral fellowship (17PRE32860001). Author contributions: DJM performed the experiments and analyzed data. DJM, CG, and PA designed the research and wrote the manuscript. We thank Dr. Luis Reuss for comments on the manuscript, and Dr. Ben Wylie and Derek Versteeg for help with 86Rb+ uptake.

Supporting Information. Includes figure S1 comparing of functional characteristics of α1β1 and α1β3


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Biochemistry

Accession codes. This article describes functional characterization of human Na+,K+ATPases formed by several mutants of the α1 subunit (uniprot P05023), and either the β1 subunit (uniprot P05025) or the β3 subunit (uniprot P54709).


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Biochemistry

Table of contents figure


K Pump Mutations Associated with Primary Hyperaldosteronism

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