Mechanism of Action of Salt Adaptation Mutations in Artemia franciscana
by
Jessica Eastman
AN HONORS THESIS
for the
HONORS COLLEGE
Submitted to the
Honors College
at Texas Tech University in
partial fulfillment of the
requirement for
the degree designation of
HIGHEST HONORS
MAY 2015
Approved by:
_________________________________________
Dr. Pablo Artigas, Ph.D., TTUHSC CPMB
_______________
Date
_________________________________________
Sukanyalakshmi Chebrolu, TTUHSC CPMB
_______________
Date
_________________________________________
Dr. Keira V. Williams, Ph.D., Honors College
_______________
Date
_________________________________________
Dr. Michael San Francisco, Ph.D., Dean, Honors College
_______________
Date
The author approves the photocopying of this document for educational purposes.
Copyright 2015, Jessica Eastman
ACKNOWLEDGMENTS
I would like to take this opportunity to express my extreme gratitude for my
mentor Dr. Pablo Artigas. Without his encouragement, constant support, and motivation,
this work would never have been possible. I would also like to thank the Department of
Cellular Physiology and Molecular Biophysics department at the Texas Tech Health
Sciences Center for the equipment and supplies that allowed me to collect all my data.
Finally, I would like to thank the Texas Tech Honors College for their support throughout
my time in the lab. This project was supported by NSF-MCB-1243842.
i
Table of Contents
Acknowledgements…...........................................................................................................i
Table of Contents.................................................................................................................ii
Abstract...............................................................................................................................iii
Preface..................................................................................................................................v
Introduction........................................................................................................................1
1. Biological/Physiological Roles and Structure of the Na+/K+ pump....................1
2. P-Type ATPase Family........................................................................................2
3. Post Albert’s Cycle..............................................................................................3
4. Artemia franciscana.............................................................................................4
Materials and Methods......................................................................................................8
1. Molecular Biology...............................................................................................8
2. Oocyte Preparation..............................................................................................9
3. Solutions..............................................................................................................9
4. Electrophysiology..............................................................................................10
5. Analysis..............................................................................................................10
Results...............................................................................................................................12
1. Continuous Traces.............................................................................................12
2. Kind in the absence of Na....................................................................................14
3. Kind in the presence of Na..................................................................................15
4. Q-V....................................................................................................................16
5. Naind...................................................................................................................19
Discussion.........................................................................................................................21
1. K+ Affinity.........................................................................................................21
2. Na+ Affinity.......................................................................................................22
3. RD/N333K/N785K............................................................................................23
Conclusions.......................................................................................................................24
Bibliography.....................................................................................................................25
ii
ABSTRACT
Nearly all animals maintain a large electrochemical gradient for Na+ across the
plasma membrane. This gradient is generated by the Na+/K+ pump, which exports 3 Na+
and imports 2 K+ per ATP molecule hydrolyzed. The ion-coordinating residues in the α
subunit of the pump are usually conserved in most species. However, the brine shrimp
(Artemia franciscana) that lives in extreme saline conditions express a pump with two
asparagine to lysine substitutions within the ion binding site region (Jorgensen and Amat,
2008). We used two-electrode voltage clamp on Na+-loaded Xenopus oocytes to evaluate
the effect of the equivalent substitutions (N333K and N785K) individually and
concurrently on the function of Xenopus Na+/K+ pumps. In particular, we studied the
effect of these mutations on activation of pump currents by eternal K+ and on voltagedependent conformational changes related to external Na+ binding (charge voltage (Q-V)
curves). The apparent affinity for K+ in the absence of Na+ was reduced (approximately
ten-fold) by the N785K, mutation while N333K and the double mutant had similar
affinity to the wild-type. The centers of the Q-V curves were displaced approximately -80
mV by both individual mutations suggesting a reduced (greater than ten-fold) external
Na+ affinity. Surprisingly, the double mutant showed a slightly smaller shift in the Q-V,
indicating non-additive effects on external Na+ affinity and energetic coupling between
the residues.
These results can be explained with recent structures of the Na+/K+ pump with
Na+ or K+ bound. N333, outside the ion-binding pocket, forms a hydrogen bond with the
ion-coordinating N785 in the Na+ bound conformation. Therefore, once the disruption of
iii
normal Na+ coordination by N785K is in place the mutation N333K does not affect Na+
binding. This contrasts with previous findings regarding internal Na+ binding.
iv
PREFACE
Cells are the basic building blocks of life and comprise all living things. In the
human body, cells provide structure and support, convert nutrients into energy the
organism can use, and carry out many specialized functions. A cell consists of many
different organelles that work together to ensure all processes are carried out correctly.
One of the most important features of a cell is its plasma membrane. The plasma
membrane lines the outside of the cells and separates it from the surrounding
environment. Materials enter and leave the cell through this membrane and it controls all
transport in and out of the cell with its selective permeability. Selective transport is made
possible by the phospholipid bilayer that contains many specialized proteins that aid the
membrane with the movement of ions and organic matter. The transport of ions across
the membrane is vital in maintaining an appropriate electrochemical gradient that drives
many vital cellular processes, such as the synthesis of ATP or the ability to generate an
action potential to send an electrical impulse to a neuron or contract a muscle. Many
different proteins maintain the electrochemical gradient and allow the cell to carry out
these functions; one of the most important of these proteins is the Na+/K+ pump.
Mutations in the Na+/K+ pump have been implicated in many different diseases, such as
rapid-onset dystonia Parkinsonism and familiar hemiplegic migraine (Ashmore et al.,
2009).
Understanding the structure, function, and kinetics of the Na+/K+ pump is vital to
understanding and treating these diseases. The aim of this project is to focus exclusively
on understanding the mechanism of action of natural amino acid substitutions made in the
v
Na+/K+ pump in animals that live in highly saline environments. More specifically, the
two asparagine to lysine substitutions within the binding site region of the Na+/K+ pump
in brine shrimp (Artemia franciscana) were examined by the use of two-electrode voltage
clamp. These mutations were tested individually and concurrently to study the effect they
had on activation of pump currents by external K+ and on voltage-dependent
conformational changes related to external Na+ binding. It is hypothesized that by
reducing the Na+/K+ pump’s affinity for external Na+ and possibly by changing the
pump’s stoichiometry, the mutations allow for better survival in an environment where
the high concentration of external Na+ would otherwise inhibit the function of “normal”
Na+/K+ pumps.
vi
INTRODUCTION
1. Biological/Physiological Roles and Structure of the Na+/K+ Pump
The Na+/K+ pump is a P-type ATPase integral membrane protein that is almost
ubiquitous in the animal kingdom (Jorgensen, Hakansson, and Karlish,, 2003) because it
is essential in maintaining the Na+ and K+ gradients across the plasma membranes in the
cell. These gradients are fundamental in many cellular processes: Na+-coupled secondary
transport of many different molecules such as H+, Ca2+, glucose, amino acids, and
neurotransmitters; the regulation of cell volume; maintenance of the resting potential and
excitability of both muscle and neuronal cells; and the ability to control osmotic activity
(Bottger et al., 2011). The Na+/K+ pump actively transports Na+ and K+ ions, using up
about 20-30% of the ATP produced by the body (Jorgensen, Hakansson, and Karlish,
2003).
Functional Na+/K+ pumps are heteromeric proteins composed of a catalytic alpha
subunit that binds the Na+ and K+ ions and ATP, a glycosylated beta subunit that affects
ion binding (Moseley et al., 2003), and, in some tissues, a FXYD protein that has
regulatory effects (Geering, 2005). The alpha subunit consists of ten transmembranespanning helices and cytoplasmic segments that contain both the N- and C-terminal. The
beta subunit has only one transmembrane segment and is responsible for the proper
targeting of the pump to the plasma membrane, for functional maturation (Song et al.
2006), and has an effect of the K+ affinity of the pump (Lutsenko and Kaplan, 1993).
In mammals, there are four different isoforms of the alpha subunit (α1-α4) and
three different beta subunits (β1-β3) that come together to form different isozymes in a
1
tissue specific manner. The presence of the α1 isoform can be found is nearly every cell
type: α2 is found in adipocytes, muscle, heart, and brain tissue; α3 is common in nerve
tissues; and α4 is exclusive to testis tissue. Similarly, the β1 isoform is also found in most
cell types, while β2 is common in skeletal muscle, the pineal gland, and nervous tissues,
and β3 is found in testis, the retina, liver, and the lung (Blanco and Mercer, 1998). Some
invertebrates, like Artemia franciscana, also present multiple genes encoding for different
Na+/K+ pump isoforms
Mutations within the genes encoding the Na+/K+ pump α subunit have been linked
to human diseases, such as familial hemiplegic migraine and rapid-onset dystonia
parkinsonism. The Na+/K+ pump is also a major target of cardiotonic steroids, such as
digitalis, that have been used for over 200 years in patients with congestive heart failure
(Sandtner et al., 2011).
2. P-Type ATPase Family
The P-type ATPase family consists of a large protein family, which includes
membrane pumps across many phyla. This family is present in virtually all living
organisms and is involved in the transport of many different materials, including metal
ions and lipids (Palmgren and Nissen, 2011). The name of the family is derived from the
fact that an acid-stable, phosphorylated aspartate (P) residue is formed during the
catalytic cycle of each member of this family. The P-type ATPase family is divided into
five different classes with the Na+/K+ pump being a P-type IIC (Kuhlbrandt, 2004).
Each P-type ATPase contains five different domains, three cytoplasmic and two
membrane-embedded. The cytoplasmic domains include the actuator domain, the
nucleotide-binding domain, and that phosphorylation domain. The membrane-embedded
2
domains are the transport and class specific support domains. The phosphorylation of the
aspartate residue is done by two of these residues: the nucleotide-binding domain acts as
a protein kinase to phosphorylate while the movement of the actuator domain
sequentially dephosphorylates the residue. The transport domain allows this process to
occur, as the movement of ions through this region is coupled with the chemical reactions
necessary for phosphorylation (Palmgren and Nissen, 2011).
3. Post Alber’s Cycle
Just like the other members of the P-type ATPase family, the Na+/K+ pump
alternates between two conformations, E1 and E2, each of which has a different affinity
for both the nucleotide and the transported ions (Jorgensen, Hakansson, and Karlish,
2003). Phosphorylation and dephosphorylation that occurs in response to ion binding and
movement drive the conformational changes in this cycle. Figure 1 depicts the
conformational changes the Na+/K+ pump undergoes and the different molecules that it
binds.
Figure 1. Post-Alber’s cycle: The cycle’s process is accompanied with a representation of
the Na+/K+ pump with ions and reactions represented (Yaragatupalli et al., 2009; Ratheal et
al., 2010).
3
In the E1 conformation the binding of three Na+ ions on the cytoplasmic side of
the pump triggers the phosphorylation of the aspartate residue, leading to a
conformational change into the E1P occluded state. The pump then opens to the
extracellular side of the membrane upon the departure of the ADP molecule. Once in the
E2 conformation, the pump releases the three Na+ ions to the space outside the cell.
Following release of the Na+ ions, it binds two extracellular K+ ions, triggering a rapid
dephosphorylation. Hydrolysis of the phosphate on the aspartic acid residue of the pump
leaves it in the E2 state with two K+ ions occluded. The binding of ATP brings the
Na+/K+ pump back to the E1 state, allowing it to open on the intracellular side of the cell
and release the K+ ions. Once the K+ ions are released, the cycle restarts (Jorgensen,
Hakansson, and Karlish,, 2003).
The Na+/K+ pump’s activity is electrogenic, producing a net current that can be
easily measured under voltage clamp conditions. In addition, because the release of the
Na+ ion to the extracellular side of the membrane is a voltage-dependent process, it is
also possible to use voltage clamp techniques to study the kinetics of the conformational
changes that the pump undergoes during its Na+-dependent partial reactions
(Yaragatupalli et al, 2009).
4. Artemia franciscana
When an organism resides in an environment with a salinity concentration higher
or lower than that of its bodily fluids, it must transport certain salts across its cellular
membranes to maintain an osmolality consistent with physiological levels. Membrane
transporters are almost always involved in this process, with the Na+/K+ pump being
among one of the most important in regards to saline transport (Sáez, Lozano, and
4
Zaldívar-Riverón, 2008). The brine shrimp (Artemia franciscana) are known for their
ability to live in up to 300 g/l of salt (Jorgensen and Amat, 2008), a concentration that is
nearing levels of saturation (Sáez, Lozano, and Zaldívar-Riverón, 2008) and can only be
tolerated by a few species of algae, bacteria, ciliates and arthropods (Jorgensen and Amat,
2008). The brine shrimp are known to have two different genes for the catalytic α subunit.
When the shrimp are exposed to high salinity, they express an α subunit with two
asparagine to lysine substitutions in the fourth and fifth transmembrane segments. This
region of the protein is otherwise extremely well conserved among a wide range of
animals, because it is involved in the recognition and binding of ions. The location of
these two asparagine residues within the crucial binding pockets of the Na+/K+ pump can
be seen in Figures 2 and 3.
TM5
N785
N333
TM4
I
+
K
II
+
K
Figure 2. E2 K+-Bound State: Enlarged view of ion binding sites in E2 with two K + bound (shown
as yellow spheres, Shinoda et al., 2009) indicating the distance between the two asparagine residues
substituted with lysine in this study and between N785 and the two K + ions in sites I & II.
5
TM5
N333
N785
III
+
Na
N78
+
Na
K
I
TM4
+
TM6
+
Na
II
Figure 3. E1 Na+-Bound State: Enlarged view of the ion binding sites in the E1 conformation with 3
Na+ ions bound (purple spheres. Kanai et al., 2013) showing the location of the two asparagine
residues substituted with lysine in this study and the distances to the Na+ ion binding sites.
These mutations were shown to only be prevalent in brine shrimp that reside in
environments with high salinity, and it was seen that the isoform of the α subunit that
contained these lysine substitutions had increased levels of expression when the external
levels of saline were increased, while the canonica α isoform of the pump was unaffected
(Jorgensen and Amat, 2008). Due to these observations and the fact that these mutations
were observed exclusively in osmoregulatory organs (gills) of the brine shrimp (Sáez,
Lozano, and Zaldívar-Riverón, 2008), it was hypothesized that the Na+/K+ pump plays a
major role in the regulatory processes behind maintaining physiological levels of
osmolality. Experiments led researchers to conclude that this double mutation could be
6
reducing the amount of positive ions bound and moved through each cycle of the Na+/K+
pump, thus leading to an overall reduction in affinity to each the Na+ and the K+ ions
(Jorgensen and Amat, 2008). In this work, we characterize the kinetics of the Na+/K+
pump with these asparagine to lysine mutations, both separately and together, in order to
shed light on the importance of this physiological adaptation from a mechanistic stand
point.
7
MATERIALS AND METHODS
1. Molecular Biology
A ouabain-resistant background was used as a template for mutagenesis for all
mutations mentioned in this work. The template was chosen because the double mutant
α1-Q120R/N131D has a lower ouabain sensitivity and thus allows for a distinction
between exogenous and endogenous pumps by the reversible inhibition of the mutant
exogenous pumps in the oocytes and the continuous inhibition of the endogenous pumps
created by a preincubation with ouabain (Ratheal et al., 2010).
Substitutions were introduced into the Xenopus alpha 1 subunit using
Quickchange site-directed mutagenesis (Stratagene). As per the Quickchange protocol,
PCR product was rid of template plasmid by treatment with DpnI for the duration of one
hour. 10 μL of the DpnI treated product is mixed with 100 μL XL-1 Blue competent cells
to begin bacterial transformation. The mixture is kept on ice for 25 minutes, heat shocked
at 42° C for 30 seconds, placed back on ice for two minutes, then mixed with 250 μL of a
pre-warmed solution of SOC for recovery. The mixture is then incubated at 37° C and
250 rpm for one hour before plating on an Ampicillin-LB agar plate. Bacterial growth is
allowed overnight at 37° C and a single colony is picked, grown in 5 mL of Terrific
Broth, and incubated at 37° C and 250 rpm overnight. After this incubation a mini prep
(Machery Nagel- Nucleospin plasmid kit) is performed to isolate DNA from the bacteria.
An automated sequencing analysis confirms the presence of the intended mutation and
the DNA is then linearized with BglII restriction enzyme. Linearization is confirmed by
8
gel electrophoresis and then purified with the Machery Nagel kit. cRNA was transcribed
using the mMessage machine SP6 kit (Ambion) (Artigas and Gadsby, 2006).
2. Oocyte Preparation
Oocytes used in this work were isolated surgically from Xenopus laevis. After
removal from the ovary lobes, oocytes were isolated enzymatically by incubation in Ca2+free OR2 solution. They are then washed three times for 30-minute intervals with Ca2+
OR2. The oocytes are then placed in SOS media and kept at 17° C.
Oocytes are injected with an equimolar mixture of the mutant α1 subunit and
Xenopus β3 cRNA. They are kept at in SOS media for two to six days to allow for protein
expression before measuring pump current (Yaragatupalli et al., 2009). One hour before
recording oocytes are incubated with a Na+-loading solution and ~10 μM of ouabain, as
less than 1 μM of ouabain is needed to completely block endogenous pumps (Horisberger,
2004).
3. Solutions
The Cl-free external solutions are made to contain 133 mM methane sulfonic acid
(MS), 10 mM Hepes, 5 mM Ba(OH)2, 1 mM Mg(OH)2, 0.5 mM Ca(OH)2, and 125 mM
of either N-methyl Glucamine (NMG+) or Na+. Solutions containing K+ are made from
adding differing amounts of a 3 M K-MS stock to the external NMG+ or Na+ solutions.
Mixing Na+ and NMG+ solutions created solutions with intermediate concentrations of
Na+ (Yaragatupalli et al., 2009).
Na+ loading solution consists of 90 mM Na-sulfamate, 20 mM Na-Hepes, 20 mM
tetraethylammonium chloride, and 0.2 mM ethylene glycol tetraacetic acid. NaOH was
used to adjust the Na+ loading solution to a final pH of 7.2. SOS media contains 100 mM
9
NaCl, 2 mM KCl, 1.8 mM CaCl2, 1 mM MgCl2, and 5 mM Hepes. SOS is also
supplemented with an anti-mycotic/antibiotic mixture (Anti-Anti; Gibco) and 1 X horse
serum (Sigma) (Ratheal et al., 2010). Ca2+-free OR2 solution is made with 82.5 mM
NaCl, 2 mM KCl, 1 mM MgCl2, and 5 mM Hepes (pH 7.4). For oocyte isolation, 0.5
mg/mL of collagenase Type IA (Sigma-Aldrich) was added to Ca2+-free OR2
(Yaragatupalli et al., 2009).
The Terrific Broth solution is made with 47 grams of Terrific Broth mixed with 4
mL of Glycerol. The solution is autoclaved for 15 minutes at 121° C and 2 mL of 100
mg/mL Ampicillin was added after the solution cooled.
4. Electrophysiology
Two-electrode voltage clamp (TEVC) recordings were performed with an OC725C amplifier (Warner Instruments), a Digidata 1440 A/D board, a Minidigi 1A, and
pClamp 10 software (Molecular Devices). Current microelectrodes had resistances of 0.51.2 MΩ and voltage microelectrodes had resistances of 0.5-2 MΩ. Microelectrodes were
filled with 3 M KCl (Ratheal et al., 2010). Oocytes were maintained at a -50 mV holding
potential and the signals obtained were filtered at 2 kHz and digitized at 10 kHz. Manual
valves were used to exchange solutions.
5. Analysis
Clampfit and Origin softwares were used to analyze results obtained by TEVC.
Na+ dependent charge analysis was seen by fitting the charge vs. voltage curves (Q-V
curves) with a Boltzmann distribution, Q = QMAX/(1 + exp((V-V1/2)/ks), where ks is the
slope factor related to the steepness of the curve, V1/2 is the half-maximal voltage and
center of the distribution, and QMAX is the total amount of charge moved.
10
Current vs. voltage (I-V) curves in the absence and presence of Na+ were fitted
with the Hill equation, I = IMAX[K]nH/(K0.5nH + [K+]nH), where IMAX is the maximal K+activated current, K0.5 shows the concentration where K+ is half-maximal, and nH is the
Hill coefficient that relates to the cooperativity of the two K+ sites (Yaragatupalli et al.,
2009).
11
RESULTS
1. Continuous Traces
Two-electrode voltage clamp was used to obtain readings of current over time for
each of the mutations described previously, both separately and concurrently. Continuous
traces of representative experiments are shown in Figure 4. These traces display the
changes in current that the mutant Na+/K+ pumps undergo in the different solutions
throughout each maneuver in an experiment.
Each experiment starts with an application of 3 mM K+. When an outward current
of more than 50 nA is observed in the K+ solution, the mutant Na+/K+ pumps are shown
to be expressing in the membrane of the oocyte. Experiments testing the Na+/K+ pump’s
affinity for K+ involved step-wise increases in K+ concentration with an application of
voltage pulses at each concentration of K+. The single mutant RD/N785K was observed
to reach its maximal outward current in higher concentrations of K+ than the RD/N333K
single mutant or the RD/N333K/N785K double mutant in the absence of Na+. K+-dose
responses were preformed in the absence (NMG solution) and presence (125 mM Na+
solution) of Na+ in order to determine if competition between the Na+ and K+ ions had an
effect on K+ affinity. The RD/N333K/N785K mutation experienced an outward current in
Na+ that was not observed in any of the other mutants.
12
A
B
C
Figure 4. Continuous Traces: Displayed are continuous two-electrode voltage clamp recordings of an
oocyte, held at -50 mV 3 days after injection of RD/N333K (A), RD/N785K (B), or RD/N333K/N785K (C)
cRNA. Vertical lines correspond to applications of brief (50 ms) pulses to different voltages to measure
voltage dependent parameters. Perfusion of the oocyte with 3 mM K + in NMG solution induced an outward
current (IP). Step increases in [K+] induced an increase in IP that saturated at high K+ concentrations. Note
that N785K required higher concentrations of K+ to activate IP, indicating a reduced apparent affinity. Doseeffect curves were constructed at each voltage and the apparent affinity K0.5 was obtained from the fits of a
Hill equation to the data.
13
2. K+-induced current (Kind) in the absence of Na+
Dose-effect curves were obtained from the two-electrode voltage clamp
experiments and fit with a Hill equation to obtain the apparent dissociation constant (K0.5)
of each mutant. The data for each mutant’s K0.5 as a function of voltage is shown in
Figure 5. Each of the mutations displayed a voltage-dependence similar to the RD
background mutant. RD/N785K showed a large reduction in affinity for external K+ in
the absence of Na+, as compared to the other three mutations.
3.
Figure 5. Kind in the Absence of Na+: Displayed are the apparent dissociation constants (K0.5) for
K+ without Na+o as a function of voltage for the RD background mutant (Mitchell et al., 2014),
RD/N333K, RD/N785K, and RD/N333K/N785K. Note that all curves present similar voltage
dependence (slope-wise) in the logarithmic scale, and that N785K has a reduced affinity for
external K+, compared to the other three.
Kind in the presence of Na+
14
K0.5 values were also obtained in the presence of sodium and can be seen in
Figure 6. The voltage dependencies of RD/N333K and RD/N333K/N785K were similar
to that of the RD background. RD/N785K displayed a voltage dependency inverse to that
of the RD background.
Figure 6. Kind in the Presence of Na+: Displayed are the apparent dissociation constants (K0.5) for
K+ with Na+o as a function of voltage for the RD background mutant, RD/N333K, RD/N785K, and
RD/N333K/N785K. The voltage dependency of RD/N785K was shown to be inverse to that of the
RD background mutant.
4. Charge-voltage curves (Q-V)
15
The changes induced in extracellular Na+ binding affinity by the mutations were
studied by measuring the transient currents elicited by voltage pulses in the presence of
Na+ without K+. The same pulse protocol was applied in 125mM Na+ solution without
ouabain and after addition of 10 mM ouabain. The Na+/K+ pump mediated transient
currents were obtained by subtracting the current in ouabain (Na+/K+ pump inhibited)
from the current in the absence of ouabain.
Figure 7 schematizes the conformational change that is being studied by this
method, where, due to the voltage dependence of external Na+ binding, the transient
currents are representations of the Na+ binding and release as the pump transitions from
the E1P with Na+-bound conformation to the Na+-free E2P conformation. Figure 8 shows
the transient current produced by the Na+/K+ pump when pulses from the holding
potential (-50 mV) to voltages ranging from -160 to +40 mV were applied in 40 mV
increments.
Figure 7. Voltage Dependence of the Na+/K+ pump: This diagram depicts the shuttling of the Na +/K+
pump from the Na+-bound E1P state at more negative voltages to the Na +-free E2P state at more
positive voltages.
16
Figure 8. Transient Currents: Ouabain-sensitive currents (current before ouabain minus current after
ouabain) in response to voltage pulses from -50 mV to voltages from -160 mV to +40 mV (40 mV
increments are displayed). Each trace was obtained in the absence of K+ with 125 mM external Na+.
These transient currents represent Na+ binding and release as the pumps transit between E1P with 3 Na +
bound at negative voltages to E2P, free of Na+ at positive potentials. Note that compared to the RD
background, all mutants, RD/N333K, RD/N785K and RD/N333K/N785K move more charge with
negative pulses. The current at the end of these pulses (OFF, indicated by an arrow) were integrated to
obtain the charge-voltage (Q-V) plots.
Charge was moved with more negative voltages in each of the brine shrimp
mutations (RD/N333K, RD/N785K, and RD/N333K/N785K) as compared to the RD
template. The transient current when the pulses are turned off was integrated to obtain the
Q-V plots. The data from each oocyte were fitted with a Boltzmann distribution and
normalized to the total charge to obtain the average Q-V plots shown in Figure 9.
17
Figure 9. Q-V Plots: Normalized QOFF-V plots from traces like those shown in Figure 8. The curves
were fitted with a Boltzmann distribution. Both the N333K mutant (blue triangles) and the N785K
(black squares) present curves shifted by approx. 100 mV to more negative voltages, consistent with a
reduced apparent affinity for Na+ (more energy is needed to push Na+ back into their binding pockets).
Each 20 mV represents a two-fold change in apparent affinity for external Na+ and thus each mutation
reduces affinity by more than ten-fold. The double mutant N333K/N785K (pink triangles) presents a
curve shifted to more negative voltages.
The center of the Q-V plots for each of the two single mutations (RD/N333K and
RD/N785K) both experienced a shift of about 80mV to more negative voltages from that
of the RD background; because every two-fold change in affinity shifts the curve around
20 mV, we conclude that around a 16-fold change in external Na+ affinity is induced by
each individual mutation. The RD/N333K/N785K mutant experienced a negative shift as
well, but to a lesser extent than the single mutants. This indicates that the two mutations
do not act independently.
18
5. Na+-induced current in the double mutant (Naind)
As mentioned before, the double mutant presents a ouabain-sensitive outward
current in the presence of extracellular Na+. To obtain the apparent affinity for this
uncommon mode of electrogenic Na+/Na+ exchange, the Na+-induced current
RD/N333K/N785K was gathered by first applying voltage pulses following step-wise
increases in Na+ concentration (5mM, 25mM, 50mM, then 125mM Na+), then by the
application of the same voltage pulses at the same Na+ concentrations in the presence of
10 mM ouabain. The steady state current at each concentration was then plotted as a
function of voltage, and can be seen in Figure 10.
Figure 10. Naind with RD/N333K/N785K: Ouabain-sensitive currents of RD/N333K/N785K plotted as
a function of current over voltage. At more positive voltages, higher concentrations induced more
current in the mutant pump, similar to wild-type isoforms of the Na+/K+ pump. At more negative
voltages, current increased with the first step-wise increase in concentration, and then began decreasing
with each subsequent increase in concentration.
19
The Na+ activated outward current of the RD/N333K/N785K mutation shares
characteristics with the K+-induced current, as at most voltages (above -50 mV)
increasing Na+ concentration increases outward current. At more negative potentials,
however, an initial increase is followed by inhibition. This is because the high Na+
concentration begins to produce voltage dependent inhibition by interaction of Na+ with
binding site III (Yaragatupalli et al, 2009).
20
DISCUSSION
1. K+ Affinity
Based on the studies performed by Jorgensen and Amat (2008), it was believed
that the N785K mutant decreased the binding affinity of ouabain by 17-fold, and the
N333K/N785K mutant decreased this binding by 26-fold (as compared to the wild-type).
In addition, they showed that the double mutant and each single mutant produced an
almost three-fold increase in their K0.5 for Tl+ binding (Tl binds to the pump like K+ with
much higher affinity), when compared with measured affinities of the wild-type
(Jorgensen and Amat, 2008). Our results supported parts of their data, especially with
respect to the RD/N785K mutant. The RD/N785K mutation displayed a ten-fold
reduction in apparent affinity for K+ when compared to the RD background. This can be
explained on the basis of recent structures that have been published of the Na+/K+ pump,
most notably the K+-bound state (Figure 2). The RD/N785K mutation is in very close
proximity to the ion-binding pocket and thus the K+ ion, which would cause the lysine
mutation to interfere with binding and reduce the Na+/K+ pump’s affinity for K+.
Our results differed from Jorgensen and Amat with the RD/N333K and the
RD/N333K/N785K mutants, both of which did not display the great reduction in affinity
that had been published previously. The RD/N333K mutant may not have this reduction
in affinity because of its greater distance from the ions in the E2 state of the Na+/K+ pump.
It is interesting that the RD/N333K/N785K mutation did not have an effect similar to that
of the RD/N785K mutant, as we had expected from published results. It is possible that
the N785K mutation is being coordinated to a different conformation when the N333K
21
mutation is present. This could cause N785K to point away from the ion-binding pocket,
thus not having an effect of K+ binding when the lysine on N333K is present. This
hypothesis is supported by data found by Jorgensen and Amat, stating that both the
N333K and N785K mutations reduced the free energy with which Tl+ ions could be
bound (5.5 kJ/mol and 13.5 kJ/mol, respectively), while the N333K/N785K mutant
favored the binding of Tl+ (-15.0 kJ/mol) even more than the wild-type. The two lysines
may be repelling one-another through an electrostatic interaction, causing the pump to
bind these ions differently and possibly changing the stoichiometry of the pump to only
bind one K+ ion (Jorgensen and Amat, 2008).
2. Na+ Affinity
Data published by Jorgensen and Amat showed a 15-fold increase in the K0.5 for
intracellular Na+ binding of the N333K mutant and a ten-fold increase in the K0.5 for both
N333K and N333K/N785K (Jorgensen and Amat, 2008). Our results were very similar to
this, in that both RD/N333K and RD/N785K experienced an 80mV shift in the centers of
their Q-V curves to more negative voltages, indicating a greater than ten-fold reduction in
affinity for external Na+. Interestingly, RD/N333K/N785K showed a lesser shift in the
center of its Q-V curve, something we had not expected and that differed from results
published by Jorgensen and Amat. A non-additive effect of the two mutations together
can be explained by the recent structure of the E1 Na+-bound state (Figure 3): when N333
is outside the ion-binding pocket, it forms a hydrogen bond with the ion-coordinating
N785 only in the Na+-bound conformation; therefore, once the disruption of normal Na+
coordination by N785K is in place, the mutation N333K does not affect Na+ binding. The
22
lessened reduction in affinity could possibly be explained by a change in stoichiometry of
the Na+/K+ pump or by transport of a Na+ ion for a Na+ ion.
3. RD/N333K/N785K
After observation of the outward current induced by Na+, a Na+-dose response
was conducted. The results show that Na+ is binding to the RD/N333K/N785K mutant
Na+/K+ pumps in a manner similar to how K+ binds. This finding could support the
hypothesis of a changed stoichiometry, as proposed by Jorgensen and Amat. They state
that the conditions of saline lakes in which brine shrimp reside increase the Na+ gradient
across the basolateral membrane, which then increases the amount of energy needed to
pump Na+ across the membrane. A change in stoichiometry, such as to one of two Na+
ions being moved per molecule of ATP, would be much more feasible under these
conditions (Jorgensen and Amat, 2008). Thus, the change in the behavior of the Na+/K+
pump we observed could be the main component in discovering the molecular
mechanism and basis behind this saline adaptation.
23
CONCLUSIONS
RD/N785K reduced the Na+/K+ pump’s apparent affinity for K+ ten-told and both
RD/N785K and RD/N333K reduced the pump’s affinity for external Na+ more than tenfold. However, when combined, RD/N333K/N785K did not experience a reduction its
apparent affinity for K+ and had a smaller reduction in its affinity for Na+. Due to the
presence of an outward current in Na+, it is likely that these mutant pumps are binding
Na+ as if it is K+. Future experiments will focus on determining the stoichiometry of the
pump and further investigating the binding of Na+.
24
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