Danielle Swadberg, Brock Roberts, Sameen Singh, Chelle Wheat

HYPERKALEMIC PERIODIC
PARALYSIS







Twenty years ago, a quarterhorse named
“Impressive” won all of the titles of his
class. He was the top-winning, top-
producing Quarter Horse stallion of all
time.

As a breeding stallion, he proved himself
equally deserving of his name, turning
out champion after champion. Many of
Impressive's offspring bore the same
dramatic physical stature as their sire -
they too went on to become outstanding
and prolific stallions and broodmares.

Of the top 15 halter horses in 1992, 13
were descendants of Impressive. Even at
the age of 23, Impressive himself was
fourth on the list. In 1993, it was
estimated that more than 55,000 Quarter
Horses, Paints, and Appaloosas bore his
pedigree.

From the American
Quarterhorse
Association





Soon his progeny were seen to be affected
by a strange muscular twitching that often
left them temporarily unable to move.

Usually mis-diagnosed as tying-up
syndrom or colic, these episodes varied
widely in degree and duration....but all had
one factor in common, their pedigree. As a
result, many people now know HYPP by
it„s more common name: Impressive
Syndrome.

This turned out to be a genetic mutation
that only recently has been implicated in
the rare but burgeoning - and sometimes
fatal - muscular disorder known as
hyperkalemic periodic paralysis.

This particular defect is a dominant
condition, meaning that at least half of
the affected horses' offspring will be
affected as well. In the words of one
prominent Quarter Horse trainer, this
discovery was "one of the most
devastating things that ever hit the horse
industry."
Hyperkalemic Period Paralysis or HYPP in horses is characterized by sporadic
attacks of muscle tremors, weakness and collapsing. These HYPP attacks can
also involve loud breathing noises from paralysis of the muscles of the upper
airway. Sudden death can occur in these horses from heart failure or respiratory
muscle paralysis. Using electromyography (EMG) to measure the electrical
activity present in certain muscles, the researchers discovered a wide variety of
abnormalities, including spontaneous activity from muscles under no
stimulation. Because these muscle tremors happen in other diseases besides
HYPP, those results were not enough evidence to give an accurate
diagnosis. Eventually, a mutation in a Na channel of the skeletal muscles was
found, which was the official diagnosis of HYPP.


The disorder involves attacks of muscle weakness or paralysis, alternating
with periods of normal muscle function. Attacks usually begin in early
childhood. Multiple daily attacks are not uncommon. Attacks typically last
only 1 to 2 hours, but can sometimes last as long as a day. They tend to occur
while resting after exercise or exertion.

Risks include a family history of periodic paralysis. Attacks may be triggered
by fasting. Attacks seldom occur during exercise but may be triggered by rest
following exercise.

Disorders that cause intermittent episodes of paralysis as their primary effect
are uncommon. More commonly, an intermittent episode of paralysis or
weakness is a symptom of another disorder. Hyperkalemic periodic paralysis
occurs in approximately 1 in every 100,000 people. Men are affected more
often than women and usually have more severe symptoms.


Humans also can have HPP (HyperKPP)
•Weakness/paralysis
•Most commonly located in the shoulders and hips
•Arms and legs may also be involved
•Occurs intermittently
•May occur on awakening
•May be triggered by rest after exercise
•May be triggered by fasting
•Usually lasts for less than 2 hours
•Spontaneous recovery of normal strength between attacks
•Normal alertness during attacks


Human Symptoms



HPP is caused by a flaw in a sodium channel in the muscle membrane.
This flaw makes the person with HyperKPP extremely sensitive to
increases in serum potassium that wouldn't bother the average person.
Anyone can be made weak by a drastic increase in serum potassium, but
the person with HyperKPP gets weak with even a slight elevation in
potassium level, and patients with HyperKPP may become profoundly
paralyzed while their potassium levels remain well within normal limits,
even when their potassium is on the lower end of normal.


The human defect is also in Na channels



Weakness most commonly affects the muscles of the arms and legs but may affect
the trunk as well. In a few patients the muscles involved in breathing can be
affected during severe episodes.

An irregular heartbeat can occur during episodes as well. Most patients have
normal muscle strength between attacks, but muscle tissue can be damaged by
attacks and this damage may eventually cause permanent weakness in some
patients once they reach their 50s and 60s.

During episodes of muscle weakness the normal flow of sodium ions is interrupted
affecting the ability of the cell to contract properly. The potassium level in the
blood may not rise during attacks, but many HyperKPP patients have a slightly
elevated potassium level between attacks.

The human defect is also in Na channels



A tablespoonful of Calcium Gluconate syrup stirred into a glass of Coca Cola or
other sweet beverage has proven an effective therapy which aborts mild episodes in
the early stages. Calcium Gluconate Syrup is a mineral supplement available off the
shelf and is found in most pharmacies.

For those who have frequent episodes and whose lives are compromised, more
aggressive treatment is advisable, especially since some patients with HyperKPP
may develop permanent muscle weakness after years of episodes.

The carbonic anhydrase inhibitor 'Diamox' (acetazolomide) is often prescribed for
HyperKPP patients. A similar drug called Daranide (diclorphenamide) is far more
potent, and often works on patients who respond poorly to Diamox, or on patients
who have been on Diamox many years and have become resistant to its effects.
About 25% of patients do not respond to Diamox and must be put on other drugs.


Treatment is to temporarily increase blood glucose
or calcium, or to use K-specific diuretics
Voltage Gated Na+ channels
Muscles are excitable cells
The Protein
• LLinker between
domains III and
IV
• SS4-S5 Linker
• SS6 segment
Purpose of this Study:
•EElucidate the Molecular
Biology of HyperKPP
csbn.concordia.ca/psyc358/ Lectures/voltgate.htm
Functional Processes of the Protein
• Activation
– This refers to the protein‟s ability to open its
activation gate (m gate) in response to
depolarizing changes in membrane potential
– This initiates the action potential when the
threshold potential is reached
– Studies revolve around quantifying the
potential where this response occurs

Functional Processes of the Protein
• Fast Inactivation
– This is the ability of the protein to close its
inactivation gate (h gate) in response to
depolarizing changes in membrane potential
– Occurs with a slight delay, so that activation
and fast inactivation work in tandem to produce
action potentials with short duration
– Studies are aimed at quantifying the degree of
depolarization required to close the h gate

Activation & Fast Inactivation

csbn.concordia.ca/psyc358/ Lectures/Nachannel.htm
Functional Processes of the Protein
• Slow Inactivation
– Refers to the protein‟s tendency to inactivate
after extended depolarization
– Involves unknown molecular mechanism
– The potential required to inactivate, as well as
the time required to recover from inactivation,
are quantifiable and of interest to researchers
Activation
25 msec
20 mV
10 mV
0 mV
-10 mV
-20 mV
-30 mV
-40 mV
-50 mV
-60 mV
-70 mV
-80 mV
-120 mV
-Depolarize to different potentials from a fixed holding potential
-Each depolarization results in a quantified current
-Current converted to conductance values and normalized
-Determines activation of channel as function of potential
Activation
• In molecular terms, this protocol is designed
to establish the potential that is required to
open the activation gate
• In effect, this determines the threshold
potential for this ion channel

Fast Inactivation
-50 mV
-60 mV
-70 mV
-80 mV
-90 mV
-100 mV
-110 mV
-120 mV
-40 mV
-30 mV
-20 mV
-10 mV
0 mV
200 msec
10 mV
20 mV
25 msec
-Depolarize to fixed potentials from varied holding potentials
-Each depolarization results in a quantified current
-Current normalized as a fraction of peak current
-Determines current as a function of holding potential
0 mV
Fast Inactivation
• This protocol is designed to determine the
degree of depolarization required to close
the inactivation gate
• If the holding potential is sufficiently
depolarized, the inactivation gate is closed
• Subsequent depolarization to a potential
where the activation gate is open generates
no current. Why?
• Answer: Inactivation gate is already closed
Slow Inactivation
50 sec
10 mV
-50 mV
-70 mV
-80 mV
-90 mV
-40 mV
-30 mV
-20 mV
-10 mV
0 mV
-60 mV
-100 mV
-110 mV
-120 mV
30 msec
20 msec
-Holding potential is varied, then hyperpolarized to a fixed
potential, then depolarized to a fixed test potential
-Varied potential is held for a much longer period of time than
other protocols, and all membrane changes are sequential
Slow Inactivation
50 sec
-130 mV
-100 mV
30 msec
20 msec
-10 mV
30 msec
20 msec
-10 mV
50 sec
Change in
holding
potential
-Emphasis on Sequential Potential Changes
Current measured
-100 mV
Slow Inactivation
• This protocol is designed to determine the
degree of depolarization necessary to
invoke slow inactivation
• Assumption: brief hyperpolarizations
designed to remove the effect of fast
inactivation have a negligible effect on slow
inactivation

Slow Inactivation Recovery
-20 mV
>15 min
-100 mV
50 sec 50 sec
-Part one: fast recovery
Increasing
recovery time
duration
50 sec
-10mV, 20 msec, current measured
-100 mV -100 mV -100 mV
Removes fast
inactivation,
allows recovery
Slow Inactivation Recovery

-20 mV
-100 mV
Part two: continuous slow recovery
50 sec
0.5 sec 5 sec 15 sec
-10mV, 20 msec, current measured
Slow Inactivation Recovery
• The slow inactivation recovery protocol monitors
the amount of time at hyperpolarized potentials
required for the slow inactivation gate to open
after extended depolarization closes it
• This is determined by monitoring the current
elicited by rapid depolarization as a function of the
amount of recovery time at hyperpolarized
potentials
RESULTS

Six different tests were conducted:

•Activation
•Fast inactivation
•Slow Inactivation
•Slow Inactivation Recovery
•Deactivation
•Persistent Current
ACTIVATION & FAST-INACTIVATION
Results:

•Slight hyperpolarizing
shift in activation curve

•No accountable change
in fast inactivation
Fast-inactivation Activation
Neurology 2002;58:p.1270
T704 MUTATION
Fast-inactivation Activation
Cannon, S.C. Neuromuscular
Disorders, 1997 p.244
SLOW INACTIVATION
Results:

•Slow inactivation does
not occur as easily in
the mutated channel
Neurology 2002;58:p.1271
SLOW INACTIVATION RECOVERY
Results:

•Slow inactivation
recovery is easier
in the mutated
channel than the
wild-type
Neurology 2002;58:p.1271
DEACTIVATION
Results:

•Deactivation was same
for mutated channel
and the wild-type
channel
Neurology 2002;58:p.1270
PERSISTENT CURRENT
Results:

•L689I mutation showed a slight current after 50ms
•Wild-type showed no persistent current
Neurology 2002;58:p.1269
SUMMARY OF RESULTS
1) Activation Slight
hyperpolarizing
shift in activation
curve
Mutated channel
can open at slightly
lower membrane
potentials
2) Fast Inactivation Not affected No accountable
difference
3) Slow
Inactivation
Less mutated
channels slow
inactivated
compared to wild-
type
Mutated channel
does inactivate as
easily as wild-type
Test Results Conclusion
4) Slow inactivation
recovery
Greater amount of
recovered mutated
channels than wild-
type
Mutated channels
can more easily
recover from slow
inactivation
5) Depolarization No difference
between mutated
channels and wild-
type channels
Depolarization is not
affected
6) Persistent
Current
Slight current
persists after 50ms
at –40mV, but not at
-10mV
Mutated channels
can open at slightly
lower membrane
potentials

Test Results Conclusion
SUMMARY OF RESULTS
(Cont’d)
•In previous studies on Hyperkalemic Periodic Paralysis
(hyperKPP) fast inactivation was thought to be the source of
symptoms.
•In fast inactivation, Na+ channels open quickly in response to
depolarization and then close quickly to a fast inactivated state
where openings are rare.
•Fast inactivation limits the duration of action potentials and
initiates repolarization of muscle fibers.
•This results in slower recovery and produces a refractory period in
which no action potentials are propagated
•It was also found in previous studies that a transversion (bases are
switched) mutation is present, which results in the substitution of a
conserved amino acid.
DISCUSSION AND CONCLUSION
•In this paper the researchers studied slow inactivation and the
mutation that disrupts the process.
•Slow inactivation occurs over seconds to minutes and affects the
availability of Na+ channels.
•In order to study and measure slow inactivation an experimental
procedure that includes prolonged depolarization is used.
•This depolarization allows slow inactivation to approach steady
state.
•Then the fraction of channels not slow inactivated is measured as
the current that recovers within 20 milliseconds at –100mV.
SLOW INACTIVATION
THE MUTATION
• The mutation found is in the SCN4A
gene which encodes the human
skeletal muscle sodium channel in
patients with hyperKPP.
• The mutation is a L689I mutation
and is close to the mutation I693T
(paramyotonia congenita) and the
frequent sodium channel mutation
(T704M).
• The amino acid that is located where
this mutation occurs is highly
conserved, which means that the
same amino acid is seen in many
different sodium channels, and that it
is important to the function.
Figure 1, pg. 1269
THE EXPERIMENT
•The experiment used the whole cell patch
clamp technique and looked at the
expression of the mutation in human embryo
kidney 293 cells.
•They found a small hyperpolarizing shift in
the activation curve, which resulted in the
overlap of the activation and inactivation
curves.
•An impairment of slow inactivation was
also discovered.
•This hyperpolarizing shift has also been
described for other related mutations in
similar diseases.
Figure 3A, pg. 1270
FAST
INACTIVATION
ACTIVATION
WINDOW CURRENTS
•Window current is the area of overlap between the
activation and inactivation curves.
•In this area, Na+ activation channels are beginning to open,
and inactivation channels are beginning to close.
•As the activation curve is shifted more to the hyperpolarized
region, both inactivation and activation gates are open at the
same time.
•When this occurs window current gets larger, due to the
large influx of Na+ charge down its concentration gradient.
RESULTS
•They also found a faster recovery
time for the mutants.
•The result is that because of the
overlap of the activation and
inactivation curves, the mutants
could produce a generation of
persistent currents which they found
to be at increased negative
potentials.
•They did not find a significant
change in fast inactivation that
could describe the overlap in curves.
Figure 4, pg. 1271
RESULTS
•The researchers found that approximately 25% of the L689I
mutants failed to slow inactivate after a long depolarization
(20 min).
•This is similar to the behavior of other mutants and in all cases
faster recovery from slow inactivation was observed, which
allows for more depolarization.
•The depolarization was found to prevent the generation of action
potentials even by direct galvanic shock, which is what causes the
episodic weakness (previous studies).
•The experimental data also shows that the linker II S4-S5 is one
of the molecular determinants of sodium channel slow
inactivation.

DISCUSSION
•Normally, in a cell, Na+ channels close at hyperpolarized potentials. When the cell is
depolarized the Na+ channels open briefly and then shut to an inactive state so that there
are no more openings.
•The membrane must then be hyperpolarized to restore the Na+ channels to a closed state
from which subsequent openings are elicited.
•In hyperKPP, Na+ channels fail to inactivate and prolonged openings and depolarization
result.
•The result is that persistent Na+ currents are witnessed, the Na+ current is closer to the
maximum, and Na+ diffuses down its gradient into the cell which results in a
depolarization and a more positive membrane potential.
•The current is larger because more Na+ is moving into the cell and the cell is
depolarizing.
•There is an overlap of the curves because the activation curve is more negative (lower
threshold, less voltage required).
•Increased extracellular K+ levels worsen the inactivation which in turn causes increased
weakness in the patient.
DISCUSSION
•Weakness in the patient is caused by the depolarization
that results from Na+ moving down its concentration
gradient and the positive membrane potential.
•The constantly depolarized membrane does not
hyperpolarize and thus can not remove inactivation from
the Na+ channels and initiate an AP.
•There still is a command from the motor neuron to
contract the muscle which causes an AP in the motor
neuron, and Ach is still secreted into the muscle cell, but
the muscle is already so depolarized that it does not fire
APs or contract.
•K+ causes the symptoms to worsen because it causes
depolarization itself. As the K+ concentration rises, the
resting potential increases and will open some of the
Na+ channels that do not inactivate in turn causing more
depolarization.
•It is a combination of these two things that make the
cell too depolarized to respond to inputs.
CONCLUSIONS
•The data shows that the hypothesis that hyperKPP does not require defective
fast inactivation is correct and that hyperKPP could be caused by the
hyperpolarizing shift in voltage dependence of activation in combination with
impairment of slow inactivation. The episodic weakness that results can thus
be attributed to a combination of these two defects.
•The mutation that was discovered is a transversion mutation that results in
the substitution of a highly conserved amino acid, showing its importance in
sodium channel function. Due to the fact that slow inactivation is implicated
as the cause of HyperKPP, and fast inactivation was proven to not play a role,
it can be deduced that because it is a different mutation and different process,
it must come from a different part of the molecule.
•This study is important because it illustrates how through different
functional expression, scientists can begin to discover the structure of a
molecule.


CONCLUSIONS
Some of the difficulties associated with this study include: It is hard
to read and the researchers do not explain why they choose certain
durations of time or measurements. It would be helpful if more
references to chosen values were included in the paper.
Lastly, it is exciting that now researchers can begin to learn about a
disease at the molecular level instead of solely through symptom
management. This gives hope that in the future, diseases such as
Hyperkalemic Periodic Paralysis can be cured.
SOURCES
Cannon, Stephen C. “From mutation to myotonia in sodium
channel disorders.” Neuromuscular Disorders. 7(1997):241-
249.
Cannon, Stephen C. “Ion-channel defects and aberrant
excitability in myotonia and periodic paralysis.” TINS. 19,
#1(1996):3-10.

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