Benke, Paul J.

Dib-Hajj, Sulayman D.

Drenth, Joost P.H.

Eastman, Emmanuella M.

Estacion, Mark

Macala, Lawrence J.

te Morsche, R.H.M.

Waxman, Stephen G.

Elsevier - Publisher Connector 

NaV1.7 Gain-of-function Mutations As A Continuum: A1632E Displays Physiological Changes Associated With Erythromelalgia And Paroxysmal Extreme Pain Disorder Mutations And Produces Symptoms Of Both Disorders 

12a Sunday, March 1, 200958-Plat
CaMKII Regulates L-type Ca2þ Current during Action Potential Plateau
and Repolarization _ a Direct Link to Arrhythmias
Tamas Banyasz1,2, Leighton T. Izu1,3, Ye Chen-Izu1,3.
1University of Kentucky, Lexington, KY, USA, 2Univ. of Debrecen,
Debrecen, Hungary, 3University of California at Davis, Davis, CA, USA.
During the cardiac action potential (AP), the L-type Ca2þ current (ICa,L) is the
main source of Ca2þ entry into the cell that triggers excitation-contraction cou-
pling, and it is also the main inward current that contributes to the depolariza-
tion during AP plateau and later phases. Derangement of ICa,L has been pro-
posed to cause early or delayed afterdepolairzation (EAD, DAD) that lead to
arrhythmias. Previous studies have characterized the steady state ICa,L activa-
tion, inactivation and recovery kinetics. However, the non-steady state dynam-
ics of ICa,L during AP still remains incompletely understood. Here we study the
dynamics of ICa,L during the cell’s own AP under physiological condition.
Method: Unlike previous studies using an averaged AP waveform, we used
each cell’s own AP to perform individualized AP-clamp (iAP-clamp) study of
ICa,L. This allows us to examine the dynamics of ICa,L during AP with physiolog-
ical ionic compositions.Results: (1) ICa,L took the form of a spike at AP phase-1,
followed by a dome at phase-2 . Now we report a newly observed inward tail
current at phases-3&4 which is composed of ICa,L and Na
þ/Ca2þ exchange
current. This inward tail current is important because it causes depolarization
and directly links ICa,L to the generation of EAD or DAD. (2) Furthermore,
Ca2þ-calmodulin dependent kinase II (CaMKII) modulates this tail current;
inhibition of CaMKII abolished the tail current. (3) The tail current was
also abolished by using exogenous Ca2þ buffer to blunt SR Ca2þ release
and prevent activation of CaMKII. Conclusion: The iAP-clamp reveals, for
the first time, an inward ICa,L associated tail current at the late phases of
AP. CaMKII modulation of this tail current should play a critically important
role in generating the afterdepolarizations that lead to cardiac arrhythmias.
59-Plat
Simultaneous Recordings of Cell Shortening and cAMP or Calcium Tran-
sients Reveal Differential Regulation of Cardiac Contractility by Specific
Phosphodiesterases
Delphine Mika, Je´ro˜me Leroy, Patrick Lecheˆne,
Gre´goire Vandecasteele, Rodolphe Fischmeister.
INSERM U769, Univ Paris-Sud 11, Fac Pharmacy, Chaˆtenay-Malabry,
France.
Multiple cyclic nucleotide phosphodiesterases (PDEs) belonging to four fami-
lies (PDE1 to PDE4) hydrolyze cAMP in cardiac cells, but the functional sig-
nificance of this diversity is not well understood. The goal of this study was to
characterize the involvement of different PDEs in excitation-contraction cou-
pling in cardiomyocytes. For this, sarcomere shortening and Ca2þ transients
were recorded simultaneously in rat ventricular myocytes field stimulated at
0.5 Hz with an IonOptix system. As shown in the figure, selective inhibition
of PDE2 with Bay 60-7550 (Bay, 100 nM) or PDE4 with Ro-201724 (Ro, 10
mM) had no effect on basal cell contraction, whereas selective inhibition of
PDE3 with cilostamide (Cil, 1 mM) or b-adrenergic stimulation with isoprena-
line (Iso, 1 nM) increased myocyte shortening. Inhibition of PDE4 potentiated
the response to Cil and Iso, showing that PDE4 becomes important when cAMP
is prestimulated. Similar results were obtained on Ca2þ transients. cAMP mea-
surements by FRET in beating cardiomyocytes indicate that Iso strongly in-
creases cAMP levels. Effects of selective PDE inhibitors are under investiga-
tion. These results show that PDE2, PDE3 and PDE4 differentially regulate
excitation-contraction coupling in cardiomyocytes.Platform F: Voltage-gated Na Channels
60-Plat
How Myasthenia Gravis Alters the Safety Factor for Neuromuscular
Transmission
Robert L. Ruff1, Vanda A. Lennon2.
1Cleveland VA Med Ctr, Cleveland, OH, USA, 2Mayo Clinic, Rochester,
MN, USA.
Myasthenia gravis (MG), the best understood autoimmune disease, is charac-
terized by antibodies directed against the skeletal muscle acetylcholine recep-
tors (AChRs). An elevated concentration of Naþ channels at the endplate en-
sures the efficiency of neuromuscular transmission by reducing the threshold
depolarization needed to trigger an action potential (EAP). Postsynaptic AChRs
and voltage-gated Naþ channels are lost from the neuromuscular junction in
MG. We studied the impact of postsynaptic voltage-gated Naþ channel loss
on the safety factor for neuromuscular transmission (SF). Comparing intercos-
tal nerve-muscle preparations from controls and MG patients, endplate AChR
loss decreased the size of the endplate potential (EPP) from 40.251.3 mV to
23.5 51.7 mV (p < 0.001). Endplate Naþ channel loss in patients with MG
increased EAP from 71.9 52.2 mV to 62.3 52.7 mV (p < 0.001). The SF
was reduced from 2.98 to 1.09. If the EPP was the same as control, the SF
for MG fibers would have been 1.86 rather than the observed value of 1.09.
If EAP for MG fibers were the same as control fibers, the SF for MG fibers
would have been 1.74. The reduction in EPP accounted for 59% of the reduc-
tion in SF and the increase in EAP produced 40% of the SF reduction. To eval-
uate whether AChR-specific antibody impaired the function of Naþ channels,
we tested omohyoid nerve-muscle preparations from rats injected with mono-
clonal myasthenogenic IgG (passive transfer model of MG [PTMG]). AChR
antibody that produced PTMG did not alter the function of Naþ channels.
We conclude that loss of endplate Naþ channels in MG is due to comple-
ment-mediated loss of endplate membrane rather than a direct effect of myas-
thenogenic antibodies on Naþ channels. Loss of endplate Naþ channels caused
40% of the SF reduction in MG.61-Plat
NaV1.7 Gain-of-function Mutations As A Continuum: A1632E Displays
Physiological Changes Associated With Erythromelalgia And Paroxysmal
Extreme Pain Disorder Mutations And Produces Symptoms Of Both
Disorders
Mark Estacion1,2, Sulayman D. Dib-Hajj1,2, Paul J. Benke3,
R.H.M. te Morsche4, Emmanuella M. Eastman1,2, Lawrence J. Macala1,2,
Joost P.H. Drenth4, Stephen G. Waxman1,2.
1VA CT Healthcare System, West Haven, CT, USA, 2Dept. of Neurology,
Yale University, New Haven, CT, USA, 3Dept of Genetics, Joe DiMaggio
Children’s Hospital, Hollywood, FL, USA, 4Dept. of Gastroenterology and
Hepatology, Radboud University, Nijmegen, Netherlands.
Gain-of-function mutations of NaV1.7 have been shown to produce two distinct
disorders: NaV1.7 mutations which enhance activation produce inherited eryth-
romelalgia (IEM), characterized by burning pain in the extremities; NaV1.7
mutations which impair inactivation produce a different, non-overlapping syn-
drome, paroxysmal extreme pain disorder (PEPD), characterized by rectal,
periocular and perimandibular pain. Here we report a novel NaV1.7 mutation
associated with a mixed clinical phenotype with characteristics of IEM and
PEPD, with an alanine 1632 substitution by glutamate (A1632E) in domain
IV S4-S5 linker. Patch-clamp analysis shows that A1632E produces changes
in channel function seen in both IEM and PEPD mutations: A1632E hyperpo-
larizes (-7 mV) the voltage-dependence of activation, slows deactivation, and
enhances ramp responses, as observed in NaV1.7 mutations that produce
IEM. A1632E depolarizes (þ17mV) the voltage-dependence of fast-inactiva-
tion, slows fast-inactivation and prevents full inactivation, resulting in persis-
tent inward currents similar to PEPD mutations. Using current-clamp, we
show that A1632E renders dorsal root ganglion (DRG) and trigeminal ganglion
neurons hyperexcitable. These results demonstrate a NaV1.7 mutant with bio-
physical characteristics common to PEPD (impaired fast-inactivation) and
IEM (hyperpolarized activation, slow deactivation and enhanced ramp cur-
rents) associated with a clinical phenotype with characteristics of both IEM
and PEPD, and show that this mutation renders DRG and trigeminal ganglion
neurons hyperexcitable. These observations indicate that IEM and PEPD mu-
tants are part of a physiological continuum that can produce a continuum of
clinical phenotypes.


Estacion, Mark

Dib-Hajj, Sulayman D.

Benke, Paul J.

te Morsche, R.H.M.

Eastman, Emmanuella M.

Macala, Lawrence J.

Drenth, Joost P.H.

Waxman, Stephen G.

University of Miami: Scholarship Miami

Biophysical Journal

NaV1.7 Gain-of-function Mutations As A Continuum: A1632E Displays Physiological Changes Associated With Erythromelalgia And Paroxysmal Extreme Pain Disorder Mutations And Produces Symptoms Of Both Disorders

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NaV1.7 Gain-of-function Mutations As A Continuum: A1632E Displays Physiological Changes Associated With Erythromelalgia And Paroxysmal Extreme Pain Disorder Mutations And Produces Symptoms Of Both Disorders

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