Volatile General Anesthetic Interactions with a Bacterial Sodium Channel


136a Sunday, February 3, 2013
stabilized by surrounding hydrophobic residues. We propose that mutations of
the asparagines affect activation-inactivation of the channels and drug action
through destabilizing the open state. Diversity of H-bonding partners explains
different effects of mutations of individual asparagines on the channel activa-
tion, inactivation, and drug sensitivity. Supported by NSERC grant to BSZ, the
RAS program ‘‘Molecular and Cell Biology’’ to DBT, and CIHR award to IB.

695-Pos Board B464
Binding of Isoflurane to a Bacterial Voltage-Gated Sodium Channel:
Structure and Accessibility of Distinct Interaction Sites
Srinivasa G. Raju1, Vincenzo Carnevale1, David N. Lebard2,
Annika F. Barber3, Michael L. Klein1.
1Temple University, Philadelphia, PA, USA, 2Yeshiva University, New
York, NY, USA,

3
Thomas Jefferson University, Philadelphia, PA, USA.

The most likely targets for volatile anesthetics (VA) are ion channels. The
mechanism of volatile anesthetic action is not completely understood. Identify-
ing the molecular pathway for drug binding is crucial to understand the effect of
VA on voltage gated sodium channels. We use Molecular Dynamics simula-
tions to identify the binding sites for a hydrophobic general anesthetic isoflur-
ane, on voltage gated bacterial sodium channel NaChBac. Apart from the
voltage sensing domain (S1-S4), linker and the pore domain (S5-S6), bacterial
sodium channels also have fenestrations, which provide a hydrophobic tunnel
through the lipid-embedded portion of the channel to the central cavity, where
the known local anesthetic site is located. Unbiased ‘‘flooding’’ simulations
were performed on the activated open confirmation of NaChBac. We per-
formed a cluster analysis to identify all the possible binding sites of isoflurane.
The three most important ones among them are: a region near the selectivity
filter, called the extracellular site, a region near the S4-S5 linker, called the
linker site, and a region within the cavity, called the cavity site. The most
important observation is that isoflurane enters the central cavity through the
fenestrations. Free energy perturbation method was employed to calculate
the binding affinities of isoflurane for each of these sites. We also studied the
interactions between isoflurane and the amino acids in these three binding sites.

696-Pos Board B465
A Key Gating Charge Interaction Required for Slow Inactivation of the
Navab Bacterial Sodium Channel
Tamer M. Gamal El-Din, Todd Scheuer, William A. Catterall.
University of Washington, Seattle, WA, USA.
Bacterial voltage-gated sodium (Nav) channels are considered an ideal
model for structure-function studies. The elucidation of the crystal structure
of the bacterial channel NavAb (Payandeh et al., Nature 486, 135-139, 2011)
opened an avenue to understand electrical signaling in excitable cells at the
structural level. NavAb expressed in Hi5 insect cells as for structural studies
has unusually negative voltage-dependent activation (Va ~ 130 mV). NavAb
also has three phases of inactivation, a biphasic early inactivation process
(t1~103 ms, t2~3.8 s, at �180 mV) followed by an unusually strong and
use-dependent slow-inactivation process. To search for the molecular basis
for negative activation and slow inactivation of NavAb, we mutated the outer-
most gating charge partner in the S2 segment, Asn49, to Lys. This mutation
shifted the activation curve ~ 75 mV toward more positive potentials. Surpris-
ingly, it also completely abolished use-dependent slow inactivation. We
showed previously that the equivalent residue in NaChBac (Asp60) interacts
with the R3 gating charge in the S4 segment during activation using the disul-
fide locking method (DeCaen et al, PNAS 2008 105 (39) 15142-15147). To test
whether this interaction between R3 and N49 was critical for slow inactivation,
we mutated NavAb R3 to Cys. The resulting mutant NavAb_R3C also had
positively shifted channel activation (þ75 mV) and no use-dependent slow
inactivation. The fact that these reciprocal mutations have the same functional
effects suggests that interaction between R3 in the S4 segment and N49 in the
S2 segment is an important link that stabilizes the activated state of the voltage
sensor and triggers the slow inactivation process.

697-Pos Board B466
Computational Study of the Prokaryotic Sodium Channel
Karen M. Callahan, Benoit Roux.
University of Chicago, Chicago, IL, USA.
Ion-selective voltage-gated ion channels allow ions of a specific element or set
of elements to pass, but exclude other ions. Recently, the crystal structure of
NavAb was published (Payandeh et al (2011) Nature 475, 353), giving an un-
ambiguous picture of the structure of the sodium channel selectivity filter (SF)
of the prokaryote Arcobacter butzleri. The filter is much wider than that of the
narrow, carbonyl-lined potassium channels, allowing ions to penetrate with
minimal dehydration, multiple pathways, and even to pass each other. Previous
computational studies have attempted to explain sodium selectivity, and
claimed consensus (Corry and Thomas (2011) JACS 134, 1840, Furini and
Domene (2012) PLOS 8, e1002476). Experimental ion selectivity determina-
tion from pore-only constructs of a sodium channel with the same SF sequence
shows stronger ion selectivity from reversal potential than from the ratio of
the fluxes of sodium and potassium (Shaya et al (2011) PNAS 108, 12313),
but selectivity, Na

þ/Kþ, was still less than five. Simulations based upon the
closed-pore crystal structure could not determine selectivity directly from con-
duction. To investigate this issue, we first calculated multi-ion potentials of
mean force in the selectivity filter of pore-domain-only NavAb with an ex-
tended equilibration period, and a replica-exchange molecular dynamics
scheme, which allows for improved sampling. We then explored the non-
equilibrium conduction of sodium and potassium in the presence of an applied
electric field through a model NavAb pore truncated to mimic the conformation
of the open pore. We also present multi-ion pmf calculations to illustrate
similarities and differences between this model and the intact pore. When taken
together, these two methods provide complementary views of ion selectivity
and conduction based upon the conformation of the NavAb crystal structure
of Payandeh et al (2011).

698-Pos Board B467
Volatile General Anesthetic Interactions with a Bacterial Sodium Channel
Annika F. Barber1, Srinivas G. Raju2, David Lebard2, Vincenzo Carnevale2,
Michael L. Klein2, Manuel Covarrubias1.
1
Thomas Jefferson University, Philadelphia, PA, USA,

2
Temple University,

Philadelphia, PA, USA.
General anesthesia results from complex interactions involving ion channels in
the brain. Voltage-gated ion channels are modulated by halogenated inhaled
general anesthetics, however the drug-channel interactions are generally
believed to be non-specific making it difficult to investigate the molecular
mechanisms of anesthetic action. We find, however, that the bacterial sodium
channel NaChBac exhibits differential regulation by different general anes-
thetics, suggesting that there may be specific interactions. Molecular dynamics
(MD) simulations have identified several possible binding sites for isoflurane in
the NaChBac channel structure. Here, we have characterized the affinity, occu-
pancy and hydrogen bonding for these sites and identified key interactions. In
addition, experimental evidence suggests that both isoflurane and sevoflurane
have dual actions on NaChBac, possibly involving action at two different sites.
Consistent with pore block, sevoflurane accelerates the decay of the current
(20% faster at 0.4 mM sevoflurane) but, at the same time, increases the peak
current (10% increase with 0.4 mM sevoflurane), which argues against the
blocking mechanism. We hypothesize that an additional mechanism involving
a distinct site is necessary to explain the latter effect. We are currently employ-
ing MD simulation and structural modeling combined with mutagenesis and
electrophysiology to test this two-site hypothesis and investigate the molecular
mechanisms of these opposing effects. Supported by NINDS F31 NS077689
(AFB) and NIGMS P01 GM0558 (MLK).

699-Pos Board B468
Polymodal, High Affinity Actions of m-Conotoxins on a Bacterial Voltage-
Gated Sodium Channel
Rocio K. Finol-Urdaneta1, Robert Glavica1, Jeff R. McArthur2,
Robert J. French1.
1University of Calgary, Calgary, AB, Canada, 2RMIT, Melbourne, Australia.
m-Conotoxins (mCTXs) are potent blockers of eukaryotic sodium channels, and
individual members of the mCTX family are highly specific for particular Nav1
isoforms. We have begun to explore mCTX interactions with NaChBac, a pro-
karyotic Nav channel closely related to NavAb, whose crystal structure was re-
cently determined (Payandeh et al, 2011, Nature 475:353). Our data reveal
actions on both ion conduction and gating, consistent with polymodal actions
on the pore domain.
Under voltage clamp, whole-cell currents from NaChBac expressed in tsA-201
cells were reduced, up to nearly complete block, by concentrations in the pM to
mM range, for wildtype toxins and derivatives of mCTX PIIIA and KIIIA. For
wildtype PIIIA, dose-response data (0.001-10,000nM) yielded the following:
IC50=0.005nM; maximal fraction of current blocked = 0.95; Hill coefficient =
0.7. Chen & Chung (2012, Biophys. J. 102:483) predicted an IC50 of 0.1nM
for PIIIA when the NavAb channel is occupied by 2 sodium ions.
Even at very low [PIIIA] (1pM), the unblocked currents showed increasing
rates of inactivation as the peptides were washed in, suggesting a gating mod-
ulation, that was not tightly associated with pore block. For mCTX PIIIA
(0.1nM), or KIIIA (30nM), inactivation accelerated by ~10-fold. Substitution
of key basic residues (PIIIA-R14A and KIIIA-K7A) reduced blocking potency
and decreased the speeding of inactivation to less than 2-fold.
Given the remarkably high selectivity and affinity that mCTXs show for certain
eukaryotic Nav channels with highly asymmetric pores, it may seem surprising
that they bind to symmetric bacterial Nav channels with such high affinity.


	Binding of Isoflurane to a Bacterial Voltage-Gated Sodium Channel: Structure and Accessibility of Distinct Interaction Sites
	A Key Gating Charge Interaction Required for Slow Inactivation of the Navab Bacterial Sodium Channel
	Computational Study of the Prokaryotic Sodium Channel
	Volatile General Anesthetic Interactions with a Bacterial Sodium Channel
	Polymodal, High Affinity Actions of μ-Conotoxins on a Bacterial Voltage-Gated Sodium Channel