69 / 119
Emerging Concepts in Ion Channel Biophysics
Poster Abstracts
64
73-POS
Board 73
Ca
2+
Permeability of Bacterial Sodium Channel Heterotetramers Can Explain the EEEE
Paradox in Voltage-gated Na
+
and Ca
2+
Channels.
Zeyu Zheng
, Olena Fedorenko, Stephen Roberts.
Lancaster University, Lancaster, United Kingdom.
The highly selective permeation of ions through voltage gated Na
+
and Ca
2+
channels can be
explained in terms of fixed negative charged in the pore region (
Q
f
). However, the
Q
f
associated
with bacterial voltage-gated Na
+
channels (bacNa
v
s) is reported to be equivalent to that found in
mammalian Ca
2+
selective channels (i.e. a pore region with an “EEEE motif”;
Q
f
= -4). This
anomaly could be explained if we consider the conserved aspartate residue located in domain II
(D
2
p
51
) of L-type Ca
2+
channels and defining the pore with an “EEEED motif” (i.e.
Q
f
= -5). To
investigate this hypothesis, we generated concatemers of bacNa
v
s to allow the asymmetrical
mutation of the selectivity filter (SF) and enable 1
e
step changes in the
Q
f
value associated with
the SF. Western blot analysis, immunofluorescence microscopy and patch clamp were employed
to evaluate the functionality of these concatenated bacNa
v
s and test the hypothesis that a
Q
f
value
of at least -5 is necessary for Ca
2+
permeation. This approach was corroborated with the use of
defined mixtures of cDNAs encoding for NaChBac mutants exhibiting different
Q
f
values.
Preliminary patch clamp investigations suggest that a |
Q
f
| ≥ 5 is necessary for Ca
2+
permeation,
consistent with the hypothesis that Ca
2+
permeation in L-type Ca
2+
channels is mediated by a
EEEED locus.
The work was supported by EPSRC (grant No.\ EP/M015831/1).