Mutations Linked to Autosomal Dominant Nocturnal Frontal
Lobe Epilepsy Affect Allosteric Ca
2+
Activation of the 42
Nicotinic Acetylcholine Receptor
□ S
Nivalda O. Rodrigues-Pinguet, Thierry J. Pinguet, Antonio Figl, Henry A. Lester, and
Bruce N. Cohen
Division of Biology, California Institute of Technology, Pasadena, California (N.O.R.-P., H.A.L., B.N.C.); Luxtera, Inc., Carlsbad,
California (T.J.P.); and Molecular Devices Corp., Union City, California (A.F.)
Received January 18, 2005; accepted May 17, 2005
ABSTRACT
Extracellular Ca
2+
robustly potentiates the acetylcholine re-
sponse of 42 nicotinic receptors. Rat orthologs of five
mutations linked to autosomal dominant nocturnal frontal
lobe epilepsy (ADNFLE)—4(S252F), 4(S256L), 4(+L264),
2(V262L), and 2(V262M)—reduced 2 mM Ca
2+
potentiation
of the 42 1 mM acetylcholine response by 55 to 74%. To
determine whether altered allosteric Ca
2+
activation or en-
hanced Ca
2+
block caused this reduction, we coexpressed the
rat ADNFLE mutations with an 4 N-terminal mutation,
4(E180Q), that abolished 42 allosteric Ca
2+
activation. In
each case, Ca
2+
inhibition of the double mutants was less than
that expected from a Ca
2+
blocking mechanism. In fact, the
effects of Ca
2+
on the ADNFLE mutations near the intracellular
end of the M2 region—4(S252F) and 4(S256L)—were con-
sistent with a straightforward allosteric mechanism. In contrast,
the effects of Ca
2+
on the ADNFLE mutations near the extra-
cellular end of the M2 region—4(+L264)2, 2(V262L), and
2(V262M)—were consistent with a mixed mechanism involv-
ing both altered allosteric activation and enhanced block. How-
ever, the effects of 2 mM Ca
2+
on the 42, 4(+L264)2, and
42(V262L) single-channel conductances, the effects of
membrane potential on the 2(V262L)-mediated reduction in
Ca
2+
potentiation, and the effects of eliminating the negative
charges in the extracellular ring on this reduction failed to
provide any direct evidence of mutant-enhanced Ca
2+
block.
Moreover, analyses of the 42, 4(S256L), and 4(+L264)
Ca
2+
concentration-potentiation relations suggested that the
ADNFLE mutations reduce Ca
2+
potentiation of the 42 ace-
tylcholine response by altering allosteric activation rather than
by enhancing block.
ADNFLE is a monogenic partial epilepsy linked to four 4
and two 2 nicotinic subunit mutations (Steinlein et al.,
1995, 1997; Hirose et al., 1999; De Fusco et al., 2000; Phillips
et al., 2001; Leniger et al., 2003). ADNFLE seizures occur
primarily during slow-wave sleep and seem to originate in
the frontal lobe (Scheffer et al., 1995). However, the physio-
logical mechanism that generates these seizures has not been
established.
At physiological concentrations (1.5–2 mM), Ca
2+
potenti-
ates the neuronal nicotinic agonist response by binding to an
N-terminal site in the receptor protein (Galzi et al., 1996; Le
Novere et al., 2002; Rodrigues-Pinguet et al., 2003). Alloste-
ric Ca
2+
activation of the neuronal nicotinic receptors is
conserved across species (rat, human, chicken) and across
nicotinic receptor subtypes (Mulle et al., 1992; Vernino et al.,
1992; Galzi et al., 1996; Steinlein et al., 1997; Liu and Berg,
1999). Adding 2 to 2.5 mM Ca
2+
to the extracellular saline
increases the wild-type 42 acetylcholine response by 300 to
500% (Steinlein et al., 1997; Figl et al., 1998; Rodrigues-
Pinguet et al., 2003). A common feature of the ADNFLE
mutations is that they reduce 2 to 2.5 mM Ca
2+
potentiation
of the 42 acetylcholine response by 50 to 72%, at acetyl-
choline concentrations 30 M (Steinlein et al., 1997; Figl et
al., 1998; Rodrigues-Pinguet et al., 2003). Because the extra-
cellular Ca
2+
concentration in the mammalian brain is nor-
mally 1.5 to 2 mM (Egelman and Montague, 1999), a decrease
in Ca
2+
potentiation of the 42 acetylcholine response could
contribute to ADNFLE seizures either by (1) reducing 42-
This work was supported by grants from the National Institute of Health
(NS0438000 and NS011756, to B.N.C. and H.A.L., respectively and a minority
supplement (NS0438000-04S1 to N.O.R.).
Portions of this work were published previously by UMI publishers in a
doctoral dissertation by N.O.R.
Article, publication date, and citation information can be found at
http://molpharm.aspetjournals.org.
doi:10.1124/mol.105.011155.
□ S The online version of this article (available at http://molpharm.
aspetjournals.org) contains supplemental material.
ABBREVIATIONS: ADNFLE, autosomal dominant nocturnal frontal lobe epilepsy; [Ca
2+
]
o
, added extracellular Ca
2+
concentration; i
s
, single-
channel current; g
s
, single-channel conductance; SSD, percentage of steady-state desensitization;
D
, time constant of desensitization.
0026-895X/05/6802-487–501$20.00
MOLECULAR PHARMACOLOGY Vol. 68, No. 2
Copyright © 2005 The American Society for Pharmacology and Experimental Therapeutics 11155/3044683
Mol Pharmacol 68:487–501, 2005 Printed in U.S.A.
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