The Timothy syndrome mutation differentially affects
voltage- and calcium-dependent inactivation of
CaV1.2 L-type calcium channels
Curtis F. Barrett* and Richard W. Tsien†
Department of Molecular and Cellular Physiology, Stanford University School of Medicine, Stanford, CA 94305-5345
Contributed by Richard W. Tsien, November 7, 2007 (sent for review September 6, 2007)
Calcium entry into excitable cells is an important physiological signal,
supported by and highly sensitive to the activity of voltage-gated
Ca2?channels. After membrane depolarization, Ca2?channels first
open but then undergo various forms of negative feedback regula-
tion including voltage- and calcium-dependent inactivation (VDI and
a rare yet devastating disorder known as Timothy syndrome (TS),
whose features include autism or autism spectrum disorder along
with severe cardiac arrhythmia and developmental abnormalities.
Most cases of TS arise from a sporadic single nucleotide change that
generates a mutation (G406R) in the pore-forming subunit of the
L-type Ca2?channel CaV1.2. We found that the TS mutation power-
fully and selectively slows VDI while sparing or possibly speeding the
was further substantiated by measurements of Ca2?channel gating
currents and by analysis of another channel mutation (I1624A) that
hastens VDI, acting upstream of the step involving Gly406. As high-
an absorbing inactivation process. Thus, the TS mutation offers a
unique perspective on mechanisms of inactivation as well as a
promising starting point for exploring the underlying pathophysiol-
ogy of autism.
autism ? autism spectrum disorder ? channelopathy ? mutation ?
disorders, typified by impaired social interaction and commu-
nication skills and restricted and repetitive behavior. Despite
great interest in ASD, their etiology remains largely unknown.
However, genetic evidence supports the notion that the roots of
the pathology will ultimately be uncovered at the level of cellular
and developmental neurobiology and that insights into funda-
mental mechanisms may emerge from studies of rare forms of
the disease with simple genetic origin. Accordingly, increasing
attention has been directed toward Timothy syndrome (TS), a
rare childhood disorder whose manifestations include a very
strong association with autism or ASD (P ? 1.2 ? 10?8) along
with abnormally prolonged cardiac action potentials and a
wide-ranging set of developmental abnormalities. TS was iden-
tified in 1992 (1–3), but its likely importance as an exemplar only
came into sharp focus a dozen years later, when Splawski et al.
(4) showed that the diverse symptoms of TS could be traced in
most cases to a single amino acid defect in a single protein
molecule. The mutation (a Gly-to-Arg missense mutation at
position 406) was identified in a well known signaling molecule,
the pore-forming subunit of the class C (CaV1.2) L-type Ca2?
channel. Recently, a mouse model of TS bearing the Gly-to-Arg
mutation was reported to exhibit behavioral characteristics
reminiscent of ASD.‡That TS arises from a mutation in CaV1.2
is particularly instructive because this L-type Ca2?channel is
utism and autism spectrum disorders (ASD) are a contin-
uum of debilitating and mysterious neurodevelopmental
critically important for electrical activity, development, and
What is the functional impact of the mutation at the cellular
level? When introduced into rabbit recombinant CaV1.2 chan-
nels, the TS mutation produced no obvious changes in either the
voltage dependence of channel activation or the level of channel
expression (4). However, the mutation greatly impaired the
ability of the channels to stop conducting during depolarization,
a process known generically as inactivation. These results raised
a series of questions about the basic mechanism by which the
G406R mutation affects channel function. First, because inac-
tivation of voltage-gated Ca2?channels is strongly affected by
the identity of the auxiliary ? subunit, is the effect of the TS
mutation on L-type channels equally prominent regardless of
whether the ? subunit is typical of those found in either heart or
brain? Second, how strong are the effects of the TS mutation
when studied in combination with other amino acid changes that
themselves affect inactivation? Third, L-type channels display
multiple forms of negative feedback, including voltage-
dependent inactivation (VDI), which can be studied with Ba2?
(CDI), observed with Ca2?as the permeant ion (5–7). Given
proposals that VDI and CDI share the same final common
pathway (8, 9), do G406R and other mutations affect both forms
of inactivation in the same general way? Answers to these
questions would help clarify mechanisms of inactivation and also
provide useful clues for future explorations of the higher-order
effects of the TS mutation in its primary organ targets, including
the autistic brain.
The TS Mutation Slows Inactivation of CaV1.2 Irrespective of the
Coexpressed ? Subunit. We began by examining the effect of the
TS mutation in the context of various calcium channel accessory
subunits. The ? subunit subtype varies widely among tissues
affected by TS and profoundly influences Ca2?channel inacti-
vation (8, 10). For example, ?2(predominant in heart) confers
much slower inactivation than ?1(prominent in brain). Whereas
Splawski et al. (4) studied ?2b, we chose ?2a, also found in heart,
Author contributions: C.F.B. and R.W.T. designed research; C.F.B. performed research;
C.F.B. analyzed data; and C.F.B. and R.W.T. wrote the paper.
The authors declare no conflict of interest.
Freely available online through the PNAS open access option.
*Present address: Departments of Neurology and Human Genetics, Leiden University
Medical Center, Leiden, The Netherlands.
Beckman Center, Room B105, Stanford, CA 94305-5345. E-mail: email@example.com.
This article contains supporting information online at www.pnas.org/cgi/content/full/
‡Wersinger SR, Hesse RA, Badura MA, Bett GCL, Rasmussen RL, 2007 Society for Neuro-
science Annual Meeting, November 3–7, 2000, San Diego, CA, abstr. 62.6.
© 2008 by The National Academy of Sciences of the USA
February 12, 2008 ?
vol. 105 ?
no. 6 ?
Hepes, 10 mM EGTA, 5 mM MgCl2, 4 mM ATP, 0.4 mM GTP, pH 7.5. The bath
solution contained 155 mM NMDG-Asp, 0.1 mM EGTA, 10 mM Hepes, 10 mM
BaCl2or CaCl2, pH 7.4. Series resistance (8.95 ? 1.1 M?) was compensated
electronically by ?90%, and membrane capacitance (19.6 ? 0.9 pF) was
corrected online; where applicable, residual linear capacitive and leak cur-
rents were subtracted by the ?P/4 method.
EGFP-positive cells were visualized by epifluorescence and selected for
recording. Cells were voltage-clamped at ?90 mV, and pulse depolarizations
Bessel filter at 1–10 kHz, digitized at 5–100 kHz with a Digidata 1320A
(Molecular Devices), and stored on a personal computer.
Data Analysis. Data were acquired and analyzed with pClamp 8.2 (Molecular
Devices). Summary data are presented as mean ? SEM, and n ? 4–12 cells per
condition. Statistical significance was tested by using a two-tailed Student’s
unpaired t test, except where indicated.
The fits in Fig. 4 were calculated by using the equation:
I ? ??1 ? Afast? ? Afaste?t/?fast? ??1 ? Aslow? ? Aslowe ?t/?slow?
where I is normalized current amplitude, t is time in ms, Afastand Asloware the
fast and slow amplitudes, respectively, and ?fastand ?sloware the fast and slow
time constants, respectively.
ACKNOWLEDGEMENTS. We thank Roger Zu ¨hlke and Harald Reuter for pro-
viding the wild-type and I1624A cDNAs, Gerald Zamponi for providing acces-
sory subunit cDNAs, and Harald Reuter and Damian Wheeler for helpful
discussions. This work was supported by National Heart, Lung, and Blood
Training Grant 5T32HL007708-14 (to C.F.B.) and National Institutes of Health
Grants 5R01NS024067-22 and 5R01GM058234-08 (to R.W.T.).
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