From the Department of Pharmacology, University of Washington, Seattle, Washington 98195-7280
During inactivation of Na+ channels, the intracellular loop connecting domains III and IV is thought to fold into the channel protein and occlude the pore through interaction of the hydrophobic motif isoleucine-phenylalanine-methionine (IFM) with a receptor site. We have searched for amino acid residues flanking the IFM motif which may contribute to formation of molecular hinges that allow this motion of the inactivation gate. Site-directed mutagenesis of proline and glycine residues, which often are components of molecular hinges in proteins, revealed that G1484, G1485, P1512, P1514, and P1516 are required for normal fast inactivation. Mutations of these residues slow the time course of macroscopic inactivation. Single channel analysis of mutations G1484A, G1485A, and P1512A showed that the slowing of macroscopic inactivation is produced by increases in open duration and latency to first opening. These mutant channels also show a higher probability of entering a slow gating mode in which their inactivation is further impaired. The effects on gating transitions in the pathway to open Na+ channels indicate conformational coupling of activation to transitions in the inactivation gate. The results are consistent with the hypothesis that these glycine and proline residues contribute to hinge regions which allow movement of the inactivation gate during the inactivation process of Na+ channels.
Key words: mutagenesis; Xenopus oocyte; ion channel; ratVoltage-gated Na+ channels are responsible for initiation of action potentials in neurons and other excitable
cells. They are activated by depolarization and are inactivated within ~1 ms by closure of their inactivation
gate. Previous results (reviewed in Kellenberger et al.,
1997 in this issue) are consistent with the hypothesis
that the inactivation gate is formed by the intracellular loop connecting domains III and IV (LIII-IV) of the Na+
channel
subunit and that a hydrophobic motif (IFM,
isoleucine-phenylalanine-methionine)1 serves as a putative inactivation particle which binds to the intracellular mouth of the pore via hydrophobic interactions and blocks it (West et al., 1992
). Based on these studies it
was proposed that LIII-IV functions as a "hinged-lid"
which closes over the intracellular mouth of the pore
(Catterall, 1992
; West et al., 1992
; Eaholtz et al., 1994
).
By analogy with the structure and function of the
hinged lids of allosteric enzymes (Joseph et al., 1990
;
Wierenga et al., 1991
; Derewenda et al., 1992
), this
model implies that flexible regions on both sides of the
IFM motif act as hinges to allow IFM to fold into the
channel structure and bind to a putative inactivation
gate receptor in order to latch the inactivation gate in
the closed position. Consistent with this hypothesis,
previous experiments have provided evidence for movement of the inactivation gate during inactivation.
Modification of the inactivation gate by binding of a
site-directed antibody (Vassilev et al., 1988
; Vassilev et
al., 1989
) or by reaction of methanethiosulfonate derivatives with a cys in the position of the essential F1489 in
the IFM motif (Kellenberger et al., 1996
) is rapid when
the channel is in the resting state but not in the inactivated state, suggesting that the LIII-IV moves toward the
body of the channel upon inactivation and becomes inaccessible to both macromolecular and small cysteine-modifying reagents.
LIII-IV contains several pro and gly residues, amino acids that are components of molecular hinges in other
proteins. Gly residues confer flexibility to polypeptides,
whereas pro residues induce bends (Creighton, 1993).
Thus, gly and pro residues could be components of the
hinges in the hinged lid. In these experiments, we
tested this idea by analysis of the functional effects of
mutations of the pro and gly residues in the inactivation gate. Some of these mutations impaired inactivation by slowing the kinetics of the transition into the inactivated state from closed and open states. In addition,
these mutations affected steps in channel gating that
occur before channel opening and inactivation, consistent with a firm linkage between conformational
changes in LIII-IV and those involved in the voltage-
dependent channel activation process.
The experimental procedures used in this study are described in
Kellenberger et al. (1997) in this issue.
Effects of Mutations of Glycine Residues in the Inactivation Gate
To examine the functional role of gly residues in the inactivation gate, we mutated each gly in LIII-IV (SCHEME I) individually to ala and, in some cases, to val or pro. In addition the double mutation GG1484/5AA was made.
1480-KKKFGGQDIFMTEEQKKYYNAMKKLGSKKPQKPIPRPANKFQGMVF-1525
(SCHEME I)
The time course of inactivation of each mutant channel was analyzed using cell-attached macropatches and
compared to wild type (WT). Averaged current traces
from several experiments (n = 3-16) at four different
test potentials are displayed in Fig. 1. The values for the
time constant for macroscopic inactivation, h, at a
range of membrane potentials and the extent of steady-state inactivation as a function of membrane potential
are plotted in Fig. 2 for selected mutants. The membrane potentials at which half-maximal activation and
steady-state inactivation were observed are presented in
Table I, and mean and SEM values of
h at +30 mV are
indicated in Table II.
Table I. Voltage-dependent Gating Parameters of Macroscopic Currents from Mutant and WT Na+ Channels |
Table II. Inactivation Time Course in Cell-attached Macropatches at +30 mV |
Among the gly residues studied, the largest effects
were observed for substitutions of G1484 and G1485 toward the NH2-terminal end of LIII-IV. Both G1484A and
G1484V slowed macroscopic inactivation at potentials
positive to 30 mV (Figs. 1 and 2 A, Table II). G1485A
slowed macroscopic inactivation at all potentials tested (Figs. 1 and 2 A, Table II), and inactivation was incomplete (Fig. 1, Table I). In the macropatches studied
here, the fraction of sustained current at the end of 11-ms pulses was 8 ± 3% for G1485A in comparison to 2 ± 1% in WT. In contrast, mutation G1485V slowed macroscopic inactivation only slightly, although significantly (Fig. 1, Table II). The double mutant GG1484/
5AA slowed macroscopic inactivation to an extent similar to the single G1485A mutation (Fig. 1, Table II), but
there was no clear increase in effect for the double mutation. Moreover, the non-inactivating current associated with the G1485A single mutation was absent in the
double mutant (3 ± 1% noninactivating current, n = 5). Thus, the impairment of stability of the inactivated
state in the G1485A channel is compensated by the additional G1484A mutation.
Macroscopic inactivation was not affected by mutation G1505A, and was slightly slowed by G1505V (Fig. 1, Table II). However, it was slowed dramatically in mutant G1505P (Fig. 1, Table II). This mutation also caused slowed macroscopic activation. Mutation G1522A at the COOH-terminal end of LIII-IV did not have a significant effect on the inactivation time course.
The mutations of gly residues caused little change in the voltage dependence of activation (Table I). However, the midpoint of steady-state inactivation was shifted positively by mutations G1484A, G1484V, and GG1484/ 5AA by 7-14 mV (Fig. 2 B, Table I). Steady-state inactivation is primarily a measure of inactivation from closed states without prior channel opening (see DISCUSSION), so these effects suggest that the closed-to- inactivated transition is also impaired by these mutations. Recovery from inactivation after repolarization to negative membrane potentials was not significantly accelerated for these mutants, except for a small effect of G1522A (Table III).
Table III. Recovery from Inactivation in Two-electrode Voltage-clamp Experiments |
Effects of Mutations of Proline Residues in the Inactivation Gate
Each of the 4 pro residues in LIII-IV (SCHEME I) was mutated, one at a time, to ala, and in addition the triple
mutation PPP1512/4/6AAA was made. Of the single
mutations the largest effect on current time course was
observed in mutant P1512A. This mutation slowed inactivation markedly at all voltages (Figs. 3 and 4 A, Table II). However, the effect was greatest at potentials
negative to 0 mV, in contrast to the gly mutations for
which effects became stronger with increasing depolarization. Mutation P1512A had little effect on the fraction
of non-inactivating current (4 ± 1% in macropatches).
In addition to its effects on macroscopic inactivation, P1512A also slowed the time course of macroscopic activation (Fig. 3). This effect may be secondary to the
slowing of inactivation at negative membrane potentials in this mutant.
Mutation P1516A slowed inactivation at all potentials tested, but its effects were smaller than those of P1512A (Fig. 3). Mutation P1514A slowed macroscopic inactivation slightly but significantly (Fig. 3, Table II). Macroscopic inactivation of the triple mutant PPP1512/4/ 6AAA was not significantly different from the single mutation P1512A. In the mutant P1509A, macroscopic inactivation was faster than in WT at potentials negative to 0 mV and not significantly different at more positive potentials (Figs. 3 and 4 A). The different voltage dependence of macroscopic inactivation in these pro mutants is considered in the DISCUSSION.
The mutations of pro residues caused little change in the voltage dependence of activation (Table I). However, the midpoint of steady-state inactivation was shifted 10 and 8 mV positively by the mutations P1512A (Fig. 4 B, Table I) and PPP1512/4/6AAA (Table I), respectively, suggesting impairment of transitions from closed to inactivated states. Recovery from inactivation was slowed for mutants P1514A, P1516A, and PPP1512/ 4/6AAA, but not for the other pro mutants (Table III). The slowing of inactivation for mutants P1514A and P1516A occurred without effects on the voltage dependence of activation or steady-state inactivation.
Single Channel Gating of the G1484A, G1485A, and P1512A Mutants
Mutations of G1484, G1485, and P1512 had the most
pronounced effects on macroscopic inactivation during depolarizing pulses. Single channel current traces
and ensemble averages of mutants to ala of these channels at 20 mV are shown in Fig. 5. The ensemble averages (final sweep for each mutant, solid traces) were derived from all single channel traces analyzed for each
channel type. Their time course corresponds well with
the macropatch current traces recorded from the corresponding mutants (Figs. 1 and 3). Thus, the behavior
of these single channels is representative of the overall
behavior of the channels conducting the macroscopic currents we have measured.
In response to the majority of depolarizations these
mutant channels opened once or twice early in the depolarization and then inactivated (Fig. 5), similarly to
WT channels (see Kellenberger et al., 1997, in this issue). Sometimes, however, inactivation was delayed or
absent, and the single channels had a much higher probability of being open (Po) late in the pulse. The exact pattern of this high Po activity varied among mutants (see trace 3 for each mutant in Fig. 5). Because of
the unusually high Po during these depolarizations, relatively few sweeps of this type can potentially have pronounced effects on the macroscopic current. Therefore, to understand the single channel basis for macroscopic current, it was important to consider such high
Po sweeps separately and assess their contribution to
the overall behavior (e.g., Patlak and Ortiz, 1985
; 1986
;
Bennett et al., 1995
; Dumaine et al., 1996
).
Sweeps were separated into two data sets depending on whether they contained high Po behavior as indicated by bursts of activity after 8 ms of depolarization. The last traces in each panel of Fig. 5 compare ensemble averages of all the current traces analyzed (solid traces) to ensemble averages from which high Po traces have been omitted, which therefore represent the predominant gating behavior (dashed traces). The dotted traces are ensemble averages from WT channels for comparison. For each of the mutants macroscopic inactivation due to the predominant gating behavior (dashed traces) was slower than in WT (dotted traces). The most notable effect of including high Po sweeps was seen for mutant G1485A, where they produced a noninactivating component that was also present in the macroscopic currents for this mutant (Fig. 1). For G1484A and P1512A, the main effects of the mutations on the macroscopic current were observed in the traces with the predominant gating behavior but were increased in magnitude by inclusion of sweeps displaying high Po gating.
Because most aspects of macroscopic currents were
reproduced by channels displaying the predominant,
low Po gating behavior, the properties of single channels during depolarizations to 20 mV were analyzed
with high Po sweeps omitted (Fig. 6). The time constants of single exponential fits to open time histograms (compared to 0.39 ms for WT, see Kellenberger
et al., 1997
, in this issue) were 0.39 ms for G1484A and
0.59 ms for G1485A. A second component with a slower
time constant represented <10% of openings in these two mutants (Fig. 6 A). The open time distribution of
P1512A channels was best fitted with the sum of two exponentials with time constants 0.30 and 1.40 and approximately equal amplitudes (Fig. 6 A). Multiple open
times would reflect multiple open states (Correa and
Bezanilla, 1994
) or the presence of undetected high Po
behavior in our sample with a longer open time. The
increased open times in G1485A and P1512A are likely
to contribute to the slowed macroscopic inactivation in
these mutants whereas the slowed macroscopic inactivation in G1484A must be primarily due to other effects.
First latency distributions measure the probability of
entering the open state after a depolarization and
therefore reflect the rate of channel activation. Cumulative first latency distributions, corrected for the number of channels in the patch (Patlak and Horn, 1982;
Fig. 6 B) were well-fit by a delay followed by a single exponential time course. Fit parameters for maximal
open probability, time constant
, and delay were 0.57, 0.45, and 0.30 ms for G1484A, 0.65, 0.67, and 0.26 ms
for G1485A, and 0.64, 0.30, and 0.21 ms for P1512A,
compared to 0.54, 0.29, and 0.27 ms for WT. Thus, in
the G1484A, G1485A, and P1512A mutants, the total
probability of opening was slightly higher than in WT.
The time course of the first latency distribution was
faster than WT for the pro mutation, due to a decrease
in the lag before the exponential increase in channel
opening. In contrast, channel opening was clearly
slower in gly mutations, due to an increase in the time constant for first opening. This shows that the mutation
of P1512 makes the channel open more easily but mutations of G1484 and G1485 make opening more difficult. The slowing of the first latency is responsible for
much of the slowed macroscopic inactivation time
course observed in G1484A and adds to the effect of increased open times in G1485A channels causing further slowing of macroscopic inactivation.
Pro and Gly Mutations Increase the Probability of High Po Gating
As described above, activity in patches containing mutant channels was characterized by many sweeps with
prolonged bursts which were ended by inactivation or
by the end of the pulse. Depolarizing pulses with such
high activity were also observed for WT channels, but
far less frequently. To characterize the frequency of
such behavior quantitatively, we measured the probability that a channel was open between the 5th and
40th ms of each depolarization (Po(ms5-40)). Diaries of
this probability from representative WT (2 channels/ patch) and P1512A (3 channels/patch) patches are
shown in Fig. 7, A-C. WT channels opened only rarely
after 5 ms, and, when they did, the openings were short
(Fig. 7 A). For P1512A, the frequency of high Po activity
did vary with time during an experiment. High Po
openings were either rare, similar to WT (Fig. 7 B) or
occurred every few sweeps (Fig. 7 C). Once or twice
during each experiment of 200-2,000 depolarizing
pulses, channels switched between having rare high Po
depolarizations (Fig. 7 B) and having them occur more
frequently (Fig. 7 C). Diaries of G1484A and G1485A
channel activity were qualitatively similar to those
shown for P1512A. The high Po behavior occurred
most often in isolated sweeps which were preceded by
and followed by sweeps without bursts of activity. Therefore, if high Po behavior represents a different
mode of channel gating, the rate of leaving that mode
was similar to the depolarization rate of 1/s.
Most high Po sweeps contained single prolonged bursts of openings that were terminated by inactivation, after which the channel remained inactive (e.g., Fig. 5, trace 3 for each mutant). Thus, the relative magnitude of Po(ms 5-40) largely reflects the duration of activity before such a final inactivation event. Po(ms 5-40) was measured from more than 1,500 depolarizing pulses for WT and for each of the three mutants. The frequencies of sweeps with Po(ms 5-40) > 0.02 and Po(ms 5-40) > 0.2 were calculated for WT and for each of the three mutants and normalized to 1 channel/patch. P(Po (ms 5-40) > 0.02) (Fig. 7D) was more than seven times that of WT for G1484A, G1485A and P1512A. P(Po (ms 5-40) > 0.2) was 62 times greater than WT for G1484A, 184 times for G1485A, and 116 times for P1512A (Fig. 7 E). Thus, the probability of sustained activity is dramatically increased over WT in all mutants tested.
Comparison of the frequencies between the different
mutants supports the qualitative observation that high
Po burst duration in G1485A > P1512A G1484A. The
bursts in G1484A and P1512A generally terminated
with inactivation before the end of the pulse and led to
an additional slowing of the macroscopic inactivation
time course in ensemble averages (Fig. 5). Many of the
bursts in G1485A continued for the duration of the 40-ms long depolarization, producing sustained current in
addition to slowed macroscopic inactivation.
Five Gly and Pro Residues May Contribute to Movement of the Inactivation Gate
The hinged-lid model of Na+ channel inactivation predicts the presence of molecular hinges in the inactivation gate which allow it to close rapidly over the intracellular mouth of the open pore during the inactivation
process. Mutation of five of the eight gly and pro residues in LIII-IV significantly slowed macroscopic inactivation during strong depolarizations: G1484, G1485,
P1512, P1514, and P1516. Single channel analysis of
G1484A, G1485A, and P1512A showed that these mutant channels have increased open times (G1485A,
P1512A) and latency to first opening (G1484A, G1485A). In addition, these mutant channels had a higher frequency than WT of depolarizations with a sustained
high Po. The high Po behavior in each of these mutants
is most likely due to a further dramatic decrease in the
O I transition rate. High Po gating was reproduced by reducing the O
I transition rate in simulations of
SCHEME I of Kellenberger et al. (1997)
for multi-channel patches.
Gly and pro residues in LIII-IV are important in inherited diseases of human cardiac and skeletal muscle.
Naturally occurring mutations of the gly analogous to
G1484 in the skeletal muscle Na+ channel cause Na+
channel myotonia, a disorder characterized by an impairment of muscle relaxation (Lerche et al., 1993;
Mitrovi'c et al., 1995
; for review see Barchi, 1995
; Hoffman et al., 1995
; Cannon, 1996
). In the cardiac Na+
channel, deletion of a pro-containing triplet of amino
acids in LIII-IV (analogous to KPQ1508-10) causes a form
of long Q-T syndrome, a disorder which increases the
duration of ventricular action potentials and can cause
sudden death due to ventricular arrhythmias (Bennett
et al., 1995
; Wang et al., 1995a
, b
; Dumaine et al.,
1996
). These mutant channels show slowed macroscopic inactivation and have a more frequent occurrence of depolarizations with high Po activity.
Mutations of gly and pro residues in LIII-IV primarily
cause slowing of transitions which lead to inactivation
of Na+ channels. These results are in striking contrast
to the effects of mutations in the putative inactivation
particle, including the hydrophobic IFM motif and
T1491, which primarily destabilize the inactivated state
and accelerate the return from the inactivated state
(Kellenberger et al., 1997). Thus, these data argue for distinct roles of these two sets of amino acid residues in
the inactivation process. We propose that G1484, G1485,
P1512, P1514, and P1516 participate in the hinge motion that accompanies closure of the inactivation gate.
No single one of these amino acid residues is absolutely
essential for this closing motion because mutations of
these single residues cause only up to threefold increases in
h for macroscopic inactivation at depolarized potentials and up to 14-mV positive shifts in steady
state inactivation at negative membrane potentials. Evidently, multiple amino acid residues participate in the
molecular rearrangements which allow closure of the
inactivation gate and can compensate for single mutations of these gly and pro residues. Nevertheless, our
results support the concept that amino acid residues on
both sides of the inactivation particle participate in the
motion required for closure of the inactivation gate
and are consistent with a hinge motion for this process.
Voltage Dependence of Effects of Gly and Pro Mutations
Although the simple model of SCHEME I in the preceding paper (Kellenberger et al., 1997) is adequate for
description of Na+ channel behavior during strong depolarizations, it is not adequate for the negative potential range because it does not contain closed inactivated states. A more realistic model (SCHEME II, Stimers et al., 1985
) incorporates the possibility of inactivation
from all closed states. We will use this model to discuss
some of the effects of the mutations on channel gating
Scheme II.
A feature of most mutations which slow macroscopic
inactivation is that their effects are greater at more positive potentials. This behavior is expected for mutants
which affect the open to inactivated transition (O O-I
in SCHEME II), which is thought to be voltage-independent
(Armstrong and Bezanilla, 1977
; Armstrong, 1981
; Aldrich et al., 1983
). In WT neuronal Na+ channels, the
rate of activation is slower than the rate of entry into
the inactivated state for small depolarizations and
strongly influences the time course of macroscopic inactivation. The inactivation rate becomes rate limiting
at more positive potentials, where activation is faster
(Aldrich et al., 1983
; Aldrich and Stevens, 1987
). Inactivation of the mutant channels cannot be accelerated by
stronger depolarizations, because the rate-limiting
transition is voltage-independent. In contrast, the effects of the P1512A and P1509A mutations were strongest at small depolarizations (Fig. 4 A). This behavior
suggests that mutations P1509A and P1512A affect voltage-dependent transitions required for inactivation. Evidence for a voltage-dependent step in inactivation also
comes from studies of a mutation in the third transmembrane segment of domain IV of the human skeletal muscle Na+ channel
subunit (Ji et al., 1996
),
which slowed macroscopic inactivation with a similar
voltage dependence as P1512A, and from the observation that
-scorpion toxins and sea anemone toxins immobilize a component of gating charge and cause slowed
inactivation without pronounced effects on channel activation (Nonner, 1979
; Neumcke et al., 1985
; Hanck and
Sheets, 1995
; Sheets and Hanck, 1995
).
Effects on Gating Transitions in Closed Channels
The mutations G1484A, G1484V, and P1512A shifted
the voltage dependence of steady-state inactivation positively without affecting the rate of recovery from inactivation. Steady-state inactivation depends on rate constants for entry into and exit from inactivated states
(transitions Cn Cn-I and Cn-I
Cn in SCHEME II).
The behavior of these mutant channels is consistent
with a reduction in the Cn
Cn-I transition rates,
while leaving the reverse transition rates unchanged.
The mutations P1512A, G1484A, and G1485A affect
the fast component of first latencies, which represents
the time between depolarization and opening of the
channel. Thus, these mutations affect transitions C1 C2
. . .
C5
O in SCHEME II which occur before
the inactivation gate closes and binds to its receptor.
The simplest interpretation of the effects on transitions
in the activation pathway is that these mutations affect
conformational changes in LIII-IV which are required for
fast inactivation and also are coupled to movements of
the voltage sensors and therefore are rate limiting for
channel activation.
In the mutant YY1497/8QQ (Kellenberger et al.,
1997), steady-state inactivation was shifted positively by
6 mV, indicating a change in the equilibrium between
closed (Cn) and closed inactivated states (Cn-I). Recovery from inactivation was accelerated, indicating an increase in the rate for leaving inactivated states at negative potentials. The faster recovery of YY1497/8QQ mutant channels from inactivation indicates destabilization
of the closed/inactivated states (C-I in SCHEME II) or an
increase in the transition rates from the open/inactivated state back to closed/inactivated states (O-I
C5-I
. . .
C1-I) in response to repolarization. The mutation analogous to YY1497/8QQ in the human heart
Na+ channel causes complete loss of the voltage dependence of the time constant for macroscopic inactivation and strong acceleration of recovery from inactivation (O'Leary et al., 1995
), consistent with the conclusion that Y1497 and Y1498 are important for normal coupling of activation to inactivation in both cardiac
and brain Na+ channels.
A Model for Na+ Channel Inactivation
Current models of Na+ channel inactivation are based
on the model of Armstrong and Bezanilla (1977) which
proposed that depolarization causes activation gates to
move, ultimately creating a favorable binding site for a
tethered cytoplasmic inactivation particle that acts as
an open channel blocker. We propose the following updated version of this model. Immediately after onset
of a depolarizing pulse, the channel protein including
the inactivation gate LIII-IV undergoes conformational
transitions. Activation and inactivation are coupled
during transitions which precede channel opening, as
indicated by the effect of mutations of G1484, G1485,
F1489, T1491, and P1512 on first latency distributions.
These transitions lead progressively to opening of the
pore and creation of a favorable inactivation gate receptor site. Conformational changes in the inactivation
gate allow its rapid movement into a closed position and binding to the inactivation gate receptor. Two
steps can be distinguished which are required for channel inactivation but not for channel opening. The first
is a voltage-dependent transition made evident by mutations P1509A and P1512A in our study, by mutations in IVS3 which show a similar behavior (Ji et al., 1996
),
and by charge immobilization by
-scorpion and sea
anemone toxins (Sheets and Hanck, 1995
). The second step is the final voltage-independent closure and
binding of the inactivation gate to its receptor. This
voltage-independent step is affected by mutations of gly and pro residues which contribute to the hinge-like
motion of the gate. Once inactivated, the inactivation
gate is bound firmly to its receptor via hydrophobic interactions with F1489 in the IFM motif and additional
interactions with T1491 (West et al., 1992
; Kellenberger et al., 1997
). Although this "hinged-lid" model for inactivation is fully consistent with extensive electrophysiological and molecular biological results, definitive
proof of this mechanism of inactivation will require structural analysis of resting and inactivated Na+ channels.
Original version received 15 October 1996 and accepted version received 25 February 1997.
Address correspondence to Dr. Todd Scheuer, Department of Pharmacology, Box 357280, University of Washington, Seattle WA 98195-7280. Fax: 206-543-3882; E-mail: scheuer{at}u.washington.edu
This research was supported by National Institutes of Health Research Grant NS 15751 to W.A. Catterall and postdoctoral fellowships from the Swiss National Science Foundation and the Schweizerische Stiftung für medizinisch-biologische Stipendien to S. Kellenberger.