From the Institute of Molecular Cardiobiology, The Johns Hopkins University School of Medicine, Baltimore, Maryland 21205
Received for publication, October 28, 2002, and in revised form, January 22, 2003
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ABSTRACT |
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If, encoded by the
hyperpolarization-activated cyclic
nucleotide-modulated (HCN) channel family, is a key player in cardiac and neuronal pacing. Although HCN channels structurally resemble voltage-gated K+ (Kv) channels, their
structure-function correlation is much less clear. Here we probed the
functional importance of the HCN1 S3-S4 linker by multiple
substitutions of its residues. Neutralizing Glu235, an
acidic S3-S4 linker residue conserved in all
hyperpolarization-activated channels, by Ala substitution produced a
depolarizing activation shift (V1/2 = If or Ih, encoded by the
hyperpolarization-activated cyclic
nucleotide-modulated
(HCN)1 or the so-called
pacemaker channel gene family, is a key contributor to spontaneous
rhythmic activity in cardiac and neuronal cells (1-9). Although HCN
channels structurally resemble voltage-gated K+ (Kv)
channels (for instance, both are tetramers made up of monomeric subunits consisting of six membrane-spanning segments) (7-10), a
distinguishing functional feature that discriminates pacemaker channels
from the Kv counterparts is their signature "backward" gating
(i.e. activation upon hyperpolarization rather than
depolarization). The molecular basis of this unique gating phenotype is
unknown. Recently, it has been suggested that the voltage-sensing
mechanism of the sea urchin sperm HCN (i.e. SPIH or spHCN)
and Kv channels may be conserved (11). In any case, the
structure-function correlation of HCN channels is much less defined
compared with the well studied Kv channels. Comparison of these related
yet functionally distinct ion channels should provide important
insights into the unique behavior of HCN channels.
Previous studies of Kv channels have demonstrated that the S3-S4 linker
influences activation gating (12-15). By analogy to Kv channels, it is
possible that the S3-S4 linker (defined as residues 229-237 here, HCN1
numbering) of HCN channels also contributes to activation gating.
However, this idea has not been tested. In this study, we probed the
functional importance of the S3-S4 linker of HCN1 channels by multiple
substitutions of its residues. We found that the acidic linker residue
Glu235, conserved among all known
hyperpolarization-activated channels (Fig.
1), prominently influences HCN gating.
Glu235 is also largely responsible for the charge-shielding
effects of external Mg2+. Novel insights into the
structural and functional roles of the S3-S4 linker of HCN channels are
discussed. A preliminary report has appeared (16).
Molecular Biology and Heterologous Expression--
Mouse
HCN1 (kindly provided by Drs. S. A. Siegelbaum and B. Santoro) was
subcloned into the pGHE expression vector (9). Mutations were created
using PCR with overlapping mutagenic primers. The desired mutations
were confirmed by DNA sequencing. cRNA was transcribed from
NheI-linearized DNA using T7 RNA polymerase (Promega, Madison, WI). HCN1 channel constructs were heterologously expressed and
studied in Xenopus oocytes. Briefly, stage IV-VI oocytes
were surgically removed from female frogs anesthetized by immersion in
0.3% 3-aminobenzoic acid ethyl ester, followed by digestion with 1 mg/ml collagenase (type IA) in OR-2 containing 88 mM
NaCl, 2 mM KCl, 1 mM MgCl2, and 5 mM HEPES (pH 7.6) for 30-60 min. Isolated oocytes were
injected with cRNA (50 or 100 ng/cell) and stored in 96 mM
NaCl, 2 mM KCl, 1.8 mM CaCl2, 1 mM MgCl2, and 5 mM HEPES (pH 7.6)
supplemented with 50 µg/ml gentamycin, 5 mM pyruvate, and
0.5 mM theophylline.
Electrophysiology, Experimental Protocols, and Data
Analysis--
Two-electrode voltage-clamp recordings were performed at
room temperature (23-25 °C) using a Warner OC-725B amplifier 1-2 days after cRNA injection as described (10). Because Xenopus oocytes are essentially a mass of yolk surrounded by an outer fenestrated membrane, membrane potential could be accurately measured only to within approximately ±2 mV. Furthermore, it was assumed that
ion conductance had reached steady-state under our experimental conditions. The regular recording bath solution contained 97.8 mM KCl, 2 mM NaCl, 10 mM HEPES, and
1 mM MgCl2 (pH 7.5). The MgCl2 concentration was increased in certain experiments as indicated.
The voltage dependence of HCN channel activation was assessed by
plotting tail currents measured immediately after pulsing to
Changes in free energy (
For simplification of kinetic analysis, the time constants for
activation ( Statistics--
Data are presented as mean ± S.E.
Statistical significance was determined using unpaired Student's
t test with p < 0.05 representing significance.
The S3-S4 Linker Residue Glu235 Influences HCN1
Activation Gating--
We first characterized wild-type (WT) HCN1
channels by examining their activation gating properties. Stepping the
transmembrane potential to voltages below
To investigate the functional roles of the S3-S4 linker in HCN1
activation, we first mutated Glu235, an acidic residue
conserved in all hyperpolarization-activated channels (cf.
Fig. 1), to Ala and Arg for net charge changes of +1 and +2 at this
channel site, respectively. The charge-neutralized substitution E235A
produced a significant depolarizing shift in steady-state channel
activation without altering the slope factor (Fig.
3C; see also Fig. 4).
Interestingly, the charge-reversed mutation E235R shifted activation
even more positively (Fig. 3D), highly suggestive of an
electrostatic role of residue 235. Similar to E235A, the slope factor
of E235R channels was also not altered (Figs. 3D and
4C).
Glu235 Influences the Charge-screening Effects of
Mg2+ on HCN1 Activation--
Because HCN channels are not
embedded in the membrane in an orientation opposite to that of
depolarization-activated channels (17), the S3-S4 linker should be on
the extracellular side. We hypothesized that if Glu235 is
externally accessible and its effects on channel activation are
electrostatic, screening surface charges should effectively shield its
anionic charge and thereby produce activation shifts similar to those
observed with the E235A and E235R mutants. Consistent with this notion,
increasing external Mg2+ gradually shifted steady-state
activation in the depolarizing direction (V1/2 = 70.6 ± 0.7 mV (n = 6), Glu235 Electrostatically Influences Steady-state
Activation from an External Location--
If the S3-S4 linker residue
Glu235 influences HCN1 activation by electrostatic means,
substitutions of residue 235 by amino acids other than Ala and Arg that
also render its charge neutralized or reversed should produce rightward
activation shifts similar to those of E235A and E235R channels. In
complete accordance with this notion, the activation midpoints of
E235K, E235H, and E235P channels were all displaced in the depolarizing
direction (Fig. 4B). The charge-reversed mutations (E235R,
E235K, and E235H) generally produced more pronounced activation shifts
than did the charge-neutralized counterparts (E235P and E235A),
although E235P was more positively shifted than E235H. In contrast to
these charge-altered substitutions, the charge-conserved substitution
E235D displayed gating properties indistinguishable from those of WT
channels. None of the Glu235 mutations significantly
altered the slope factor (p > 0.05) (Fig. 4C).
To determine whether a correlation between steady-state activation and
the charge of residue 235 (Q235) exists, we
plotted the energetic changes ( Asp233 Is Not Externally Accessible, and Its Mutations
Do Not Alter Gating--
We also investigated the effects of
substituting another anionic residue, Asp233, within the
same linker (cf. Fig. 1). In contrast to Glu235
mutations, however, none of the D233A, D233G, D233E, and D233R channels
exhibited gating properties different from those of WT HCN1 (Fig.
6, A-C) despite the close
proximity of residues 233 and 235. These results indicate that the
functional changes observed with Glu235 substitutions are
site-specific. We also probed the external accessibility of residue 233 by examining the sensitivity of D233C channels (in the background of
C318S to eliminate the intrinsic sensitivity of WT channels to
MTS compounds) (17) to the hydrophilic sulfhydryl modifier
MTSEA. Like C318S channels, D233C/C318S channels were not
reactive to external application of 2.5 mM MTSEA (Fig. 6D). E235C/C318S did not lead to functional expression of
measurable currents, rendering the assessment of its sensitivity to MTS
agents not possible. Combining the S3-S4 linker mutations D233A and
E235A with the S4-S5 linker substitution W270A, whose equivalent
mutation in HCN2 (i.e. W323A) is known to shift activation
positively (18), produced an activation shift more positive than either
of the individual mutations, indicating that the effects from these
single substitutions were largely additive (Fig.
7). These results suggest that the
external S3-S4 linker and the cytoplasmic S4-S5 linker are likely to
exert their effects on activation via mechanisms that are independent
of each other.
The S3-S4 Linker Residue Glu235 Acts as an External
Surface Charge--
Previous studies have demonstrated that the
extracellular S3-S4 linker is a determinant of activation in various
K+ and Ca2+ channels (12-15, 19). Our results
indicate that the S3-S4 linker of HCN channels also influences
activation gating. The charge-neutralizing and charge-reversing
mutations of linker residue 235 positively shifted steady-state
activation in a charge-dependent fashion (as reflected by
the linear
Theoretically, change of a surface charge should produce concomitant
shifts in the voltage dependence of both steady-state activation
and gating kinetics. Indeed, this was observed with our
Glu235 mutants (Fig. 5B, inset),
consistent with the effect of a surface charge. Taken collectively, our
results suggest that Glu235 is largely responsible for the
surface charge-shielding effects of Mg2+, probably at an
externally accessible position (although the Mg2+ effects
could be indirect). Interestingly, the attenuated charge-screening effects of external Mg2+ on Glu235 mutant
channels mirror the abolition of surface charge-shielding effects of H+ observed with the Shaker mutant,
whose S3-S4 linker (including the acidic residues Glu333,
Glu334, Glu335, and Asp336) has
been deleted (14).
Structural and Functional Roles of the HCN1 S3-S4
Linker--
Mechanistically, it is tempting to speculate that the
S3-S4 linker of HCN1 channels does not undergo significant
conformational changes during activation (e.g. movements in
the direction opposite to that of the positively charged S4) (15)
because of its apparent lack of contribution to the effective gating
charge (as reflected by the relatively unchanged slope factors of
Glu235 mutants). Because Glu235 is separated by
only two residues from the first basic S4 residue (i.e.
Lys238), this native glutamate may serve as a surface
charge that influences HCN activation by shaping the local electric
field sensed by the positively charged S4 (thereby shifting the
steady-state V1/2). Theoretically, neutralization of
a negative surface charge will always have the same effect, independent
of the voltage-sensing mechanism, by altering the transmembrane voltage
gradient: the voltage dependence should be shifted to more depolarized
potentials such that a stronger depolarization will restore the
original voltage profile. In this regard, the S3-S4 linker does not
need to directly participate in gating to exert its effects. However, based on the data presented, we cannot exclude the possibility that the
S3-S4 linker could undergo major conformational changes (e.g. horizontal movements would not alter the slope
factor). Although
It should be noted that the gating parameters studied here can also be
modulated by other mechanisms. For instance, it has been demonstrated
that cAMP binding to the cyclic nucleotide-binding domain of HCN
channels shifts the voltage dependence of steady-state activation to
more depolarizing potentials and accelerates activation kinetics by
relieving an intrinsic inhibitory influence of the cyclic
nucleotide-binding domain on basal gating (20). Although mutating the
S3-S4 linker and/or Mg2+ application may have indirect or
long-range allosteric effects on this modulatory mechanism, it is
reassuring that a highly correlated linear relationship was observed
with multiple Glu235 mutations carrying different charges;
indeed, the isoform used in our study, HCN1, is less sensitive to
allosteric modulation by the cyclic nucleotide-binding domain than
HCN2 (8, 9, 20-24).
Although Glu235 is likely to influence activation gating by
acting as an external surface charge of the channel, none of the Asp233 mutations studied altered HCN gating properties.
Furthermore, Asp233 was not externally accessible, although
it is possible that Cys233 was successfully modified, but
without functional changes. These observations could be readily
explained by assuming that the HCN S3-S4 linker also forms a helical
structure. Such an arrangement of S3-S4 linker residues would place
Asp233 on a side of the helix opposite to that of
Glu235, shielding the aspartate side chain from the S4
voltage sensor. Alternatively, Asp233 could be farther away
from the S4 segment, thereby minimizing the effects of charge
neutralization at this channel site. Clearly, additional experiments
are needed to distinguish between these possibilities.
Insights into Hyperpolarization-activated Gating--
Previous
studies with depolarization-activated K+ channels have
established that channel regions other than the S4 segments are also
involved in the process of voltage sensing (25-37). Although deletion
of the S3-S4 linker in Kv channels slows gating kinetics, voltage-dependent movements of S4 remain (12). This finding led to the conclusion that S4 moves only a short distance during activation, although many other experiments suggest that S4
translocates a large distance during the process of activation gating
(23, 24, 38). Indeed, it is possible that S4 undergoes
voltage-dependent movements without requiring linker motion
(15). Despite these discrepancies, the consensus is that S4 moves
outward upon depolarization to induce channel opening of Kv channels
(and also other depolarization-activated channels).
Using sulfhydryl modification of cysteine-substituted sea urchin sperm
SPIH mutant channels, Larsson and co-workers (11) recently
provided evidence that the voltage-sensing mechanism of Kv channels is
also conserved in this hyperpolarization-activated channel
(i.e. outward movements of S4 upon depolarization). Because the voltage-sensing mechanisms are conserved between depolarization- and hyperpolarization-activated channels, the mechanisms that couple
the voltage sensor and the activation gate must be different. Although
the present study did not address the molecular mechanism underlying
the difference in coupling that contributes to the backward
gating phenotype of HCN channels, emerging evidence appears to support
the notion that Kv and HCN channels share significant structural and
functional similarities, e.g. both are tetramers whose
fundamental building blocks are six-transmembrane subunits (7-10); the
GYG motif found in HCN and K+-selective pores (39) is
prerequisite for ion conduction (10); the voltage-sensing S4 segments
are positively charged (40, 41); S4 moves outward during depolarization
(11); alteration of S4 charges shifts activation; the pore and the gate
of HCN channels are coupled (42); and the S3-S4 linker influences
gating, etc. Therefore, it is becoming increasingly apparent that
subtle differences in structural design or coupling mechanism (albeit to be identified) may explain their distinctive activation phenotypes (11, 16).
65.0 ± 0.7 versus
70.6 ± 0.7 mV for wild-type
HCN1); the charge-reversed mutation E235R shifted activation even more
positively (
56.2 ± 0.5 mV). Increasing external
Mg2+ mimicked the progressive rightward shifts of E235A and
E235R by gradually shifting activation (V1/2 = 1 < 3 < 10 < 30 mM);
V1/2 induced by 30 mM
Mg2+ was significantly attenuated for E235A (+7.9 ± 1.2 versus +11.3 ± 0.9 mV for wild-type HCN1) and
E235R (+3.3 ± 1.4 mV) channels, as if surface charges were
already shielded. Consistent with an electrostatic role, the energetic
changes associated with
V1/2 resulting from
various Glu235 substitutions (i.e. Asp, Ala,
Pro, His, Lys, and Arg) displayed a strong correlation with their
charges (
G =
2.1 ± 0.3 kcal/mol/charge; r = 0.94). In contrast, D233E, D233A, D233G, and D233R
did not alter activation gating. D233C (in C318S background) was also not externally accessible when probed with methanethiosulfonate ethylammonium (MTSEA). We conclude that the S3-S4 linker residue Glu235 influences activation gating, probably by acting as
a surface charge.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
Putative transmembrane topology of HCN1.
A, the six putative transmembrane segments (S1-S6) of a
monomeric HCN subunit. The approximate locations of the S3-S4
residues investigated (i.e. Asp233 and
Glu235, HCN1 numbering) are highlighted. The GYG
selectivity motif and the cyclic nucleotide-binding domain
(CNBD) are also shown. B, sequence comparison of
the S3-S4 linkers of HCN1-4 with those of hyperpolarization-activated
sea urchin sperm (SPIH) and plant Arabidopsis
thaliana (KAT1) channels and depolarization-activated
Shaker and HERG K+ channels. Although the
S3-S4 linker is generally variable even within the
hyperpolarization-activated channel family, Glu235 is
absolutely conserved among HCN1-4, SPIH, and KAT1 channels.
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
140 mV
as a function of the preceding 3-s test pulse voltage normalized to the
maximum tail current recorded. Data were fit to the Boltzmann functions
using the Marquardt-Levenberg algorithm in a nonlinear least-squares
procedure: m
= 1/(1 + exp((Vt
V1/2)/k)),
where Vt is the test potential;
V1/2 is the half-point of the relationship; and
k = RT/zF is the slope factor,
where R, T, z, and F have
their usual meanings.
G) associated with
steady-state activation shifts caused by amino acid substitution were
calculated using the following equation:
G = RT(V1/2(mutant)/k(mutant)
V1/2(WT)/kWT).
act) and deactivation (
deact)
were estimated by fitting macroscopic and tail currents, respectively,
with a monoexponential function. However, it should be noted that the onset of HCN1 currents shows sigmoidicity and that tail currents also
exhibited an initial delay. The mechanism underlying such complex
kinetic behavior of HCN channels is not understood; further analysis
using multiple exponential components was beyond the scope of the this
study. For estimating open and closed rates of channels, the
bell-shaped distribution of
act and
deact
was fitted to the following relation:
= 1/(
0e
Vm/Vo +
0eVm/Vo),
where
0 and
0 reflect the open and closed
rates at zero voltage, respectively.
RESULTS
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
40 mV activated typical
time-dependent inward currents whose time constants
(
act) became faster with progressive hyperpolarization
(Fig. 2, A and D).
The midpoint (V1/2) and slope factor (k)
derived from the steady-state activation curve were
70.6 ± 0.7 mV and 9.5 ± 0.5 (n = 6), respectively (Fig.
2B). We also studied the deactivation properties of WT
channels by examining the voltage dependence of the rate of tail
current decay following maximum channel opening by hyperpolarizing to
140 mV (Fig. 2, C and D). Unlike
act, the deactivation time constant
(
deact) became faster with increasing depolarization.
Plotting these time constants together against the test potential
revealed that the voltage dependence of
act and
deact had a bell-shaped form (Fig. 2D);
0 and
0 for WT HCN1 channels derived from
this curve were (3.6 ± 0.5) × 10
1 and
(2.3 ± 0.2) × 10 s
1, respectively (see
"Experimental Procedures"). The peak of the
curve coincided
with the steady-state activation midpoint.
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Fig. 2.
WT HCN1 activation gating. A,
representative traces of hyperpolarization-activated currents through
WT HCN1 channels. The electrophysiological protocol used to elicit
currents is displayed above. The tail currents recorded at 140 mV
(boxed) are magnified to the right. Activation
kinetics (
act) were obtained by fitting the initial
macroscopic current with a monoexponential function. B,
steady-state activation relationship. C, typical tail
currents (magnified to the right) obtained by pulsing the
oocyte to a series of potentials from
100 to +40 mV after a 3-s
prepulse to
140 mV. Fitting tail currents with a monoexponential
function allows estimation of the deactivation kinetics
(
deact). D, summary of
act
(
) and
deact (
). The distribution of
was
bell-shaped. The rate constants
0 and
0,
which reflect the open and closed rates, respectively at zero voltage,
were derived from the
curve.
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Fig. 3.
E235A and E235R shift activation and
attenuate the charge-shielding effects of Mg2+.
A, representative traces of hyperpolarization-activated
currents through E235A and E235R HCN1 channels recorded under standard
conditions. The same protocol described in the legend to Fig. 1 was
used to elicit currents. B, steady-state activation curves
of WT HCN1 recorded with 1, 3, 10, and 30 mM external
Mg2+. The activation curves displayed a progressive
positive shift with increasing Mg2+ concentration.
C and D, steady-state activation curves of E235A
and E235R, respectively, recorded with 1 ( ) and 30 (
)
mM external Mg2+. Both E235A and E235R
mutations produced significant depolarizing activation shifts without
altering the slope factor compared with WT channels (- - -; 1 mM Mg2+). E, bar graph
summarizing steady-state V1/2 shifts in response to
30 mM Mg2+ (versus 1 mM)
for WT, E235A, and E235R channels. Notably, activation shifts caused by
30 mM Mg2+ were significantly attenuated for
E235A channels and almost abolished for E235R channels. *,
p < 0.05.
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Fig. 4.
Effects of various Glu235
mutations on HCN1 activation gating. A, representative
currents through E235D, E235P, E235H, and E235K channels. B
and C, summary of steady-state activation midpoints and
slope factors, respectively, of various Glu235 mutants
(i.e. E235D, E235A, E235P, E235H, E235R, and E235K).
68.9 ± 1.1 mV
(n = 3),
66.0 ± 0.4 mV (n = 3),
and
60.1 ± 1.5 mV (n = 8) for 1, 3, 10, and 30 mM Mg2+, respectively) (Fig. 3B),
mimicking the progressive rightward shifts caused by mutations E235A
and E235R. As extra support for the external electrostatic role of
residue 235, Fig. 3 (C and D) shows that the
response of E235A and E235R channels to Mg2+ was
significantly attenuated. 30 mM Mg2+ induced
only a 7.9 ± 1.2 mV (n = 5) depolarizing shift of
the steady-state activation midpoint for E235A channels
(versus
V1/2 = 11.3 ± 0.9 mV
(n = 8) for WT channels; p < 0.05),
whereas that for E235R channels was virtually not shifted at all
(
V1/2 = 3.3 ± 1.4 mV (n = 3); p > 0.05), as if surface charges were already
shielded. Activation shifts of WT, E235A, and E235R HCN1 channels
caused by 30 mM Mg2+ are summarized and
compared in Fig. 3E.
G) associated with the
activation shifts resulting from the various substitutions studied
relative to WT channels (i.e. Glu235) against
their own charges. Fig. 5A
indicates that
G and Q235 were
linearly correlated (r = 0.94) with a slope dependence
of 2.1 ± 0.3 kcal/mol/charge. Taken collectively, our results
were consistent with an external electrostatic role of residue 235. In
comparison with WT channels, all Glu235 mutants displayed
bell-shaped
curves, except with peaks shifted in the depolarizing
direction that paralleled the corresponding
V1/2
(see Fig. 5B (inset) for an example). Neither
0 nor
0 (derived from
curves)
displayed any obvious correlation with the charge of residue 235. Fig.
5B shows that, whereas
0 was not affected by
the side chain volume at all, there was a trend that
0
tended to accelerate with increasing side chain bulk, but the
correlation was weak (r = 0.44).
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Fig. 5.
Energetic and kinetic analyses of various
Glu235 mutations. A, energetic
changes ( G) in steady-state activation shifts
associated with various Glu235 substitutions relative to WT
channels were plotted against their own charges (i.e.
Q235). E235H was assigned a charge of 0.8 (estimated from the Henderson-Hasselbalch equation assuming that the
pKa of His235 is the same as that of
free histidine in solution under our recording conditions). A strong
linear correlation (r = 0.94) with a slope dependence
of 2.1 ± 0.3 kcal/mol/charge was observed between
G associated with Glu235 mutations and
Q235, consistent with an electrostatic role of
residue 235. B,
0 (
) and
0 (
) of various Glu235 mutants were
plotted against the corresponding side chain volume. The
inset shows that the peak of gating kinetics of E235R
channels (solid arrow) was shifted in a depolarizing
direction relative to that of the WT
curve (dashed
arrow). Other constructs were shifted similarly.
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Fig. 6.
Effects of Asp233 mutations on
HCN1 activation gating. A, representative records of
currents through D233E, D233A, D233G, and D233R channels. B
and C, steady-state activation curves and gating kinetics,
respectively, of the same channels displayed in A. All of
the Asp233 mutants studied displayed gating properties
that were not different from those of WT HCN1 channels.
D, effects of external application of 2.5 mM
MTSEA on D233C/C318S channels. D233C/C318S channels were not reactive
to MTSEA indicating that residue 233 is not functionally modified by
this sulfhydryl modifier from the extracellular side.
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Fig. 7.
Effects of W270A and D233A/E235A/W270A on
HCN1 activation gating. A, representative currents
through W270A and D233A/E235A/W270A channels. B,
steady-state activation curves of the same channel constructs in
A. W270A alone already produced significant depolarizing
activation shifts compared with WT channels. The triple mutation
D233A/E235A/W270A shifted activation even more positively, suggesting
the S3-S4 and S4-S5 linkers operate independently.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
G versus
Q235 relationship). Surface charge
shielding by external Mg2+ mimics our charge-changing
mutations by shifting the WT activation curve in the depolarizing
direction; such Mg2+-induced shifts were reduced by
mutation E235A, as if the channels were already partially screened by
this charge-neutralized substitution. The charge-screening effects of
Mg2+ were further attenuated by E235R, although there was a
modest residual response (
V1/2 ~ 3 mV) to the
addition of 30 mM Mg2+. Although the activation
shift and the decreased Mg2+ effect caused by the
neutralization of Glu235 could be most easily explained by
residue 235 functioning as a surface charge, the additional decreased
Mg2+ effect caused by the charge-reversed E235R mutation
could result from the screening of an additional negative surface
charge of the channel by the substituted arginine. Indeed, this notion
is consistent with our finding that neutralizing Glu235
eliminated ~50% of the Mg2+ effect and that
Mg2+ can screen only negative surface charges. Further
studies are required to identify this additional endogenous surface charge.
0 and
0 of HCN1 were
not drastically altered by Glu235 mutations, other S3-S4
linker residues may alter the energy barriers separating the channel
transitions required for channel openings (i.e. gating
kinetics). Clearly, additional experiments are needed to further
explore the functional role of this channel region.
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FOOTNOTES |
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* This work was supported in part by National Institutes of Health Grant R01 HL52768 and a research career development award from the Cardiac Arrhythmias Research & Education Foundation, Inc. (to R. A. L.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Supported by NHLBI Training Grant T32-HL07227-26 from the National
Institutes of Health.
§ Holds the Michel Mirowski, M.D. Professorship of Cardiology of The Johns Hopkins University.
¶ To correspondence should be addressed: Inst. of Molecular Cardiobiology, The Johns Hopkins University School of Medicine, 720 Rutland Ave., Ross 844, Baltimore, MD 21205. Tel.: 410-614-0035; Fax: 410-955-7953; E-mail: ronaldli@jhmi.edu.
Published, JBC Papers in Press, February 11, 2003, DOI 10.1074/jbc.M211025200
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ABBREVIATIONS |
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The abbreviations used are: HCN, hyperpolarization-activated cyclic nucleotide-gated; Kv, voltage-gated K+; WT, wild-type; MTSEA, methanethiosulfonate ethylammonium.
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