A Single Histidine Residue Determines the pH Sensitivity of the
Pacemaker Channel HCN2*
Xiangang
Zong
§,
Juliane
Stieber
,
Andreas
Ludwig
,
Franz
Hofmann
, and
Martin
Biel§¶
From the
Institut für Pharmakologie und
Toxikologie der Technischen Universität München,
Biedersteiner Str. 29, 80802 München and the
§ Department Pharmazie-Zentrum für Pharmaforschung,
Ludwig-Maximilians-Universität München, Butenandtstrasse 7,
81377 München, Germany
Received for publication, November 14, 2000
 |
ABSTRACT |
Hyperpolarization-activated cyclic
nucleotide-gated (HCN) cation channels control the rhythmic
activity of heart and neuronal networks. The activation of these
channels is regulated in a complex manner by hormones and
neurotransmitters. In addition it was suggested that the channels may
be controlled by the pH of the cytosol. Here we demonstrate that HCN2,
a member of the HCN channel family, is directly modulated by the
intracellular pH in the physiological range. Protons inhibit HCN2
channels by shifting the voltage dependence of channel activation to
more negative voltages. By using site-directed mutagenesis, we have
identified a single histidine residue (His-321) localized at the
boundary between the voltage-sensing S4 helix and the cytoplasmic S4-S5
linker of the channel that is a major determinant of pH sensitivity.
Replacement of His-321 by either arginine, glutamine, or glutamate
results in channels that are no longer sensitive to shifts in
intracellular pH. In contrast, cAMP-mediated modulation is completely
intact in mutant channels indicating that His-321 is not involved in
the molecular mechanism that controls modulation of HCN channel
activity by cyclic nucleotides. Because His-321 is conserved in
all four HCN channels known so far, regulation by intracellular pH is
likely to constitute a general feature of both cardiac and neuronal
pacemaker channels.
 |
INTRODUCTION |
Cationic inward currents activated upon hyperpolarization of the
plasma membrane, termed Ih (h for
hyperpolarization) or If (f for funny), are
found in heart and in a variety of central and peripheral neurons
(1-2). In spontaneously firing cells like sinoatrial node cells of the
heart (3-5), thalamic relay neurons (6), and respiratory neurons of
the brainstem (7), Ih contributes to the
pacemaker depolarization that generates rhythmic activity. In cell
types that do not reveal pacemaker activity Ih
fulfills diverse functions. It helps to determine the resting potential (2), provides rebound depolarizations in response to pronounced hyperpolarizations (8-9), and is involved in the control of synaptic plasticity (10) and the integration of synaptic inputs (11).
Recently, a family of four Ih channel
genes, designated as
HCN1-41 for
hyperpolarization-activated cyclic
nucleotide-gated cation channels, has been cloned (12-19).
HCN1-4 are structurally related to voltage-dependent
K+ channels and cyclic nucleotide-gated (CNG) channels
(20-21). HCN channels contain six transmembrane helices (S1-S6),
including a voltage-sensing S4 segment, a pore region between S5 and S6 and a cyclic nucleotide-binding domain (CNBD) in the C terminus.
Ih channels are tightly regulated by
neurotransmitters and metabolic stimuli. It is well known that cAMP
enhances the activation of Ih by directly
binding to the CNBD of the channel and thereby shifting the voltage
dependence of its activation curve to more positive values and speeding
up its activation kinetics (22). Up-regulation of
Ih by cAMP underlies the positive chronotropic effect of catecholamines in heart as well as the regulation of oscillation patterns of neuronal networks by neurotransmitters that
stimulate cAMP synthesis (1-2). Other factors including NO/cGMP (23),
GTP-binding proteins (24) and Ca2+ (25-26) have also been
reported to modulate Ih; however, in most cases
the physiological relevance of these modulatory mechanisms is a matter
of controversy.
Recently, Munsch and Pape (27-28) provided first evidence for an
additional kind of Ih regulation. They showed
that Ih of thalamocortical neurons is sensitive
to shifts of the intracellular pH (pHi). Alkalinization
induced an increase of the Ih amplitude, whereas acidification inhibited the current. Although it could not be unequivocally decided whether protons act directly on the
Ih channel or modulate Ih
via an indirect pathway, these findings suggested that
pHi-mediated modulation of Ih may
provide an important mechanism to control the activity of neuronal
networks in vivo. A possible modulation of
Ih by protons would also have profound
implications concerning the role of Ih under
pathophysiological conditions in a variety of cell types and tissues.
As a first step to address this important issue, we have investigated
the molecular basis of pH sensitivity of the heterologously expressed
HCN2 channel. Within the HCN channel family HCN2 is the most abundantly
expressed member. It is found in heart cells (14-15) and in most parts
of the brain including the thalamus (29-30). In particular, the
heterologously expressed HCN2 channel reveals all functional properties
attributed to native Ih, i.e. activation by
hyperpolarization, permeation of Na+ and K+,
block by Cs+, and activation by cAMP (14-15, 30-31). In
the present study, we have demonstrated that protons reversibly inhibit
the HCN2 current by shifting the activation curve of the channel to
more hyperpolarizing membrane potentials. We identified a highly
conserved histidine residue localized at the C-terminal end of the S4
segment that confers most if not all of the pH effect.
 |
EXPERIMENTAL PROCEDURES |
Construction of HCN2 Mutants and Functional Expression in HEK293
Cells--
All channel mutants were constructed in the expression
vector for murine HCN2 (mHCN2, former designation HAC1 (14)). Point mutations were introduced with polymerase chain reaction. The correctness of the introduced mutations and all sequences amplified by
polymerase chain reaction were verified by sequencing. HEK293 cells
were transiently transfected with expression vectors encoding either
wild-type or mutant HCN2 channels using conventional calcium-phosphate transfection methods.
Electrophysiology--
Currents were measured at room
temperature 2-3 days after transfection using either whole cell,
outside-out, or inside-out patch clamp techniques. The extracelluar
solution was composed of (in mM): 110, NaCl; 0.5, MgCl2; 1.8, CaCl2; 5, HEPES; 30, KCl. The
intracellular solution contained (in mM): 130, KCl; 10, NaCl; 0.5, MgCl2; 1, EGTA; 5, HEPES. The pH of
intracellular (pHi) and extracellular (pHe)
solutions was adjusted to various pH values by using KOH for
pHi and NaOH for pHe.
Data were acquired at 10 kHz using an Axopatch 200B amplifier and
pClamp 7 (Axon Instruments) and were low-pass filtered at 2 kHz with an
8-pole Bessel filter (LPBF-48DG, npi). Voltage clamp data were stored
on the computer hard drive and analyzed off-line by using Clampfit
(Axon Instruments). Steady-state activation curves were determined by
hyperpolarizing voltages (
150 to
95 mV for inside-out and
outside-out patch measurements, respectively;
150 to
30 mV for
whole cell recording) from a holding potential of
40 mV for 2.8 s followed by a step to
150 mV. Tail currents measured immediately
after the final step to
150 mV were normalized by the maximal current
(Imax) and plotted as a function of the preceding membrane potential. The data points were fitted with the
Boltzmann function:
(I-Imin)/(Imax-Imin) = 1/(1-exp[(Vm - V0.5)/k)] where
Imin is an offset caused by a nonzero holding current, Vm is the test potential, V0.5 is the
membrane potential for half-maximal activation, and k is the
slope factor. As has been described earlier for expressed HCN2 channel
(14) and native Ih (32), V0.5 was
dependent on the configuration of the electrophysiological measurement.
At pH 7.4, V0.5 of HCN2 was
104 ± 1.6 mV
(n = 6; see Fig. 5B),
117 ± 1.5 mV
(n = 6; see Fig. 4B) and
125 ± 1.3 mV (n = 16; see Fig. 5C) in whole-cell, outside-out, and inside-out mode, respectively. Similarly, in inside-out patches, V0.5 of mutant channels was also
shifted by about 20-25 mV to more hyperpolarizing voltages with
respect to the V0.5 values measured in whole cell mode (see
Fig. 5, B and C).
Time constants of channel activation (
act) of wild-type
and mutant HCN2 channels were determined in excised inside-out patches by fitting the current evoked during hyperpolarizing voltage pulses of
2.8-s duration with monoexponential functions. As has been described
earlier (15), the initial lag in the activation of HCN channel was
excluded from the fitting procedure. All values are given as mean ± S.E.; n is the number of experiments.
 |
RESULTS |
HCN2 Current Is Modulated by Internal pH in the Physiological
Range--
To test for a possible modulation of HCN channels by shifts
in intracellular pH (pHi), we transiently expressed the
murine HCN2 channel in HEK293 cells. Inward currents were activated
from excised inside-out patches of transfected cells by subsequently stepping from a holding potential of
40 mV to
130 mV, a voltage at
which the channel is about half-maximal activated, and then to
150
mV, a voltage at which the channel is fully activated. Fig.
1A shows current traces
obtained from an inside-out patch measured at different pHi
values in the bath solution. Whereas the maximal current amplitude at
150 mV was clearly not altered by switching between pHi
6.4 and pHi 8.4; the current amplitude measured at-130 mV
was strongly dependent on pHi being smaller under acidic
conditions and bigger at alkaline pHi. The effect of
pHi on the current amplitude was fully reversible and not
because of a time-dependent increase or rundown of the
current (Fig. 1B).

View larger version (20K):
[in this window]
[in a new window]
|
Fig. 1.
Modulation of HCN2 current by shifts in
pHi. A, current traces generated in
response to hyperpolarizing voltage steps in an excised inside-out
patch of a HEK293 cell expressing wild-type HCN2. The holding potential
was 40 mV and the voltage was stepped for 2.8 s to 130 mV and
then to 150 mV, respectively. The steady-state current was measured
at the end of each 2.8-s hyperpolarizing pulse as indicated by the
filled circles and squares. Shown are examples of
current traces evoked at different pH values in the bath solution
(pHi) as indicated. B, the modulation of HCN2
current by pHi is voltage-dependent and fully
reversible. Time course of the steady-state current amplitudes at 130
and 150 mV is measured from the patch shown in A. Each
symbol represents the current measured at the end of a 2.8-s voltage
step to either 130 mV (circles;
I 130) or 150 mV (squares;
I 150). The pulse protocol as indicated in
A was applied every 10 s. The patch was clamped to a
holding potential of 40 mV between the individual pulses.
pHi was changed at the indicated time points.
|
|
We next determined the voltage dependence of the HCN2 activation for a
range of pHi values (Fig. 2).
Acidic pHi (6.0) led to a leftward shift of the
half-maximal activation voltage (V0.5) to about 10 mV more
negative potentials (V0.5(pHi 6.0) =
134 ± 1.3 mV,
n = 11), whereas alkaline pHi (9.0)
resulted in a shift of V0.5 to 10 mV more positive
potentials (V0.5(pHi 9.0) =
114 ± 2.9 mV,
n = 6), as compared with V0.5 at the
control pHi of 7.4 (V0.5(pHi 7.4) =
125 ± 1.6 mV, n = 12). In contrast, shifts in
pHi had no major influence on the steepness of the
activation curve (kpHi 6.0 = 4.3 ± 0.4 mV;
kpHi 7.4 = 4.4 ± 0.5 mV;
kpHi 9.0 = 6.3 ± 0.8 mV). By plotting
V0.5 versus pHi and fitting the data with a sigmoidal equation, a titration curve for the pHi
dependence of HCN2 was obtained (Fig. 2B). The
pHi effect on V0.5 was most pronounced in the
range between pHi 7.0 and pHi 8.0 whereas
further acidification or alkalinization had significantly less effect. The calculated pKa value was 7.6, which is within
the range of normal cellular pHi. This finding indicated
that modulation of HCN channel by protons may be physiologically
important.

View larger version (15K):
[in this window]
[in a new window]
|
Fig. 2.
pHi affects voltage dependence
and kinetics of HCN2 channel activation. A, voltage
dependence of HCN2 channel activation measured from inside-out patches
at pHi 6.0 (circles), pHi 7.4 (squares), and pHi 9.0 (triangles).
The solid lines represent fits to the Boltzmann function
(see "Experimental Procedures") with the following factors: pH 6.0, V0.5 = 134 mV, k = 4.8 mV; pH 7.4, V0.5 = 125 mV, k = 5.2 mV; pH 9.0, V0.5 = 114 mV, k = 7.2 mV. Number of
experiments (n) = 11, 12, and 6 for pHi
values of 6.0, 7.4, and 9.0, respectively. B, proton
titration curve of V0.5. V0.5 was determined
for various pHi values from Boltzmann fits as described in
A and plotted as a function of pHi. Solid
line is a fit to a sigmoidal function with a
pKa value of 7.58; n = 3-12.
C, dependence of current activation kinetics on voltage and
pHi. Current traces were evoked at different
pHi in excised inside-out patches by hyperpolarizing pulses
from a holding potential of 40 mV to either 150 mV or 130 mV.
Activation constants ( act) were obtained by fitting the
current traces with a monoexponential function (see "Experimental
Procedures"). The number of experiments is indicated on top of the
bars.
|
|
Single-exponential fits to current traces recorded at different
pHi values indicated a strong dependence of the kinetics of channel opening on pHi (Fig. 2C). At
130 mV,
the time constant (
act) of channel activation decreased
significantly with an increase in pHi. At pHi
6.0, the time constant was 6.8 ± 1.6 s (n = 12), whereas it was 2.2 ± 0.6 s (n = 12) at
pHi 7.4 and 0.3 ± 0.02 s (n = 6)
at pHi 9.0. The pHi effect on activation
kinetics was profoundly influenced by the membrane potential. It was
very pronounced in the voltage range of V0.5 but
disappeared completely at
150 mV when the channel was fully
activated (
act(pHi 6.0): 0.28 ± 0.02 s;
act(pHi 7.4): 0.28 ± 0.02 s;
act(pHi 9.0): 0.26 ± 0.05 s).
The modulation of HCN current by shifts in pHi resembled
the cAMP-mediated modulation of Ih in two major
characteristics. 1) It affected the voltage dependence of channel
activation, and 2) it affected the speed of channel activation. We
therefore tested whether both kinds of channel modulation interfered
with each other. Fig. 3 shows activation
curves determined at different pHi in the absence and
presence of a saturating cAMP concentration (10 µM) in
the internal solution. At each pHi, cAMP induced a profound
shift of the V0.5 to more positive values. The shift was
strongest at acidic pHi (
V0.5 (pH 7.0) = +22 mV, shift from
132 ± 1.8 mV (n = 3) to
110 ± 1.0 mV (n = 3)) and became increasingly
weaker when pHi was altered to physiological
pHi (
V0.5 (pH 7.4) = +18 mV, shift from
128 ± 1.1 mV (n = 4) to
110 ± 3.8 mV
(n = 3)) and to alkaline pHi (
V 0.5
(pH 7.9) = +12 mV, shift from
118 ± 1.2 mV
(n = 5) to
106 ± 2.6 mV (n = 4)). On the other hand, although the channel was very sensitive to
shifts in pHi under control conditions (shift of
V0.5 of +14 mV between pHi 7.0 and
pHi 7.9 in the absence of cAMP), it became rather
insensitive to pHi when it was fully activated by a
saturating cAMP concentration (shift of V0.5 of +4 mV
between pHi 7.0 and pHi 7.9 at 10 µM cAMP). This result suggested that modulation of the
channel by intracellular protons critically depends on the basal
activity of the channel.

View larger version (28K):
[in this window]
[in a new window]
|
Fig. 3.
Cylic AMP induces a positive shift in the
voltage dependence of HCN2 activation at various pHi
values. Shown are activation curves measured from inside-out
patches at pHi values of 7.0, 7.4, and 7.9 in the absence
and in the presence of 10 µM cAMP in the bath solution,
as indicated. Solid lines represent fits to Boltzmann
function with the following parameters: pH 7.0, V0.5 = 132 mV, k = 3.4 mV (n = 3); pH 7.0 (+cAMP), V0.5 = 110 mV, k = 4.5 mV
(n = 3); pH 7.4, V0.5 = 128 mV,
k = 3.9 mV (n = 4); pH 7.4 (+cAMP),
V0.5 = 110 mV, k = 5.7 mV
(n = 3); pH 7.9, V0.5 = 119 mV,
k = 4.8 mV (n = 5); pH 7.9 (+cAMP),
V0.5 = 107 mV, k = 4.6 mV
(n = 4).
|
|
Finally we tested whether HCN2 currents could be modulated by shifts in
external pH (pHe). Fig.
4A shows current traces evoked at three different pHe values from the same outside-out
patch by stepping to
125 mV and
150 mV. It is evident that both
current amplitudes and kinetics were unaffected by acidification or
alkalinization. Similarly, the activation curves determined at
different pHe values (Fig. 4B) revealed no shift
in V0.5 or alteration of the slope factors (pHe
6.0: V0.5 =
116 ± 1.7 mV, k = 3.9 ± 0.5 mV, n = 6; pHe 7.4:
V0.5 =
117 ± 1.5 mV, k = 5.1 ± 0.8 mV, n = 6; pHe 9.0: V0.5 =
119 ± 4.1 mV, k = 3.4 ± 0.7 mV,
n = 4). Thus, modulation of the HCN2 channel by pH
revealed a profound site preference being restricted to shifts in
internal pH.

View larger version (16K):
[in this window]
[in a new window]
|
Fig. 4.
The HCN2 channel is not sensitive to shifts
in external pH (pHe). A, current traces
evoked from an excised outside-out patch by stepping from a holding
potential of 40 mV to 125 mV and then to 150 mV. The current
amplitude and kinetics was not different for pHe values of
6.0, 7.4, and 9.0, respectively. B, voltage dependence of
channel activation as determined from outside-out patches. Solid
lines are fits to the Boltzmann equation with the following
parameters: pH 6.0, V0.5 = 117 mV, k = 4.5 mV (n = 6); pH 7.4, V0.5 = 116 mV,
k = 4.6 mV (n = 6); pH 9.0, V0.5 = 119 mV, k = 5.5 mV
(n = 4).
|
|
Histidine 321 Is a Crucial Determinant of Sensitivity to
pHi--
Our experiments suggested that protons inhibit
HCN2 by binding to a titratable acceptor side of the channel, which
faces the intracellular environment. Because the pKa
deduced from the HCN2 titration curve was 7.6, we postulated that a
histidine residue (pKa = 6.0) would be the most
likely candidate for providing a proton binding site. By contrast, the
pKa values of lysine (pKa = 10.5)
and arginine (pKa = 12.5) are far away from the
observed range of pH regulation. HCN channels contain several histidine
residues that principally could confer the pH effect. The finding that
protonation induced a shift in the activation curve prompted us to
focus on residues in the cytoplasmic S4-S5 linker, which has been shown
to control together with the S4 segment the activation of
K+ channels (33-34). The sequence of the S4 segment and
the S4-S5 linker is completely conserved in HCN1-4 (Fig.
5A). A histidine residue in
this sequence, His-321, is positioned "in-frame" with the nine
regularly spaced arginines and lysines of the S4 segment suggesting
that it may be part of the voltage-sensing and/or gating machinery of
the channel. To investigate the role of His-321, we replaced the
residue with either a positively charged arginine (H321R), a neutral
glutamine (H321Q), or an acidic glutamate (H321E). Replacement by
arginine did not significantly alter the voltage dependence of
activation at physiological pHi as determined in whole cell
mode (Fig. 5B) or in excised inside-out patches (Fig. 5C). Similarly, mutation of His-321 to glutamine only weakly
affected the activation curve. It induced a slight shift of the
V0.5 to about a 5 mV more positive voltage with respect to
the wild-type channel (Fig. 5, B and C). By
contrast, the V0.5 was profoundly shifted to the right
(
V0.5 = +20 mV) when His-321 was exchanged by the
negatively charged glutamate residue (Fig. 5, B and
C). Determination of reversal potentials from
current/voltage relationships of the fully activated channels (14)
revealed that mutations at position 321 did not alter the ion
selectivity of the channels with respect to wild-type HCN2 (not
shown).

View larger version (23K):
[in this window]
[in a new window]
|
Fig. 5.
Functional characterization of mutant HCN2
channels. A, structural model of the HCN channel and
sequence of the C-terminal part of the S4 segment and the S4-S5 linker
of mHCN2. In HCN2 mutants His-321 is replaced by either arginine
(H321R), glutamine (H321Q), or glutamate (H321E). CNBD,
cyclic nucleotide-binding domain. B, activation curves
determined at physiological pHi, 7.4, from whole cell
currents of wild type (wt) and mutant HCN2 channels.
Solid lines are fits to the Boltzmann equation with the
following parameters: wt, V0.5 = 103 mV,
k = 9.1 mV (n = 6); H321R,
V0.5 = 101 mV, k = 10.1 mV
(n = 6); H321Q, V0.5 = 96.5 mV,
k = 9.4 mV (n = 8); H321E,
V0.5 = 84.8 mV, k = 9.2 mV
(n = 6). C, activation curves determined at
physiological pHi, 7.4 from inside-out patches of cells
expressing wild-type and mutant HCN2 channels. Symbol usage is as in
Fig. 5. Solid lines are fits to the Boltzmann equation with
the following parameters: wt HCN2, V0.5 = 126 mV,
k = 5.2 mV (n = 16); H321R,
V0.5 = 126 mV, k = 6.2 mV
(n = 17); H321Q, V0.5 = 120 mV,
k = 4.6 mV (n = 8); H321E,
V0.5 = 106 mV, k = 5.3 mV
(n = 6). Please note that in inside-out patches
V0.5 of both wild-type and mutant channels are shifted by
about 20-25 mV to more hyperpolarizing voltages with respect to
V0.5 measured in whole cell mode (see "Experimental
Procedures").
|
|
We next asked whether HCN2 mutants were sensitive to alterations of
pHi (Fig. 6). Activation
curves determined from inside-out patches revealed that pHi
sensitivity was almost completely lost in H321R (Fig. 6A),
H321Q (Fig. 6B), and H321E (Fig. 6C). Again, the
activation curves of the H321E mutant were significantly shifted to
more depolarizing voltages when compared with H213R, H321Q, and
wild-type channels. The loss of pHi-dependent
shifts in V0.5 of mutant channels was mirrored by a loss of
pHi effect on activation kinetics (Fig. 6D).
Whereas in wild-type channel alkalinization led to a pronounced
decrease of
values at
130 mV (Fig. 2C), activation
constants of H321R and H321Q were not significantly altered by shifts
in pHi under the same conditions. However, as in wild-type,
channel activation constants of HCN2 mutants were dependent on the
membrane potential reaching a minimal value at voltages that fully
activated the channel (
150 mV) and increasing with membrane
depolarization. Similar results were obtained with the H321E mutant
(not shown).

View larger version (33K):
[in this window]
[in a new window]
|
Fig. 6.
Modulation of mutant HCN2 channels by shifts
in pHi. A-C, activation curves of H321R
(A), H321Q (B), and H321E (C) mutants
as determined in inside-out patch mode at pHi values of 6.0 (circles), 7.4 (squares), and 9.0 (triangles). Solid lines are fits to the
Boltzmann function with the following parameters: H321R:
pHi 6.0, V0.5 = 131 mV, k = 4.9 mV (n = 5); pHi 7.4, V0.5 = 126 mV, k = 5.4 mV (n = 7);
pHi 9.0, V0.5 = 132 mV, k = 8.8 mV (n = 3). H321Q: pHi 6.0, V0.5 = 122 mV, k = 6.3 mV
(n = 8); pHi 7.4, V0.5 = 120
mV, k = 4.6 mV (n = 8); pHi
9.0, V0.5 = 120 mV, k = 8.5 mV
(n = 5). H321E: pHi 6.0, V0.5 = 106 mV, k = 6.1 mV (n = 5);
pHi 7.4, V0.5 = 106 mV, k = 5.3 mV (n = 6). D, activation time constants
( act) of H321R and H321Q mutants. Constants were
determined for pHi 6.0, 7.4, and 9.0 as described under
"Experimental Procedures" by fitting current traces evoked in
inside-out patches during hyperpolarizing pulses to either 150 mV
( act( 150)) or 130 mV ( act( 130)).
H321R: pHi 6.0, act( 150) = 239 ± 35 ms (n = 7); act( 130) = 1.4 ± 0.5 s (n = 7); pHi 7.4, act( 150)=244 ± 16 ms (n = 20);
act( 130) = 1.3 ± 0.2 s (n = 20); pHi 9.0, act( 150) = 267 ± 75 ms
(n = 3); act( 130) = 1.1 ± 0.2 s (n = 3). H321Q: pHi 6.0, act( 150) = 167 ± 22 ms (n = 11);
act( 130) = 0.8 ± 0.2 s (n = 11); pHi 7.4, act( 150) = 205 ± 21 ms
(n = 15); act( 130) = 0.7 ± 0.1 s (n = 15); pHi 9.0, act( 150) = 219 ± 33 ms (n = 9);
act( 130) = 0.5 ± 0.2 s (n = 9). Error bars of individual experiments are
indicated.
|
|
We finally investigated whether the replacement of His-321 in HCN2
interefered with the cAMP modulation of the channels. Activation curves
were measured at pHi7.4 for wild type (Fig.
7A), H321R (Fig.
7B), H321Q (Fig. 7C), and H321E (Fig.
7D) in whole cell mode under control conditions and after
intracellular perfusion with 1 mM cAMP. Cyclic AMP shifted
the V0.5 of all three mutants to 20-25 mV more positive
values. The V0.5 values for H321R, H321Q, and H321E were
102 ± 2.6 mV (n = 6),
96.7 ± 1.7 mV
(n = 8) and
85.2 ± 1.7 mV (n = 8) in the absence of cAMP and
76.8 ± 1.4 mV (n = 3),
73.3 ± 4.1 mV (n = 5) and
66.4 ± 4.1 mV (n = 4) in the presence of cAMP, respectively.
These values are in the same range as the shift observed for wild-type
HCN2 (V0.5 =
104 ± 1.6 mV (n = 6)
in the absence, and
75.5 ± 3.6 mV (n = 5) in the presence of cAMP) indicating that mutation of His-321 does not
alter the cAMP-mediated activation of HCN2.

View larger version (23K):
[in this window]
[in a new window]
|
Fig. 7.
Mutation of His-321 does not affect
modulation by cAMP. A-D, activation curves of
wild-type (A), H321R (B), H321Q (C),
and H321E (D) mutants measured in whole cell current mode
under control conditions (circles) or after pipette
perfusion with 1 mM cAMP for at least 1 min
(squares). The pHi was 7.4 in all experiments.
Cyclic AMP induced a positive shift of the voltage dependence for both
wild-type and mutant channels. Solid lines are fits to the
Boltzmann function with the following parameters: wt HCN2,
V0.5 = 103 mV, k = 9.0 mV
(n = 6); wt HCN2 (+cAMP), V0.5 = 75.4 mV,
k = 8.8 mV (n = 5); H321R,
V0.5 = 101 mV, k = 9.9 mV
(n = 6); H321R (+cAMP), V0.5 = 76.6 mV,
k = 7.7 mV (n = 3); H321Q,
V0.5 = 96.4 mV, k = 9.6 mV
(n = 8); H321Q (+cAMP), V0.5 = 73.1 mV,
k = 9.4 mV (n = 5); H321E,
V0.5 = 84.8 mV, k = 9.2 mV
(n = 8); H321E (+cAMP), V0.5 = 66.0 mV,
k = 8.8 mV (n = 4).
|
|
 |
DISCUSSION |
Modulation of HCN2 by Shift in pHi--
This study
demonstrates for the first time the modulation of a heterologously
expressed HCN channel by protons. Like native Ih, HCN2 is specifically modulated by shifts in
cytosolic pH but is not sensitive to changes in external pH. The
modulation by internal protons refers to two aspects of HCN2 channel
activation, namely its voltage dependence and its kinetics.
Intracellular acidification induces a down-regulation of the current by
shifting the activation curve to the left and also slows down the speed of activation. Alkalinization enhances the current by shifting the
activation curve to more depolarized voltages and in addition accelerates opening kinetics. Our results strongly support the hypothesis that protons modulate Ih by directly
binding to the HCN channel molecule. By contrast, there is no evidence
that additional factors or auxiliary channel subunits are necessary to
confer this effect.
Site of Action of Internal Protons and Mechanism of
Modulation--
By using site-directed mutagenesis we have identified
a single amino acid in the HCN2 primary sequence (His-321) that is a major determinant of the pHi modulation. Replacement of
this histidine residue by a nontitratable residue (H321Q) or by
residues that at physiological pHi are either positively
(H321R) or negatively (H321E) charged almost completely abolished
sensitivity to pHi. By contrast, these mutations did not
alter the ion selectivity and the kinetics of the fully activated
channels. Whereas all three mutants lacked pHi sensitivity,
they also differed from each other in their respective V0.5
values. The activation curve was shifted to more and more positive
values in the order of H321R, H321Q, and H321E. This finding suggests
that the electrical charge at position 321 may be involved in the
mechanism that regulates HCN channel activity. However, charge effects
alone are not sufficient to explain the inhibitory effect of protons.
If the electrical charge at position 321 would be the major determinant
of V0.5 one would expect the V0.5 of the H321R
mutant to be more negative than the V0.5 of the wild-type
channel. This is clearly not the case. In contrary, protons even shift
the V0.5 of the wild-type channel to values that are
significantly more negative than the V0.5 of the H321R mutant.
How might His-321 control the voltage dependence of channel activation?
His-321 is separated by two neutral amino acid residues from the last
positive charged residue of S4. Thus, His-321 could either constitute
the C terminus of the S4 helix or the N terminus of the S4-S5 linker.
Recent studies indicate that the C-terminal end of S4 of Shaker
K+ channel subunits is accessible by thiol-reactive
reagents from the cytoplasm making it impossible to precisely define
the boundary between S4 helix and S4-S5 linker (35). In any case, the
localization of His-321 is very consistent with a possible role in
modulating channel activation. The S4 segment has been demonstrated to
constitute the main component of the voltage sensor in calcium, sodium,
and potassium channels (36). Recently, it was shown that replacement of
charged residues in the S4 of HCN channels profoundly influences the
voltage dependence of channel activation (37-38). Thus, conformational changes induced by the protonation or deprotonation of His-321 could
well interfere with the voltage-dependent movement of the S4 helix. There is also evidence that the S4-S5 linker is an important determinant of voltage-dependent gating in other channels.
Mutations in the S4-S5 linker of the KCNQ1 channel associated with long QT syndrome induce shifts in the voltage dependence of channel activation (33). Similarly, the S4-S5 linker was shown to be a crucial
component of the activation gate of HERG K+ channel (34).
It is thus tempting to speculate that as in these K+
channels, the S4-S5 linker of HCN channels transduces movement of the
voltage-sensing S4 helix into opening of the channel. Our data indicate
that His-321 could play a key role in this transduction process.
Mutant channels that had lost sensitivity to shifts in pHi
could still be activated by cAMP indicating that cAMP-mediated modulation of voltage dependence of channel activation is not controlled by His-321. In addition, wild-type HCN2 is up-regulated by
cAMP over a broad range of pHi supporting the notion that
cAMP-mediated modulation works independent of pHi. On the
other hand, pHi-mediated shifts in V0.5 are
profoundly influenced by the intracellular cAMP concentration being
significantly larger in the absence than in the presence of the cyclic
nucleotide. This finding could be explained by the presence of
allosteric interactions between the molecular pathways that confer
proton- and cAMP-mediated channel modulation.
Physiological Implications of HCN Channel Modulation by
pHi--
Given the very abundant expression of HCN2 in
most parts of brain and heart, modulation of this channel by
pHi is of significant physiological importance. Because the
S4-S5 region including the His-321 residue is completely conserved in
all other mammalian HCN channels cloned so far, sensitivity to
pHi presumably is a general feature of HCN channels.
Thalamic neurons produce a range of complex firing patterns such as
tonic bursts and slow
oscillations that control brain functions
like the phases of sleep and the information flow through the thalamus
toward the cerebral cortex (2, 21). It is well established that
modulation of Ih by neurotransmitters that up- or down-regulate cellular cAMP or cGMP concentrations plays a key role
in the control of thalamic oscillations (23, 39-40). Our data indicate
that shifts in pHi may provide an additional regulatory
pathway of neuronal network activity. The sensitivity of
Ih to shifts in pHi may also be
important in neurological diseases. Acidification as occurring during
epileptiform discharges (41) could inhibit Ih,
thereby counteracting the Ca2+-mediated positive shift in
the Ih activation curve (40) and, hence,
prolonging the duration of burst activity. In accordance with this
model, it was recently postulated that the anticonvulsant action of
acetazolamide is related to an intracellular alkalinization of thalamic
neurons causing an up-regulation of Ih (28).
It is well known that the respiratory frequency is increased in
response to acidosis and hypoxia. Recently, it was shown in inspiratory
neurons of the brainstem that an increase in the respiratory frequency
can be induced by the blockade of Ih by
Cs+ or the specific Ih blocker
ZD7288 (7). Thus, it is tempting to assume that protons may control
respiratory frequency by controlling the activity of HCN channels.
Modulation of HCN channels by pHi may also be important in
non-neuronal cell types. It was speculated that the motility of sperm
cells may be controlled by up-regulation of the HCN4 channel because of
intracellular alkalinization (18). Although we have not tested whether
or not HCN4 is sensitive to shifts in pHi, the presence of
a residue equivalent to His-321 in HCN4 is in favor of such a modulation.
Finally, pHi may control the activity of HCN channels in
heart. Such a regulation could be of particular importance under pathological conditions such as heart failure and myocardial
infarction. During acute ischemia accompanying these diseases,
CO2 production and the net production of protons shift
pHi to values as low as 6.0 (42). Our data suggest that
inhibition of HCN channels by increases in H+ concentration
could disturb pacemaker activity in sinoatrial node and other parts of
the cardiac conduction tissue and, hence, contribute significantly to
the generation of arrhythmia.
 |
ACKNOWLEDGEMENT |
We thank A. Ebner for technical support.
 |
FOOTNOTES |
*
This work was supported by grants from the Deutsche
Forchungsgemeinschaft and Bundesministerium für Bildung
und Forschung.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.
¶
To whom correspondence should be addressed. Tel.:
49-89-2180-7327; Fax: 49-89-2180-7326; E-mail:
mbiel@cup.uni-muenchen.de.
Published, JBC Papers in Press, November 28, 2000, DOI 10.1074/jbc.M0010326200
 |
ABBREVIATIONS |
The abbreviations used are:
HCN, hyperpolarization-activated cyclic nucleotide-gated cation channel;
HEK, human embryonic kidney cells.
 |
REFERENCES |
1.
|
DiFrancesco, D.
(1996)
in
Molecular Physiology and Pharmacology of Cardiac Ion Channels and Transporters
(Morad, M.
, Ebashi, S.
, Trautwein, W.
, and Kurachi, Y., eds)
, pp. 31-37, Kluwer Academic Publishers, Dordrecht, NL
|
2.
|
Pape, H.-C.
(1996)
Annu. Rev. Physiol.
58,
299-327[CrossRef][Medline]
[Order article via Infotrieve]
|
3.
|
Brown, H. F.,
Giles, W.,
and Noble, S. J.
(1977)
J. Physiol. (Lond.)
271,
783-816[Medline]
[Order article via Infotrieve]
|
4.
|
Yanagihara, K.,
and Irisawa, H.
(1980)
Pfluegers Arch.
388,
11-19[Medline]
[Order article via Infotrieve]
|
5.
|
DiFrancesco, D.
(1981)
J. Physiol. (Lond.)
314,
359-376[Abstract]
|
6.
|
McCormick, D. A.,
and Pape, H.-C.
(1990)
J. Physiol. (Lond.)
432,
291-318
|
7.
|
Thoby-Brisson, M.,
Telgkamp, P.,
and Ramirez, J.-M.
(2000)
J. Neurosci.
20,
2994-3005[Abstract/Free Full Text]
|
8.
|
Fain, G. L.,
Quandt, F. N.,
and Bastian, B. L.
(1978)
Nature
272,
467-469
|
9.
|
Wollmuth, L. P.,
and Hille, B.
(1992)
J. Gen. Physiol.
100,
749-765[Abstract]
|
10.
|
Beaumont, V.,
and Zucker, R. S.
(2000)
Nat. Neurosci.
3,
133-141[CrossRef][Medline]
[Order article via Infotrieve]
|
11.
|
Magee, J. C.
(1999)
Nat. Neurosci
2,
508-514[CrossRef][Medline]
[Order article via Infotrieve]
|
12.
|
Santoro, B.,
Grant, S. G. N.,
Bartsch, D.,
and Kandel, E. R.
(1997)
Proc. Natl. Acad. Sci. U. S. A.
94,
12815-14820
|
13.
|
Santoro, B.,
Liu, D. T.,
Yao, H.,
Bartsch, D.,
Kandel, E. R.,
Siegelbaum, S. A.,
and Tibbs, G. R.
(1998)
Cell
93,
717-729[Medline]
[Order article via Infotrieve]
|
14.
|
Ludwig, A.,
Zong, X.,
Jeglitsch, M.,
Hofmann, F.,
and Biel, M.
(1998)
Nature
393,
587-591[CrossRef][Medline]
[Order article via Infotrieve]
|
15.
|
Ludwig, A.,
Zong, X.,
Stieber, J.,
Hullin, R.,
Hofmann, F.,
and Biel, M.
(1999)
EMBO J.
18,
2323-2329[Abstract/Free Full Text]
|
16.
|
Gauss, R.,
Seifert, R.,
and Kaupp, U. B.
(1998)
Nature
393,
583-587[CrossRef][Medline]
[Order article via Infotrieve]
|
17.
|
Ishii, T. M.,
Takano, M.,
Xie, L.-H.,
Noma, A.,
and Ohmori, H.
(1999)
J. Biol. Chem.
274,
12835-12839[Abstract/Free Full Text]
|
18.
|
Seifert, R.,
Scholten, A.,
Gauss, R.,
Mincheva, A.,
Lichter, P.,
and Kaupp, U. B.
(1999)
Proc. Natl. Acad. Sci. U. S. A.
96,
9391-9396[Abstract/Free Full Text]
|
19.
|
Vaccari, T.,
Moroni, A.,
Rocchi, M.,
Gorza, L.,
Bianchi, M. E.,
Beltrame, M.,
and DiFrancesco, D.
(1999)
Biochim. Biophys. Acta
1446,
419-425[Medline]
[Order article via Infotrieve]
|
20.
|
Biel, M.,
Ludwig, A.,
Zong, X.,
and Hofmann, F.
(1999)
Rev. Physiol. Biochem. Pharmacol.
136,
165-181[Medline]
[Order article via Infotrieve]
|
21.
|
Santoro, B.,
and Tibbs, G. R.
(1999)
Ann. N. Y. Acad. Sci.
868,
741-764[Abstract/Free Full Text]
|
22.
|
DiFrancesco, D.,
and Tortora, P.
(1991)
Nature
351,
145-147[CrossRef][Medline]
[Order article via Infotrieve]
|
23.
|
Pape, H.-C.,
and Mager, R.
(1992)
Neuron
9,
441-448[Medline]
[Order article via Infotrieve]
|
24.
|
Yatani, A.,
Okabe, K.,
Codina, J.,
Birnbaumer, L.,
and Brown, A. M.
(1990)
Science
249,
1163-1166[Medline]
[Order article via Infotrieve]
|
25.
|
Ingram, S.,
and Williams, J.
(1996)
J. Physiol. (Lond.)
492,
97-106[Abstract]
|
26.
|
Budde, T.,
Biella, G.,
Munsch, T.,
and Pape, H.-C.
(1997)
J. Physiol. (Lond.)
503,
79-85[Abstract]
|
27.
|
Munsch, T.,
and Pape, H.-C.
(1999)
J. Physiol. (Lond.)
519,
493-504[Abstract/Free Full Text]
|
28.
|
Munsch, T.,
and Pape, H.-C.
(1999)
J. Physiol. (Lond.)
519,
505-514[Abstract/Free Full Text]
|
29.
|
Moosmang, S.,
Biel, M.,
Hofmann, F.,
and Ludwig, A.
(1999)
Biol. Chem.
380,
975-980
|
30.
|
Santoro, B.,
Chen, S.,
Lüthi, A.,
Pavlidis, P.,
Shumyatsky, G.,
Tibbs, G. R.,
and Siegelbaum, S. A.
(2000)
J. Neurosci.
20,
5264-5275[Abstract/Free Full Text]
|
31.
|
Moroni, A.,
Barbuti, A.,
Altomare, C.,
Viscomi, C.,
Morgan, J.,
Baruscotti, M.,
and DiFrancesco, D.
(2000)
J. Physiol. (Lond.)
439,
618-626
|
32.
|
DiFrancesco, D.,
and Mangoni, M.
(1994)
J. Physiol. (Lond.)
474,
473-482[Abstract]
|
33.
|
Franzqueza, L.,
Lin, M.,
Splawski, I.,
Keating, M. T.,
and Sanguinetti, M. C.
(1999)
J. Biol. Chem.
274,
21063-21070[Abstract/Free Full Text]
|
34.
|
Sanguinetti, M. C.,
and Xu, Q. P.
(1999)
J. Physiol. (Lond.)
514,
667-675[Abstract/Free Full Text]
|
35.
|
Baker, O. S.,
Larsson, H. P.,
Mannuzzu, L. M.,
and Isacoff, E. Y.
(1998)
Neuron
20,
1283-1294[Medline]
[Order article via Infotrieve]
|
36.
|
Horn, R.
(2000)
Neuron
25,
511-514[CrossRef][Medline]
[Order article via Infotrieve]
|
37.
|
Vaca, L.,
Stieber, J.,
Zong, X.,
Ludwig, A.,
Hofmann, F.,
and Biel, M.
(2000)
FEBS Lett.
479,
35-40[CrossRef][Medline]
[Order article via Infotrieve]
|
38.
|
Chen, J.,
Mitcheson, J. S.,
Lin, M.,
and Sanguinetti, M. C.
(2000)
J. Biol. Chem.
275,
36465-36471[Abstract/Free Full Text]
|
39.
|
Bal, T.,
and McCormick, D. A.
(1996)
Neuron
17,
297-308[Medline]
[Order article via Infotrieve]
|
40.
|
Lüthi, A.,
and McCormick, D. A.
(1998)
Neuron
20,
553-563[Medline]
[Order article via Infotrieve]
|
41.
|
De, Curtis, M,
Manfridi, A.,
and Biella, G.
(1998)
J. Neurosci.
18,
7543-7551[Abstract]
|
42.
|
Carmeliet, E.
(1999)
Physiol. Rev.
70,
917-1017
|
Copyright © 2001 by The American Society for Biochemistry and Molecular Biology, Inc.