From the Department of Physiology and Biophysics, The University of Iowa, Iowa City, Iowa 52242
Dihydropyridines (DHPs) are well known for their effects on L-type voltage-dependent Ca2+ channels. However, these drugs also affect other voltage-dependent ion channels, including Shaker K+ channels. We examined the effects of DHPs on the Shaker K+ channels expressed in Xenopus oocytes. Intracellular applications of DHPs quickly and reversibly induced apparent inactivation in the Shaker K+ mutant channels with disrupted N- and C-type inactivation. We found that DHPs interact with the open state of the channel as evidenced by the decreased mean open time. The DHPs effects are voltage-dependent, becoming more effective with hyperpolarization. A model which involves binding of two DHP molecules to the channel is consistent with the results obtained in our experiments.
Key words: potassium channels; dihydropyridinesDihydropyridines (DHPs)1 with a 1,4 dihydropyridine
ring and a phenyl ring are clinically used to treat a variety of cardiovascular disorders, primarily acting as vasodilators. Effects of the DHPs on L-type Ca2+ channels
are especially interesting in that the DHPs can be either agonists or antagonists (Reuter et al., 1988; Hess,
1990
). These drugs appear to act allosterically to modify the channel function by promoting different modes
of gating (Hess, 1990
). They have greater affinities for
the inactivated state of the channel, which is favored by
depolarization (Hess, 1990
).
Although DHPs have profound effects on L-type Ca2+
channels, it was shown that these compounds also affected voltage-dependent Na+ channels (Yatani et al.,
1988) and voltage-dependent K+ channels (Jacobs and
DeCoursey, 1990
; Gotoh et al., 1991
). Using the whole
cell voltage-clamp method, Jacobs and DeCoursey (1990)
tested a series of clinically relevant agents, including
nifedipine, on the K+ currents in alveolar epithelial
cells and found that nifedipine produced apparent inactivation. They suggested that nifedipine, at least in
part, works by acting as an open channel blocker. Gotoh et al. (1991)
studied the effect of nicardipine on the
K+ channel current in rabbit heart cells. They found
that nicardipine accelerated the time course of the outward K+ current decline with the 50% inhibitory concentration of 630 nM. This is fourfold higher than that
for the Ca2+ current in the same cells (160 nM).
The structural elements involved in the DHP binding
to the Ca2+ channels are not thoroughly understood.
Biochemical experiments (Nakayama et al., 1991; Regulla et al., 1991
) using antibody mapping indicated that
DHPs may bind to the amino acid residues in the S6
segment of the
1 subunit of the Ca2+ channel. Some
investigators (Glossmann et al., 1984
; Triggle et al.,
1989
) have suggested that Ca2+ channels may have two
DHP binding sites, a high-affinity site (nM range) and a
low-affinity site (µM range). A mutagenesis study using
chimeric channels based on two distinct Ca2+ channels
with different DHP affinities shows that the S5, Pore, and S6 segments may be important in determining the
DHP sensitivity (Grabner et al., 1996
). A molecular simulation study showing how a DHP may bind to the Ca2+
channel pore with Ca2+ ions bound suggests that DHPs
occlude the channel pore (Zhorov and Ananthanarayanan, 1996
).
We investigated the effects of DHPs on the Shaker K+
channels expressed in Xenopus oocytes. Native cells may
express more than one class of K+ channels and the interpretations of the results of the drug action are usually not straightforward. Heterologous expression of a single class of channel can largely overcome this problem. Since the biophysical gating properties of the
Shaker channel have been studied extensively (Bezanilla
et al., 1994; Hoshi et al., 1994
; Stefani et al., 1994
; Zagotta et al., 1994
a,b), any alterations in the channel gating can be more easily detected and studied. Furthermore, molecular manipulations of the Shaker channel
affecting some of the gating transitions, such as inactivation rates, have been described (Hoshi et al., 1990
,
1991
; Zagotta et al., 1990
; Lopez-Barneo et al., 1993
),
and they can be used to probe the interactions of the
channel with DHPs. The results obtained in this study
indicate that DHPs interact with the open state of the
Shaker channel inducing apparent inactivation although
the drugs do not act as simple open-channel blockers.
The results also show that the overall DHP efficacy can
be modulated by a single amino acid mutation in the
S6 segment.
Channel Expression
Wild-type and mutant Shaker K+ channels were expressed in Xenopus oocytes essentially as described previously (Hoshi et al., 1990). The following channels were used in this study. ShD (Timpe et al., 1988
) is characterized by intact N-type inactivation and C-type inactivation. ShB
6-46 contains a 41-residue deletion in the amino terminus and lacks N-type inactivation (Hoshi et al., 1990
). In addition to the amino terminus deletion, ShB
6-46
T449V contains a single amino acid substitution at position 449 (using the ShB numbering) from T to V. The ShB
6-46 T449V
channel has disrupted N-type inactivation and drastically slowed
or disrupted C-type inactivation (Lopez-Barneo et al., 1993
).
ShB
6-46 T449K has a point mutation at position 449 from T to
K to accelerate C-type inactivation to a more experimentally manageable range (Lopez-Barneo et al., 1993
). ShB
6-46 T449V:
A463I has an additional point mutation at position 463 that has
been shown to be involved in C-type inactivation (Hoshi et al.,
1991
). The RNAs were transcribed using T7 RNA polymerase
(Ambion, Austin, TX) and injected into the oocytes (40 nl/cell).
Recordings were typically made 1 to 14 d after injection.
Electrophysiological Recording
In most experiments, the single channel and macroscopic patch
currents were recorded using excised configurations of the patch-clamp technique (Hamill et al., 1981; Methfessel et al., 1986
)
with an AxoPatch 200A amplifier (Axon instruments, Foster City,
CA). The macroscopic patch currents were low-pass filtered
through an eight-pole Bessel filter unit with 2 kHz corner frequency (Frequency Devices, Haverhill, MA) and digitized at 10 kHz using an ITC16 computer interface (Instrutech, Great Neck,
NY). The whole oocyte currents were recorded using a Warner
OC-725B (Warner, Hamden, CT) two-electrode voltage clamp
amplifier. The electrodes filled with 3 M KCl had a typical resistance of less than 0.5 M
. The data were collected and analyzed
using Pulse/PulseFit (HEKA, Lambrecht, Germany), IGOR PRO
(Wavemetrics, Lake Oswego, OR), TAC (SKALAR, Seattle, WA),
and DataDesk (DataDescriptions, Ithaca, NY) programs running
on Apple Macintosh computers. Unless otherwise indicated, the
linear capacitative and leak currents have been subtracted from
the macroscopic currents presented using a modified P/n protocol. The single channel data were idealized using the half amplitude threshold method. For the analysis of the single channel
dwell times, additional exponential components were included
only if the probability of requiring one additional component
was >0.95 using the likelihood ratio test (Hoshi et al., 1994
). Numerical simulations of the ionic currents predicted by the kinetic
schemes were performed using the Q-matrix approach (Colquhoun and Hawkes, 1995
) implemented in IGOR. The parameters
of the schemes were optimized by minimizing the chi-squared
value using the downhill simplex algorithm (Press et al., 1994
)
implemented in IGOR. When appropriate, the data values are
presented as mean ± standard deviation. The error bars are not
shown when smaller than the symbol size. All experiments were
performed at room temperature (20-24°C).
Solutions
The intracellular solution typically contained (in mM): 140 KCl, 11 EGTA, 10 HEPES, pH 7.2 (titrated with n-methyl-D-glucamine [NMG]). The extracellular solution typically contained (in mM): 140 NaCl, 2 MgCl2, 2 KCl, 10 HEPES (NMG), pH 7.2. The high K+ extracellular solution contained (in mM): 140 KCl, 2 MgCl2, 10 HEPES (NMG), pH 7.2. Other solutions used are indicated in the legends.
Nifedipine (Sigma Chemical Co., St. Louis, MO), Bay K 8644 (RBI, Natick, MA), and nimodipine (Sigma) were dissolved in 100% ethanol (10 mM). Nicardipine (Sigma) was dissolved in 100% methanol (5 mM). The stock solutions were kept in the dark at <4°C. The drug solutions were prepared fresh from these stock solutions and vortexed immediately before each use.
The ShB6-46 T449V channel contains a deletion in
the amino terminus to disrupt N-type inactivation
(Hoshi et al., 1990
). In addition, threonine at position
449 (in ShB numbering) in the P-segment is mutated to
valine to disrupt or drastically slow C-type inactivation
(Lopez-Barneo et al., 1993
). Effects of DHPs were primarily assayed using this ShB
6-46 T449V channel since the gating transitions are simplified with the N- and
C-type inactivation mechanisms disrupted. Because internal Mg2+ ions are known to induce voltage-dependent block of currents through Shaker -like potassium
channels (Ludewig et al., 1993
), all data presented were
recorded in the absence of internal Mg2+ ions to exclude interference with divalent block in interpreting the DHP effects.
Nifedipine Induces Apparent Inactivation
Application of nifedipine to the intracellular side induced a time-dependent decline in the ionic currents
through the ShB6-46 T449V channels. Fig. 1 A shows
the effects of intracellular nifedipine (50 µM) on the
ShB
6-46 T449V K+ currents elicited in response to depolarizing voltage pulses. The amplitudes of the control
ShB
6-46 T449V currents did not decline appreciably
with maintained depolarization. Most of the reduction
in the control current amplitude observed with maintained depolarization is attributable to accumulation of
K+ ions in the extracellular space immediately adjacent
to the channels as judged by the changes in the reversal
potential of the tail currents with time (data not shown).
In the presence of nifedipine, the currents underwent
marked apparent inactivation at the positive voltages
where the activation process is fast, suggesting that nifedipine exerts its action after the channel opens. The
current-voltage (I-V) curves indicate that the reduction
of the current amplitude by nifedipine is apparently
voltage dependent (Fig. 1 B).
The effect of nifedipine application to the intracellular side to induce apparent inactivation had a very fast
onset, much faster than the limit of the manual bath
perfusion system employed. The effect was readily and
fully reversible. Applications of nifedipine to the extracellular side were less effective than those to the intracellular side (Fig. 2 A). Extracellular applications of nifedipine (100 µM) often induced a time-independent decrease
in the current amplitude (inset traces in Fig. 2 A). These
observations indicate that nifedipine works preferentially from the intracellular side. Considering the nonpolar nature of nifedipine, the effects of extracellular
application were probably mediated by its movement through the cell membrane.
DHPs are light-sensitive and the effects on voltage-
dependent Ca2+ channels can be markedly diminished
by UV-light (Meyer et al., 1984). We examined whether
the nifedipine effects on the K+ channels could be diminished by UV-light. We found that UV-treated nifedipine (254 nm, 18 W, for 90 min at 1 cm) was much
less effective in producing the apparent inactivation of
the ShB
6-46 T449V currents (Fig. 2 B). The vehicles
(ethanol, methanol) did not induce the inactivation at
the concentrations used.
Other Dihydropyridines Are Also Effective
In addition to nifedipine, other DHPs are also effective
in inducing apparent inactivation in the Shaker channels.
Fig. 3 compares the intracellular applications of nimodipine, nicardipine, and BAY K 8644 (+,) on the
ShB
6-46 T449V currents elicited in response to the
voltage pulses to +50 mV. As with nifedipine, these
DHPs induced apparent inactivation in a reversible
manner when applied to the intracellular side. Extracellular applications of these drugs were also much less
effective (data not shown).
Concentration Dependence of DHP Block
The currents recorded at +50 mV in the presence of
various concentrations of nifedipine are shown in Fig. 4
A. Fig. 4 C-F show the concentration dependence of
the block of the macroscopic currents at +50 mV by
nifedipine and nimodipine. The effect of nifedipine at
+50 mV became noticeable at 3 µM with the half-maximal reduction in the steady-state current occurring at
~30 µM (Fig. 4 C). For nimodipine, the half-maximal
reduction was observed at a slightly lower concentration
(Fig. 4 E). With greater concentrations, the apparent
inactivation time course became faster and the steady-state current became progressively smaller. These results are again consistent with the drugs exerting the
action after the channel opens. The fractions of the
currents blocked at the end of the sweep are shown in
Fig. 4 C (nifedipine) and E (nimodipine). The time
constants of the current decline are shown in Fig. 4 D
(nifedipine) and F (nimodipine). These plots illustrate
a slight difference in the efficacies of nifedipine and nimodipine. The Hill plot transformations of the block
data (Fig. 4 B) indicate that the concentration dependence of both nifedipine and nimodipine block has the
Hill coefficient of 1.5, consistent with more than one DHP molecule binding to the channel.
Nifedipine Decreases the Mean Open Time
If nifedipine interacts with the open state of the channel, the mean open time should decrease in a concentration-dependent manner. To examine the effect of
nifedipine on the single channel level, a mutant Shaker
channel, ShB6-46 T449V:A463I, which contains a single amino acid substitution at position 463 (Hoshi et al., 1991
) in the ShB
6-46 T449V background was used. The
A463I mutation increases the single channel amplitude
by 50%, and it increases the mean open time by 20-30-fold. Thus, this channel is well suited to assay the DHP
effects. Representative openings of a single ShB
6-46
T449V:A463I channel recorded at 0 mV are shown in
Fig. 5 A. Application of nifedipine decreased the mean
open time in a concentration-dependent manner (Fig.
5, B and C). The reduction of mean open time is consistent with nifedipine interacting with the open state of
the channel. The amplitude of the main conductance state of the channel was not markedly affected by nifedipine application. Although we observed the substates
more frequently in the presence of nifedipine, we did
not systematically investigate this issue.
The closed durations recorded from the data shown
in Fig. 5 are compared in Fig. 6. In the absence of the
drug, the closed durations were described by a sum of
at least three exponentials. The shortest exponential
component with a time constant value of 200-300 µs
reflects the short burst closures as observed for the
ShB6-46 channel, Cf (Hoshi et al., 1994
). The second
component with a time constant value of 2-4 ms reflects the intermediate closed state, Ci (Hoshi et al., 1994
).
In addition to these closed duration components, similar to those already documented for the ShB
6-46 (Hoshi
et al., 1994
), the closed durations from the ShB
6-46 T449V:A463I channel also showed an additional component with a time constant value of 50-200 ms with a
small fractional amplitude (3% in the data shown).
Nifedipine (100 µM) did not introduce an additional
closed duration component in a statistically significant way but noticeably increased the relative amplitude of
the third component (from 3% to 11% in Fig. 6). It is
possible that nifedipine did induce an extra component, but the new time constant was too similar to one
of the existing time constants to be detected. Similar results were obtained from two other single-channel
patches.
Interaction between the Nifedipine Block and N- and C-type Inactivation
Since nifedipine induces apparent inactivation, we studied whether nifedipine interacts with N-type or C-type
inactivation. We first examined the possible interaction
between nifedipine and the N-type inactivation mechanism by using the ShD channel (Timpe et al., 1988),
which exhibits fast N-type inactivation. As described
earlier (Choi et al., 1991
), in the presence of internal tetraethylammonium (TEA) (2 mM), the current decline is slower since the TEA-bound ShD channels cannot undergo N-type inactivation, and a cross-over of the
scaled current time courses was observed (Fig. 7 A). In
the absence of TEA, the time course of the current decline was well fitted by a single exponential with a time
constant of 9 ms whereas with TEA, a sum of two exponentials with time constant values of 2 and 26 ms was
required to fit the current decline. In contrast, decline
of the ShD current in the presence of nifedipine was
not slower than the control current and the scaled currents superimposed well (Fig. 7 B). Because the apparent on and off rates of nifedipine are slower than those
of TEA (see Fig. 1), the results do not necessarily show
that N-type inactivation and nifedipine do not compete.
We further examined whether nifedipine and the
C-type inactivation mechanism compete by using the
ShB6-46 T449K channel. This channel mutant has a
41-amino acid deletion in the amino terminus to disrupt N-type inactivation (Hoshi et al., 1990
). In addition, it also contains a single amino acid mutation at position 449 from threonine to lysine to accelerate C-type
inactivation (Lopez-Barneo et al., 1993
). If C-type inactivation and nifedipine compete, it is expected that the
time course of the current decline in the presence of
nifedipine is slower because the nifedipine-bound channels are prevented from undergoing C-type inactivation. Representative currents through the ShB
6-46
T449K channels in control and in presence of 100 µM
nifedipine are shown in Fig. 7 C and rate constants obtained from two exponential current fits are shown in
Fig. 7 D. In the presence of nifedipine (100 µM), the
current decline was faster than that in the absence of
the drug. The results suggest that nifedipine and C-type
inactivation do not compete.
If nifedipine induces the apparent inactivation in the
ShB6-46 T449V channel by enhancing the residual
C-type inactivation mechanism, the nifedipine-induced
inactivation should display properties similar to those of
C-type inactivation. For example, the nifedipine-induced
inactivation should slow down in the presence of high
external K+ as C-type inactivation is slowed by high external K+ (Lopez-Barneo et al., 1993
). We tested this
hypothesis by comparing the efficacies of the nifedipine block in the presence of different external K+
concentrations. As compared in Fig. 7 E, the time
course of the nifedipine block with 140 mM external
K+ was not slower than that observed with 2 or 50 mM
external K+. Thus, the nifedipine-induced inactivation
and C-type inactivation differ in their sensitivities to external K+, suggesting that they are mediated by distinct
mechanisms. This does not, however, rule out the possibility that nifedipine and C-type inactivation interact
as suggested by the results obtained with the ShB
6-46
T449K channel (Fig. 7, C and D).
Voltage Dependence of the DHP Block
Because nifedipine is not a charged molecule, it was
not expected that its action would be voltage dependent even if it might position itself in the membrane
electric field. We examined the possible voltage dependence of the DHP action as shown in Fig. 8. The channels were opened first in response to a voltage pulse to
+50 mV to allow the nifedipine molecules to block the
channels. Then, the membrane voltage was changed to
a variety of voltages where the steady-state probability
of the channel being open is still saturated (Zagotta et
al., 1994a). If the DHP action is voltage independent,
the only instantaneous and time-independent changes
in the current, reflecting the changes in the driving force, should be observed, as recorded under control
conditions (Fig. 8 A). The presence of the time-dependent relaxation of the current would suggest that the
drug action is voltage dependent. The results in Fig. 8,
B-D do show such time-dependent relaxation. The
time courses of the current relaxations were fitted with
single exponentials and the ratios of the extrapolated
instantaneous current amplitudes to the steady state
currents are shown in Fig. 8 E. DHPs became less effective in blocking the current with greater depolarization. As a data description parameter for the voltage dependence, we chose the number of apparent equivalent charges (n). The value n was obtained by fitting
the relative block data (Fig. 8 E) with the following
function
![]() |
where V is membrane voltage, other constants have
their usual meanings. Examples of this fit for 25 and 50 µM of nifedipine block of ShB6-46 T449V current are
shown in Fig. 8 E as the solid lines. The apparent
charge number is plotted against the DHP concentration in Fig. 8 F. The voltage dependence increased with
the drug concentration. The data also show a difference in the voltage dependence of nifedipine and nimodipine blocks of the ShB
6-46 T449V currents as
well as a difference in the voltage dependence of nifedipine block of the ShB
6-46 T449V current (Fig. 8 F,
circles) and the ShB
6-46 T449V:A463I current (squares).
Nifedipine Interaction with Ion Flow through Channel
The results presented so far suggest that nifedipine interacts with the open state of the channel to impede
the ion flow. One possible mechanism is that nifedipine physically occludes the ion conducting pore,
acting as an open channel blocker. Several molecules
have been shown to act as open channel blockers for voltage-dependent K+ channels, including TEA, and
the Shaker inactivation peptide (Demo and Yellen, 1991).
If nifedipine acts as an open channel blocker, K+ flux
through the channel should regulate the block efficacy.
The effects of the direction of the net K+ flow on the
nifedipine efficacy are shown in Fig. 9. With 140 mM
K+ outside, the effects of nifedipine on the net inward
and outward currents were measured at +30 mV with 0 mM and 140 mM K+ inside, respectively. The reduction
in the steady state current caused by nifedipine was not
markedly altered by the changes in the net current
flow, suggesting that DHP efficacy is independent of
the direction of the net current flow.
Regulation of Nifedipine Efficacy by Mutation A463I in the S6 Segment
Residue 463 in the S6 segment of the ShB channel has
been shown to be involved in regulation of C-type inactivation (Hoshi et al., 1991; Lopez-Barneo et al., 1993
).
In ShB
6-46 T449V, where both the N- and C-type inactivation mechanisms are disrupted, mutating the residue
463 from A to I (ShB
6-46 T449V:A463I) causes the
mean open time and the single channel amplitude to
increase (see Fig. 5). Consistent with the slow channel
closing rate, the macroscopic G(V) curve of this channel is shifted to a more negative direction by ~40 mV
compared with that of ShB
6-46 or ShB
6-46 T449V
(data not shown). We found that the effects of nifedipine on ShB
6-46 T449V:A463I are distinct from those
on ShB
6-46 T449V. The effects of nifedipine on the
ShB
6-46 T449V:A463I channel differ in three major
areas: steady-state fraction of the current blocked, recovery from the nifedipine block, and voltage dependence of the block. Nifedipine (100 µM) was less effective in blocking the ShB
6-46 T449V:A463I channel
than the ShB
6-46 T449V channel. Fig. 10 B compares
the concentration dependence of nifedipine block of
the ShB
6-46 T449V (circles) and ShB
6-46 T449V:
A463I (squares) currents, also showing that nifedipine is
less effective on the ShB
6-46 T449V:A463I channel. A
difference in the voltage dependence of nifedipine
block of these two channel types is shown in Fig. 8 F
using the same symbols.
Nifedipine differentially altered the tail currents
through the ShB6-46 T449V and ShB
6-46 T449V:
A463I channels. The tail current was slowed by nifedipine in the ShB
6-46 T449V channels (Fig. 10 C). In
contrast, nifedipine accelerated the time course of deactivation in the ShB
6-46 T449V:A463I channel at all
the voltages examined (
100 to
140 mV).
The results presented show that DHPs, such as nifedipine
and nimodipine, reduce the ionic currents through the
Shaker K+ channels. Although higher concentrations of
DHPs are required for inhibition of the Shaker channels
than for some voltage-dependent Ca2+ channels (Hess,
1990), the DHP effects on the Shaker channels are still
specific and require the intact dihydropyridine structure since the UV-treated DHPs largely lose their ability
to reduce the current. The DHP efficacy is dependent
on voltage, but independent of the direction of the K+
flow.
Our consideration of the possible biophysical and
molecular mechanisms underlying the DHP action on
the Shaker channels is based on the description of their
gating process in Zagotta et al. (1994b). This scheme
can be presented in a simplified form as the following:
Scheme I.
where CR represents the numerous closed states that
the channel visits before opening, and Cf represents the
closed state(s) that the channel enters after opening.
At positive voltages, the value of the reverse rate constant * is small, and the fast transitions between Cf and
O can be assumed to be at equilibrium. This scheme Is
well suited for the description of the gating behavior of
ShB
6-46 T449V, which shows neither C- or N-type inactivation in Xenopus oocytes (Hoshi et al., 1990
; Lopez-Barneo et al., 1993
). Although in this scheme, activation of the channel is represented with a single transition for simplicity, in numerical simulations we used
the complete model as described in Zagotta et al.
(1994
b). Values of all the rate constants were taken
from Zagotta et al. (1994
b). All the blocking schemes
considered below are based on SCHEME I as they are reduced to this scheme In the absence of drug.
The simplest hypothesis that the DHP molecule binds to the open conducting state of the channel (O) only can be represented by the following scheme:
[View Larger Version of this Image (132K GIF file)]Scheme II.
where DHP·O represents the drug-bound non-conducting open state. According to this scheme, the mean open time should decrease in a concentration-dependent manner in the presence of nifedipine. The mean open time was indeed decreased by nifedipine as shown in Fig. 5. Furthermore, according to SCHEME II, the reciprocal of the mean open time should be linearly related to the DHP concentration by the following relationship:
![]() |
assuming that * = 0 at positive voltages. This linear
prediction did not hold for all the concentrations of
nifedipine examined (Fig. 5 C). At the high concentrations tested (>40 µM), the block was more than predicted, raising some implications about the nature of
the block (see below). Furthermore, this model does
not provide any mechanism for the voltage dependence of the block. To improve this model, we could
assume that kon and/or koff in SCHEME II are voltage dependent. Alternatively, we can assume that the voltage dependence of the block comes from a voltage-dependent gating property of the channel rather than voltage-dependent binding of the drug. The rate constant
for the Cf to O transition is voltage dependent (Zagotta
et al., 1994
b), decreasing with hyperpolarization with
an equivalent charge of 0.17, similar to the voltage dependence of the DHP action (Fig. 8 F). Thus, the following scheme where the DHP molecule binds to the
Cf state from which the exit rate constant
is voltage
dependent could be considered:
Scheme III.
An obvious drawback of this scheme Is that it does not predict the concentration-dependent mean open time. This can be resolved by combining SCHEMES II and III into a single scheme:
[View Larger Version of this Image (135K GIF file)]Scheme IV.
This model has four more free parameters than SCHEME I, and it can account for the effect of DHP on the mean open time and the voltage dependence of the block. This model, however, does not describe the concentration dependence curve well (Fig. 4) and it predicts a Hill coefficient of 1 as opposed to the observed higher value (Fig. 4 B), which requires binding of more than one DHP molecules to the channel. Thus, we introduce the following model:
[View Larger Version of this Image (314K GIF file)]Scheme V.
(SCHEME v)
Time constants of the current decline, voltage dependence of the block and the concentration dependence
predicted by SCHEMES II and V were simulated and
shown superimposed on the experimental data (Figs. 4,
8, and 10). For these computations, all parameters in
the absence of drug were fixed at the values given in
Zagotta et al. (1994b). Only the block/unblock rates included in each scheme were adjusted by minimizing
the chi square value to simultaneously fit the steady-state fractional block (Fig. 4, C and E) and the time
constant of the current decline in the presence of nifedipine and nimodipine (Fig. 4, D and F). Table I shows
the parameter values used to simulate the data. The
predictions of SCHEME V with two DHP binding steps
are markedly better than those of SCHEME II with one
binding step (Fig. 4). With the rate constant values obtained from fitting the concentration dependence,
SCHEME V also reproduces the voltage dependence of
the block even though the parameters were not specifically optimized to fit the voltage dependence (Fig. 8 F).
Table I. Numerical Values of the Rate Constants Used to Simulate DHP Block |
The prediction of SCHEME V about the concentration dependence of the mean open time, which is determined by constant konA, is consistent with initial slope of the curve shown in Fig. 5 C. The DHP binding to the fast closed state Cf in SCHEME V could also account for the non-linear concentration dependence of the mean open time (Fig. 5 C). Because the mean dwell time in Cf is short, many of the transitions to this state are unresolved. The DHP binding to the Cf state will increase its mean dwell time, thus reducing the number of unresolved events, which in turn could result in an apparent non-linear dependence of the mean open time on DHP concentration at high concentrations.
The model in SCHEME V can be used to explain the altered drug sensitivity of the ShB6-46 T449V:A463I
channel. In the ShB
6-46 T449V:A463I channel, the occupancy probability in the Cf state is lower than that in
the ShB
6-46 T449V channel as this channel has a
greater mean open time. Thus, according to SCHEME V,
where the second DHP molecule binds only to the Cf
state, the ShB
6-46T449V:A463I channel should be less
sensitive to DHP than the ShB
6-46 T449V channel, as
opposed to SCHEME ii, in which the increased occupancy of the open state should favor increased blocking. This prediction is consistent with the data obtained
(see Fig. 10), where ShB
6-46T449V:A463I is less sensitive to DHP. The shift in G(V) curve of the ShB
6-46T449V:A463I channel could be well represented by
decreasing the rate constants of the transitions away
from the open state (
and O-Cf as given in the model
of Zagotta et al., 1994
b) by a factor of 10, thus stabilizing the open state by ~1.3 kcal/mol. The concentration dependence given by SCHEME V with the block
rates obtained from the ShB
6-46T449V data with the
open state stabilized by 1.3 kcal/mol is shown in Fig. 10
B. With those values, SCHEME V produces less block,
which is consistent with the observed effect. We consider the results of the simulations based on SCHEME V
to be in qualitative agreement with the observed effects. SCHEME V reasonably describes the concentration
and voltage dependence of DHP block as well as the effect of A463I mutation. There are ways to further increase the agreement between the model prediction
and the experimental data. For example, it is possible
to improve the general fit by assuming that allosteric interactions increase the rate of DHP binding to the Cf
(DHP
B) state compared with that to the O state. Since
the available experimental data did not specifically discriminate the various possible allosteric models, we did
not consider them further.
The observation that DHP is not likely to compete
with C-type inactivation (Fig. 6) suggests that C-type inactivation itself can occur from the fast closed state,
which is stabilized by the bound DHP molecule. Because C-type inactivation is slowed by K+ ions (Lopez-Barneo et al., 1993; Baukrowitz and Yellen, 1996
), it is
reasonable to speculate that C-type inactivation occurs from the fast closed state or other nonconducting states
(Baukrowitz and Yellen, 1996
). Baukrowitz and Yellen
(1996)
showed that internal TEA derivatives enhance
C-type inactivation by emptying K+ ions from the pore.
Similarly, DHP may have two effects on the Shaker channel. First, binding of the DHP itself to the channel decreases the K+ flux. The reduced K+ ion occupancy in
the channel pore in turn increases the residual C-type
inactivation rate.
Although a mutation in the S6 segment affected the
DHP efficacy (Fig. 10), it is likely that other amino acid
residues are also involved in regulating the DHP efficacy. For example, considering that DHP preferentially
works from the intracellular side, the residues in the internal mouth of the channel may also be involved. Those amino acid residues in the S5, P, and S6 segments shown to be important for the 4-aminopyridine
block (Kirsch et al., 1993) are also good candidates for
involvement in the DHP sensitivity. However, since the
DHP efficacy does not appear to be markedly influenced by the net K+ flux (Fig. 9), the DHP molecules
probably do not lie directly in the K+ flux pathway.
A large number of dihydropyridines have been synthesized and these drugs appear to have different potencies in blocking the Shaker potassium channel (Fig. 3; unpublished observation). At the same concentration, nimodipine appears be more effective than nifedipine (Fig. 4). However, minor quantitative changes in the rate constants of the model in SCHEME V can account for the observed differential potency, suggesting that the underlying biophysical mechanisms are the same. Furthermore, by comparing the effects of different dihydropyridines with different side groups, it should be possible to gain information on which structural components of the drug are critical in determining the different rate constants of the model.
Several µM of nifedipine was required to observe noticeable effects on the ShB channel currents at +50 mV whereas the drug's effects on L-type Ca2+ channels may be observed in the nM range. Despite the higher concentration required to affect the Shaker potassium channels, the effects are specific in that the UV-treated nifedipine is ineffective. Because the DHP action on Shaker channels is enhanced by hyperpolarization, it is possible that the efficacy may be greater under physiological conditions where the transmembrane potential may not reach +50 mV. It is also possible that other homologues of Shaker channels show much higher DHP sensitivities than the channels examined in this study. Given the structural similarities found in different voltage-dependent ion channels, it will be interesting to rigorously compare the biophysical and molecular mechanisms of the DHP action on voltage-dependent Ca2+ and K+ channels.
Original version received 9 May 1996 and accepted version received 21 October 1996.
Address correspondence to Toshinori Hoshi, Department of Physiology and Biophysics, The University of Iowa, Bowen 5660, Iowa City, IA 52242. Fax: 319-335-7330; E-mail: Toshinori-Hoshi{at}uiowa.edu
T. Hoshi was supported in part by Klingenstein Foundation, McKnight Foundation and American Heart Association (96014400). E.F. Shibata is an Established Investigator of the American Heart Association.We thank J. Kabat and J. Thommandru for technical assistance. We also thank K. Larson and A. Kamath for performing some of the early experiments and Ray Dietrich for the instrument design.