From the Department of Cell Biology and Physiology, Washington University School of Medicine, St. Louis, Missouri 63110
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ABSTRACT |
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Inward rectification induced by mono- and diaminoalkane application to inside-out membrane
patches was studied in Kir2.1 (IRK1) channels expressed in Xenopus oocytes. Both monoamines and diamines block Kir2.1 channels, with potency increasing as the alkyl chain length increases (from 2 to 12 methylene
groups), indicating a strong hydrophobic interaction with the blocking site. For diamines, but not monoamines,
increasing the alkyl chain also increases the steepness of the voltage dependence, at any concentration, from a
limiting minimal value of ~1.5 (n = 2 methylene groups) to ~4 (n = 10 methylene groups). These observations
lead us to hypothesize that monoamines and diamines block inward rectifier K+ channels by entering deeply into
a long, narrow pore, displacing K+ ions to the outside of the membrane, with this displacement of K+ ions contributing to "extra" charge movement. All monoamines are proposed to lie with the "head" amine at a fixed position in the pore, determined by electrostatic interaction, so that z is independent of monoamine alkyl chain length.
The head amine of diamines is proposed to lie progressively further into the pore as alkyl chain length increases,
thus displacing more K+ ions to the outside, resulting in charge movement (z
) increasing with the increase in
alkyl chain length.
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INTRODUCTION |
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The last five years have seen much progress in our understanding of inward rectifier K+ channel function.
Beginning with the cloning of ROMK1 (Kir1.1; Ho et al.,
1993) and IRK1 (Kir2.1; Kubo et al., 1993
), several related families of inward rectifier K+ (Kir)1 channel subunit genes have been cloned (see Nichols and Lopatin, 1997
). These channel subunits differ in structure from
the previously characterized voltage-gated (Kv) channel subunits in possessing only two, rather than six,
transmembrane segments and lacking the highly positively charged S4 transmembrane segment, which serves
as a voltage sensor in the Kv channels (Liman et al.,
1991
). Inward rectification is distinct from the voltage
dependence of Kv channels in that conductance is not
a function of the absolute membrane potential, but is
instead related to the K+ reversal potential and is conferred by diffusible intracellular compounds, in particular the polyamines spermine and spermidine (and to
a lesser extent putrescine and Mg++) (Matsuda et al.,
1987
; Vandenburg, 1987; Fakler et al., 1994
; Ficker et al.,
1994
; Lopatin et al., 1994
, 1995
). Strong rectification conferred by these compounds correlates with the presence of negatively charged residues in certain positions
in the second (M2) transmembrane segment and in
the COOH-terminal region (Taglialatela et al., 1995
;
Yang et al., 1995
). Mutating these residues to uncharged residues decreases or abolishes polyamine-induced rectification (Yang et al., 1995
). Likewise, mutation of corresponding neutral residues to negatively charged residues confers polyamine-induced rectification on channels that originally do not display strong rectification
(Fakler et al., 1994
, 1995
; Lopatin et al., 1994
; Glowatzki et al., 1995
; Shyng et al., 1997
). Experiments that reduce the levels of endogenous polyamines in situ reduce the rectification of Kir currents expressed in
those cells (Bianchi et al., 1996
; Shyng et al., 1996
),
confirming the physiological relevance of polyamine-induced rectification.
Much evidence is consistent with the concept that
rectification results from positively charged ions binding to negatively charged residues in the channel. However, not all charged or polar molecules can block Kir
channels. Lopatin et al. (1994) examined several related compounds and found that only the polyamines
conferred strong rectification, while other bulkier, dipolar or nonlinear molecules (e.g., GABA, creatinine,
lysine) failed to block at a concentration of 100 µM.
These results indicate that a molecule must possess
both the correct structure and charge density or distribution to confer strong rectification. Because the most energetically favorable conformation of endogenous
polyamines in free solution is an extended linear chain
(Romano et al., 1992
), it seems likely that these molecules enter the long pore of the channel and lie in the
pore to block it. In the present study, we have systematically measured the ability of a series of alkylamine analogues to block current flow through Kir channels to
determine relevant structural features of polyamines
in inducing inward rectification. We have examined
monoaminoalkanes and diaminoalkanes with alkyl
chains composed of 2-12 methylene groups, and find that changes in molecular length and charge have profound effects on channel block, with longer chain
lengths increasing blocking affinity. Thus, the increased blocking affinity of spermine and spermidine,
when compared with diamines, may not be attributed solely to increased blocker charge. Instead, we propose
a model in which aminoalkanes act as "long pore
plugs," displacing K+ ions to the outside as they enter
the pore and binding both by electrostatic and hydrophobic interactions.
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METHODS |
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Expression of Channels
Kir 2.1 was propagated in pBluescript SK(). cRNA was prepared using Message Machine kits (Ambion Inc., Austin, TX). Xenopus oocytes were isolated using conventional techniques and
pressure injected with (50-100 nl) cRNA (1-100 ng/ml). Oocytes were maintained at room temperature in ND96 solution
(2 mM KCl, 96 mM NaCl, 1 mM MgCl2, 5 mM Na-HEPES) with
1.8 mM CaCl2, supplemented with penicillin (100 U/ml) and
streptomycin (100 µg/ml). Channels were allowed to express for
at least 18 h after injection. Before recording, each oocyte was incubated in a hypertonic solution (60 mM KCl, 10 mM EGTA, 40 mM HEPES, 250 mM sucrose, 8 mM MgCl2, pH 7.0) for ~10 min,
and the vitelline membrane was removed manually.
Electrophysiology
Oocytes were placed in a chamber continuously perfused with
"KINT" solution (see below). Recording pipettes of ~20-µm tip diameter were pulled from soft glass (World Precision Instruments, Inc., Sarasota, FL), fire polished, and coated with a mixture of mineral oil and paraffin to reduce capacitative currents. Pipettes were
filled with KINT solution and typically had series resistances from
0.2 to 0.9 M. Recordings were made with an Axopatch 1D patch clamp amplifier (Axon Instruments, Foster City, CA). Data were filtered with the amplifier's integral four-pole Bessel filter (typically at
5 kHz), digitized via a Digidata 1200 interface (typically at 10 kHz),
and stored on the hard disk of a 486 microcomputer (Gateway
2000, North Sioux City, SD) for off-line analysis. Data were also displayed on a Gould chart recorder and stored on videotape using a
Neurodata PCM digitizing interface. Data acquisition was controlled by pClamp 6 software (Axon Instruments).
After the pipettes were sealed to the oocytes, patches were excised and moved to a subchamber with a separate perfusion inlet, ensuring that endogenous polyamines released from the oocyte
did not confer rectification on the channels in the excised patch. Patches were held at 0 mV and washed with fresh KINT solution until endogenous rectification was largely washed out. Patches expressing from 0.5 to 15 nA current at 50 mV were used in
these experiments. Because several of the alkylamines tested appeared to adhere to the tubing of the perfusion system, test solutions were applied by pipetting 100-200 µl of the solution directly into the subchamber containing the patch (perfusion of
the subchamber with KINT was stopped during test solution application). When possible, several test solutions were applied to
the same patch (either different concentrations of the same compounds or different compounds). Before a new compound was
applied to the same patch, the previous compound was thoroughly washed from the patch (and currents were measured after washing to verify that wash was complete). Because inward
rectifier current "ran down" after patch excision, and because intrinsic rectification continued to wash out of the patch for several
minutes after excision, control measurements were made frequently throughout the course of an experiment.
To test the blocking affinity of a given compound, patches
were typically held at 0 mV and briefly pulsed to a "conditioning potential" (e.g., 80 mV, 5 ms) to relieve voltage-dependent
channel block, and then through a series of test potentials (e.g.,
up to 70 mV in 10-mV increments). In experiments where block
kinetics were measured, additional voltage pulses of equal amplitude were made to 0 mV after the test voltage pulses. The currents
recorded during these voltage steps were used for subtracting capacitive artifacts from the beginning of the test voltage step.
Solutions and Chemicals
KINT solution was composed as follows (mM): 140 KCl, 10 K-HEPES, 1 K-EGTA, 1 K-EDTA, pH 7.35. KD98 solution was composed as follows (mM): 98 KCl, 5 K-HEPES, 1 MgCl2, pH 7.5. Alkylamine compounds were purchased from commercial sources (Sigma Chemical Co., St. Louis, MO, and Aldrich Chemical Co., Milwaukee, WI), or were obtained from Dr. Carl Romano (Washington University School of Medicine). All compounds tested in this study contained amine groups attached to terminal methylene groups of the molecule's alkyl chain. To simplify the presentation, we refer to these compounds by using a nomenclature that indicates the number of amine groups present on the compound and the number of carbons in the alkyl chain. A given monoamine compound is referred to as MAn, and a given diamine compound is referred to as DAn, where n is the number of carbons in the alkyl chain. For example, the compound 1-aminohexane is referred to as MA6, while the compound 1,5-diaminopentane is referred to as DA5. Other compounds examined in this study included the monoamines benzylamine (phenyl methylamine, PhMA), phenylethylamine (PhEA), and the diamine p-phenylenediamine (pPhDA). Compounds were prepared as 100 mM concentrated stock solutions in H2O (pH ~7.3), and then diluted into working stock solutions of 1 mM in KINT. Serial dilutions of the 1 mM stocks were used in these experiments.
Data Analysis
To measure kinetics of channel block and unblock, data were fit
to functions of the form I = Kofs + K*exp(t/
) (single exponential) or I = Kofs + K1*exp(
t/
1) + K2*exp(
t/
2) (double exponential), where I is the current amplitude, Kofs the offset, t is time,
a time constant, and K (or K1, K2) a scaling factor. Offset coefficients were included to correct for incomplete subtraction of
leak conductances. When appropriate, capacitance currents and other time-dependent currents not associated with channel block were subtracted from the data before fitting, either by subtracting residual currents after complete channel block or rundown,
or by subtracting capacitance transients from equivalent voltage steps to 0 mV where no ionic current flows. Single exponential equations produced good fits to all relevant time-course data, except to unblock of the compound MA9, which followed a more
markedly sigmoidal time course. The data were in this case fit by
assuming four independent blocking molecules {i.e., I = [Kofs +
K*exp(
t/
)]4}. For a given concentration of a compound, relative blocking affinity was determined by fitting data to a Boltzmann equation of the form GRelative = 1/[1 + exp(Vm
V1/2)/
KSlope] + Kofs (V1/2, KSlope, and Kofs left as free coefficients),
which is equivalent to the Woodhull equation GRelative = 1/
[1+([X]/Kd(0mV))*exp(Vm
ZF/RT)] + Kofs, where
is the "electrical distance" into the transmembrane electrical field from the
cytoplasmic face, Z is the charge of the blocking particle, and Kd,
R, F, and T have their usual meanings. In these experiments, the
effective valence (Z
) was thus 25.5 mV/KSlope, and Kd(0mV) = [X]*exp(V1/2/Kslope), where [X] is the concentration of the test
compound. These equations are most useful for describing channel block by a simple first order kinetic scheme. While these
equations will not accurately describe channel block occurring by
more complicated kinetic schemes, they provide useful empirical
descriptions of channel block.
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RESULTS |
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Block of Kir2.1 Channels by Endogenous Polyamines
Kir2.1 channels were expressed at high levels in Xenopus oocytes (whole cell current >20 µA at 30 mV in
KD98). Kir2.1 current was recorded using macropatch
pipettes containing KINT solution (~150 mM K+). In
the cell-attached configuration, strongly inwardly rectifying currents were observed in response to voltage
ramps from
50 to +50 mV (Vhold = 0 mV, ramp duration = 160 ms). Immediately after patch excision, current amplitude increased and rectification decreased,
as described previously (Lopatin et al., 1994
). As patches were moved from the vicinity of the oocyte into
a fresh stream of KINT, rectification continued to decrease. The decrease of rectification was a continuous
process, and in some patches the current measured in
response to the voltage ramp became essentially ohmic
(between
50 and +50 mV). To measure channel
block by applied compounds, patches were held at 0 mV,
stepped briefly to a negative potential (typically
80 mV
for 5 ms) to relieve channel block, and then stepped to
test potentials between
80 and +70 mV in 10-mV increments, repeating every 0.5 s. Currents typically
showed some deactivation on stepping to potentials below
50 mV. Such deactivation is frequently seen in inside-out patches. Since K+ is the only monovalent ion
in the solutions, it does not appear to result from extracellular monovalent ion block (Harvey and Ten Eick,
1989; see also Nichols and Lopatin, 1997
). Deactivation occurred with a simple exponential time course, occurred more rapidly at more negative potentials, and
was not obviously affected by the presence or absence
of di- or monoamines. For test potentials at and above
+20 mV, a variable, but very slow, current inactivation was observed that was also well fit by a single exponential decay (see Fig. 1). There remains some controversy
as to whether this residual rectification results from an
incomplete washout of endogenous polyamines (Lopatin et al., 1994
, 1995
) or from a gating process that is independent of polyamine block (Aleksandrov et al.,
1996
; Shieh et al., 1996
). However, the residual process
is well resolved from the exogenous polyamine block
that we examine in the present experiments, and this
question is not considered in this paper.
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In addition to these voltage-dependent inactivation
processes, channels also commonly displayed "rundown," which occurred with variable time course from
patch to patch (as fast as 2 min for complete rundown
of current, not present at all in some patches). As shown
previously, different endogenous polyamines block Kir channels with different affinities and kinetics (Lopatin
et al., 1995). Fig. 1 illustrates channel block by 10 µM
putrescine, spermidine, and spermine. Block by these
compounds is highly voltage dependent. As considered
in detail in Lopatin et al. (1995)
, there is both a shallow
voltage dependence at very negative voltages and a
steeper component at more depolarized potentials,
which may indicate multiple binding sites; i.e., one at the
inner entrance to the channel pore, and one deeper site
within the pore. A single exponential function provides
a reasonable fit to the steeper component and may provide a reasonable quantitative description of the deep block at a single site. As shown in the relative conductance (GRelative)-voltage relations (Fig. 1 C), the steepness of block increases from putrescine (z = +2) to spermidine (z = +3) to spermine (z = +4). The following experiments used a series of mono- and diaminoalkanes
to examine the role of polyamine structure and charge
in determining biophysical blocking properties.
Alkylmonoamine Block of Kir2.1 Channels
Cytoplasmic application of short chain monoamines
(MA1-MA4) had no obvious effect on Kir2.1 currents
at concentrations up to 1 mM (at test potentials as depolarized as 70 mV). Compounds with longer alkyl chains
(5-12 methylene groups) produced voltage-dependent block of Kir2.1 current at concentrations below
100 µM. Fig. 2 illustrates block of Kir2.1 currents by
100 µM MA5, MA6, and MA7. At depolarized potentials, the time dependence of block was clearly resolved. Longer monoamines (MA8, MA9, and MA10)
also blocked Kir2.1 current in a voltage dependent
fashion, although block was increasingly more potent
with increasing alkyl chain length, and unblock at negative potentials occurred at a much slower rate. The
slower kinetics of these compounds required that different voltage protocols be used to study their blocking
properties (Fig. 3). By using longer duration conditioning pulses (20 ms) to a more negative potential (120
mV), complete channel unblock at negative potentials
was achieved, allowing kinetic and steady state measurements. Single Boltzmann functions were fit to relative conductance (GRelative)-voltage plots for 100 µM
monoamines (Fig. 4 A). The data demonstrate a very
clear dependence of blocking affinity on chain length,
V1/2 increasing by ~
10 mV per additional methylene
group (Fig. 4 B), whereas the apparent effective valence (i.e., voltage dependence) of monoamine block
(z
) was relatively constant at a value ~2.2 (Fig. 4 C).
The marked increase in affinity with chain length indicates a very significant hydrophobic component in
polyamine block.
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The current decay from residual endogenous rectification was generally well fit by a single exponential function, and the resulting time constants were voltage dependent (but typically in excess of 10 ms at +70 mV, not shown). Application of monoamines (longer than MA4) increased the rate of current decay at depolarized potentials. Blocking time constants derived from single exponential fits were voltage dependent (decreasing at depolarized potentials), and approached a minimum value at the most depolarized potentials (Fig. 5). Despite the differences in blocking affinity for the different compounds, there was little difference between the blocking rates, the block time constant at a given potential increasing slightly as the alkyl chain length increased (Fig. 5), and decreasing as blocker concentration increased (data not shown).
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The increase in blocking potency, as alkyl chain
length increases (Fig. 4), results primarily from a decreased off rate as the chain length is increased. To
compare unblock kinetics, patches were stepped from a
holding potential of 0 mV to a positive "blocking potential" (+50 mV for 10 ms), and then back to a series of
test potentials (0 to 140 mV in 20-mV increments,
Fig. 6 A). For the shortest monoamine (MA5), current
activation (i.e., unblock) was almost instantaneous
upon repolarization. For medium length monoamines (MA6, MA7, and MA8), time-dependent relaxations resulting from unblock were apparent between 0 and
40 mV (Fig. 6 A). Because little or no endogenous
current deactivation occurred at these potentials, single exponential functions could be fit directly to the
current traces. At more negative potentials, however,
current relaxations were superimposed on the endogenous voltage-dependent deactivation. Assuming that
deactivation was independent of the unblock process,
time constants for unblock were estimated after dividing the current measured in the presence of blocker by
the current measured in the absence of blocker, and
fitting a single exponential.2 Unblock time constants
decrease as an exponential function of voltage, as
shown in Fig. 6 B. Increase in chain length is accompanied by increases in unblock time constants at a given
potential. Addition of one methylene group to the alkyl
chain increases the time constant (i.e., slows unblock)
by a factor of ~10.
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Block by these alkyl monoamine compounds was fully reversible, with observed decreases in current after MA application attributable to channel rundown. MA10 also caused voltage-dependent block of Kir2.1 channels, with relief of channel block at very negative potentials. In this case, however, recovery from block was incomplete (<50%, n = 3) after washing. MA11 and MA12 also potently inhibited Kir2.1 current, but the block appeared to be voltage independent, and the current amplitude did not recover after washing the compounds from the solution (not shown). It is not clear whether the irreversibility resulted from extremely slow unblock kinetics, or from current inhibition by a distinct mechanism. MA11 and MA12 are less soluble than the shorter monoamines, are very amphipathic in nature, and could conceivably be disrupting the activity of the channels by interaction with the lipid bilayer.
Diaminoalkane Block of Kir 2.1 Channels
All diamines tested (DA2-DA12) blocked Kir2.1 channels at 100 µM. Representative currents, and current-
voltage (I-V) relations in the absence and presence of
shorter chain diamines (DA2-DA9) are shown in Fig. 7.
As with monoamines, increasing the alkyl chain length
increased the blocking affinity (Fig. 8, A and B). However, the kinetics of block and unblock were much
faster, and this was particularly obvious for longer
chain derivatives (compare DA8 in Fig. 7 with MA8 in
Fig. 3). In contrast to the behavior of monoamines, the
effective valence of diamine block increased steeply
with increases in chain length (Fig. 8 C), the longest compound (DA12) having an effective z > 4. This increase in effective z
was not obviously an artifact related to the shifted voltage dependence resulting from
the accompanying increase in potency. As shown in
Fig. 9 C, for a patch exposed to different [DA10], the
fitted z
did not change for concentrations between 1 and 100 µM. Since each diamine is divalent, the approximately monotonic increase of z
with alkyl chain
length is striking, and suggests that increasing diamine
length causes additional (i.e., non-amine) charges to
move progressively further in the voltage field.
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As shown in Fig. 9, unblock time constants were about two orders of magnitude faster for DA8 than for MA8, over a wide voltage range. At the same concentration, block time constants were also at least two orders of magnitude faster for DA8 than for MA8. Reducing DA8 concentration by 100-fold (from 100 to 1 µM) results in block time constants that are similar to the block time constants of 100 µM MA8 (Fig. 9 A). Extrapolation of block time constants to zero applied voltage suggests that even in the absence of an applied field, and where the potencies of block are similar, the relaxation rate should be about two orders of magnitude higher for the diamine. For short to medium length diamines (DA2-DA7), channel block occurred almost instantaneously upon depolarization. As the alkyl chain length of diamines was increased, however, block did occur more slowly, as seen with monoamines (Fig. 9 B). Short and medium length diamines (<DA7) unblocked very rapidly (<500 µs for complete unblock) so that unblock kinetics were only measurable for DA8-DA12 (Fig. 10 A). While diamine unblock was faster than unblock of the corresponding monoamine, a similar relationship was seen between chain length and the rate of unblock, with longer diamines unblocking more slowly than shorter diamines (Fig. 10 B).
Block of Kir2.1 by Phenyl-substituted Mono- and Diamine Derivatives: The Effect of Bulky Hydrocarbon Groups
While more than one polyamine molecule may be involved in channel block (Lopatin et al., 1995; Yang et al.,
1995
), we suggest that the steep voltage-dependent
component results primarily from a single polyamine
molecule entering deeply into the channel pore as an
extended linear molecule. However, because the alkyl
chains of mono- and diamino alkanes are not rotationally constrained, we cannot draw strong inferences
about the physical dimensions of the pore from the
effect of these compounds (since their conformations may be dramatically altered in the process of binding).
Arylamines and arylalkylamines contain aromatic groups
that confer some structural rigidity to the molecules.
PhMA (NH3-CH2-C6H5) blocked Kir2.1 current with
properties similar to the linear monoamines. Fig. 11
shows the voltage dependence of current block by 100 µM PhMA. The block was reasonably well fit by V1/2 = +36 mV. As with linear monoamines, 100 µM PhEA
was more potent (V1/2 = +28 mV) without any obvious
increase in steepness of voltage dependence (z
= 2.5 and 2.3, respectively). In contrast, pPhDA was a very
weak blocker of Kir2.1 channels. Fig. 11 shows original
current records and GRelative-V plots for currents in the
presence of pPhDA. 1 mM pPhDA inhibited currents
with z
= 1.2 and V1/2 = +56 mV.
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DISCUSSION |
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Alkylamine-induced Rectification of Kir Potassium Channels
Since the original descriptions of inward rectification
of potassium conductance (Katz, 1949; Noble, 1965
), it
has been realized that rectification is a time-dependent
process and that "activation" of the current at negative
voltages can follow a multiexponential time course
(Hagiwara et al., 1976
; Stanfield et al., 1981
; Kurachi, 1985
; Ishihara et al., 1989
; Oliva et al., 1990
). Recent
studies have shown that this intrinsic rectification is
conferred by polyamines (spermine, spermidine, and
putrescine), which block the channel from the intracellular side of the membrane (Fakler et al., 1994
, 1995
;
Ficker et al., 1994
; Lopatin et al., 1994
, 1995
; Bianchi et al., 1996
; Ishihara, 1996
; Lopatin and Nichols, 1996
;
Shyng et al., 1996
; Yamashita et al., 1996
; Oliver et al.,
1998
). The strong rectification and time-dependent activation conferred by polyamines is relieved by increase
of [K+] on the outside of the membrane (Lopatin and
Nichols, 1996
; Oliver et al., 1998
), and generally correlates with the presence of negatively charged residues
at a given position in the second (M2) transmembrane segments (Fakler et al., 1994
; Ficker et al., 1994
; Lu and
MacKinnon, 1994
; Lopatin et al., 1994
; Shyng et al.,
1997
). While at least an additional residue in the
COOH-terminal region is involved in polyamine binding (Taglialatela et al., 1995
; Yang et al., 1995
), mutating the critical M2 residues to a neutral glutamine generally decreases or abolishes polyamine-induced rectification. The potency and voltage dependence (z
) of
block increases from putrescine (z = +2) to spermidine (z = +3) to spermine (z = +4), consistent with an
electrostatic component to the blocking process. The
first, and most consistent, result to come from the
present study is that there is also a significant hydrophobic nature to the interaction. At 0 mV, the addition
of one methyl group to either mono- or diaminoalkanes increases the blocking potency ~10-fold, corresponding to ~2.4 kJ/mol. This is similar to the effect of
increasing alkyl chain length on bis-quaternary amine block of Ca-activated and sarcoplasmic reticular K+
channels (e.g., French and Shoukimas, 1981
; Miller,
1982
). Studies in squid axon by Armstrong (1969)
and
on Shaker channels by Choi et al. (1993)
, using TEA analogues with one alkyl chain of variable length, also
found that increasing the alkyl chain length increased the blocking affinity of the analogue, and that block
was decreased by increasing the extracellular K+ concentration, indicating binding in the pore.
The Mechanism of Polyamine-induced Inward Rectification: The Kir Channel Has a "Long Inner Pore" that May Accommodate Several K+ Ions, and Even the Longest Polyamines
Biophysical and theoretical analyses of inward rectifier
K channels have suggested that these channels consist
of a long narrow pore (Hille and Schwartz, 1978; Hille,
1992), and this is now confirmed by the crystal structure of the bacterial KcsA K+ channel (Doyle et al.,
1998
). We previously suggested that "long pore plugging" by polyamines may account for rectification induced by the natural polyamines (Lopatin et al., 1995
).
The simple hypothesis was that, in addition to possible
peripheral polyamine binding sites, a single polyamine
should lay "vertically" inside the long narrow pore,
binding through electrostatic interaction with a negatively charged binding site, and giving rise to the
steeply voltage-dependent component of block. As we
show here, both monoamines and diamines block
Kir2.1 channels, with potency increasing as the alkyl
chain length increases. This finding is inconsistent with
the notion that block results from a purely electrostatic interaction with residues in the channel pore, and implies that some hydrophobic interaction of the alkyl
chain stabilizes binding in the pore. Although somewhat of a surprise, the crystal structure of KcsA reveals
that the inner vestibule of the channel is actually very
hydrophobic (Doyle et al., 1998
), and this hydrophobic
vestibule is indeed large enough to accommodate a
molecule as large as spermine, in extended linear conformation (Nichols and Lopatin, 1997
).
To account for the very high z (~5.4) of spermine
block, we previously suggested that two polyamine molecules may sequentially enter the channel pore to
reach the fully blocked state. However, the strong interaction of polyamine block with external K+ ions (Lopatin and Nichols, 1996
; Oliver et al., 1998
) suggests an
alternative probability, namely that the polyamine entry into, and binding in, the channel pore "sweeps" K+
ions outwards, contributing extra charge movement to
the binding process (Ruppersberg et al., 1993). The
present results indicate a large and gradual increase in
z
(Fig. 8) of the steep part of the G-V curve, from a
limiting minimal value of z
~1.5 (n = 2 methylene
groups) to z
~4 (n = 10 methylene groups) as diamine alkyl chain length increases. It is not likely that
the number of blocking particles changes (i.e., increases) as the blocking particles become larger, so the
increase in z
cannot result from more charge being
contributed by the blocking ion. Instead, the increase
in charge associated with the blocking process may result from progressively more charge (i.e., permeating
K+ ions) being swept out of the pore as the blocking
particle increases in size (Ruppersberg et al., 1993).
Although the increase of potency of MA and DA
compounds with increasing alkyl chain length is reminiscent of the block of delayed rectifier, Ca-activated,
and sarcoplasmic reticulum K channels by substituted
quaternary ammonium ions (e.g., Armstrong, 1969;
French and Shoukimas, 1981
; Miller, 1982
), the significantly steeper voltage dependence of the block of the
Kir channels (limiting z
= ~5.4 for spermine, effective
~1.3, compare
~0.3 for quaternary ammonium
block of non-inward-rectifying K channels) may indicate significant mechanistic differences.3 The results
further contrast with those of Miller (1982)
, who found
that for a series of bis-quaternary amines (similar structures to diamines, but with trimethyl amine groups replacing the amines), z
of block initially decreased as n
increased from 2 to 5, and then increased again from 8 to 10 before saturating.
Miller's (1982) results with bis-quaternary (bisQ)
compounds could be accounted for by assuming that
the "head" charge reaches a fixed binding site within the
field, and that as the alkyl chain length increases, the
"tail" charge is pushed further and further out of the field
until it is long enough and flexible enough to bend
around and allow the tail charge to come back into the
field. The much weaker voltage dependence of bisQ
block of Maxi-K channels might suggest that bisQ compounds do not penetrate deeply into the Maxi-K channel pore. To account for the present results, we suggest
that the relevant region of the pore of the Kir channel may be much narrower than in Ca-activated K channels, such that there is a longer physical distance of restricted ion flow, and over which the electrical field
drops (Fig. 12). We hypothesize that this long pore allows even the longest DAs to remain in the field, and
does not allow the "bend-over" phenomenon seen in
the Maxi-K channel. In this case, in the absence of
polyamines, the long pore may also accommodate
more K+ ions than the short length of the pore that is
bisQ accessible in the maxi-K channel. We further suggest that the DA charges may not occupy a single point
in space or in the electric field, but may occupy a balanced position relative to the negative charges at the
"rectification controller;" i.e., the two diamine-positive charges will be equidistant from the center of the ring
of negative charges. Thus, as the alkyl chain length increases, a single DA will occupy more and more of the
available space, with more and more K+ ions being displaced to the outside of the cell, moving more charge
outwards and thereby increasing the net charge movement associated with channel block (Fig. 12). A similar
suggestion could account for the results of Fakler et al.
(1997) who observed that the apparent electrical distance for tetraalkylammonium block of Kir1.1 channels
increased from 0.83 to 0.93 to 1.44 as the alkyl chain
length increased from 2 to 3 to 4. In this case, the excess voltage dependence resulted from a slowed
blocker off rate. The maximum diameter of the inner
vestibule of the inward rectifier KcsA channel is in the
order of 10 Å, and is accessed from the cytoplasm
through a "tunnel" that is 18 Å long and ~5 Å wide
(Doyle et al., 1998
). The impotency of pPhDA (Fig. 11)
compared with hexyl-1,6-diamine (DA6, Figs. 7 and 8)
may be in part due to the steric restrictions on access of
the phenyl ring structure through the tunnel. The diameter of extended hexylamine is ~3 Å, whereas the
minimum diameter of the phenyl ring is ~5 Å, similar
to the dimensions of the access tunnel in the KcsA crystal structure (Doyle et al., 1998
).
|
If the mechanism we propose is indeed causing increased steepness of voltage dependence with increase in
diamine length, then how can we account for monamine
block having a steepness of voltage dependence that is
independent of alkyl chain length? We hypothesize that the major determinants of blocker depth within
the inner pore are the charged groups on the blocker.
Thus, for MAs, the single charge stabilizes at essentially
the same depth (i.e., at the level of the "rectification
controller" (the ring of four negative charges in the M2
segments). The increasing alkyl chain then stretches
further and further out of the pore, just as with monoQ
block of the maxi-K channel, and the K+ ion displacing
effect of increasing chain length observed with the DAs
is absent (Fig. 12). Since the major determinant of the charge associated with block by long compounds is
then due to sweeping out of K+ ions, so the present hypothesis predicts that block by triple or tetravalent species should differ from the divalent of the same length
only by the additional ~0.5 z resulting from moving the extra "middle" charge into the field. Indeed, we
may compare the block produced by DA8 to the block
of spermidine (which contains a total of seven methylene and one additional amine group). At the same concentration, the blocking potency and steepness are indeed comparable for both molecules (Fig. 8).
Conclusions
The present results, examining channel block by cytoplasmic alkylamines of different alkyl chain length, indicate that polyamine-induced steeply voltage-dependent rectification of inward rectifier channels has a significant hydrophobic component, in addition to electrostatic binding of the charged amines. Moreover, the length of the alkyl chain is an important determinant of the charge movement associated with channel block by n-alkyl-1,n-diamines. We propose a hypothesis to explain this phenomenon whereby the increasing alkyl chain displaces increasing numbers of K+ ions towards the outside of the membrane.
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FOOTNOTES |
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Address correspondence to C.G. Nichols, Department of Cell Biology and Physiology, Washington University School of Medicine, 660 South Euclid Avenue, St. Louis, MO 63110. Fax: 314-362-7463; E-mail: cnichols{at}cellbio.wustl.edu
Original version received 7 May 1998 and accepted version received 7 July 1998.
Many of the mono- and diaminoalkanes were gifts from Dr. Carl Romano. The Kir2.1 (IRK1) clone was a gift from Lou Philipson and Dorothy Hanck (University of Chicago, Chicago, IL).This work was supported by the National Institutes of Health (NIH) grant HL/NS54171 to C.G. Nichols, and an NIH Cardiovascular Training Grant Fellowship to W.L. Pearson.
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Abbreviations used in this paper |
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DA, diamine; Kir, inward rectifier K+; MA, monoamine; PhEA, phenylethylamine; PhMA, phenyl methylamine; pPhDA, p-phenylenediamine.
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