From the UCLA Cardiovascular Research Laboratory, * Department of Medicine (Cardiology) and Department of Physiology, University of California, Los Angeles, School of Medicine, Los Angeles, California 90095
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ABSTRACT |
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Inward rectifying K channels are essential for maintaining resting membrane potential and regulating excitability in many cell types. Previous studies have attributed the rectification properties of strong inward rectifiers such as Kir2.1 to voltage-dependent binding of intracellular polyamines or Mg to the pore (direct open channel block), thereby preventing outward passage of K ions. We have studied interactions between polyamines and the polyamine toxins philanthotoxin and argiotoxin on inward rectification in Kir2.1. We present evidence that high affinity polyamine block is not consistent with direct open channel block, but instead involves polyamines binding to another region of the channel (intrinsic gate) to form a blocking complex that occludes the pore. This interaction defines a novel mechanism of ion channel closure.
Key words: spider venom toxin; polyamine; inward rectification; K channels ![]() |
INTRODUCTION |
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Strong inward rectifier K channels such as Kir2.1
(IRK1) play a key role in setting the resting membrane
potential and regulating excitability in heart, neurons,
and many other cell types (Doupnik et al., 1995). The
strong inward rectification property has been previously attributed to intracellular Mg and polyamines directly plugging the channel pore at membrane potentials positive to the K equilibrium potential, causing
voltage-dependent open channel block (Matsuda et al.,
1987
; Vandenberg, 1987
; Ficker et al., 1994
; Lopatin
et al., 1994
; Stanfield et al., 1994
; Taglialatela et al.,
1994
; Fakler et al., 1995
). Detailed analysis of block by
spermine, however, requires a fairly complex model to
account for all the voltage dependence and kinetic features (Lopatin et al., 1995
). Moreover, open channel
block as the sole mechanism of inward rectification has
been questioned in a recent study in which Kir2.1 channels were reconstituted into lipid bilayers (Aleksandrov
et al., 1996
). In the absence of Mg or polyamines, the
channels still exhibited rectification. Mg or polyamines
enhanced inward rectification, but the mechanism of
block was more complex than could be modeled by
simple open channel block.
We have previously described an intrinsic pH-sensitive gating mechanism of inward rectification in Kir2.1
in the absence of Mg or polyamines (Shieh et al.,
1996). This was manifested as a persistent voltage-
dependent inactivation of outward currents in excised
patches after prolonged washout in polyamine- and
Mg-free solution, which was accelerated by raising intracellular pH to 9.0. The intrinsic gating mechanism is
kinetically much slower than polyamine or Mg block
and is most obvious only with large depolarizations (to
more than +40 mV), explaining why it was only subtly
apparent in some previous studies and assumed to represent residual polyamine block (Ficker et al., 1994
; Lopatin et al., 1994
).
Spider venom polyamine toxins, philanthotoxin and
argiotoxin, have been shown to block N-methyl-D-aspartate receptors extracellularly (Brackley et al., 1993; Williams, 1993
; Donevan and Rogawski, 1996
). Since
polyamines cause inward rectification in Kir channels, we investigated the effects of these toxins on the strong
inward rectifier channel Kir2.1 (IRK1) to obtain further mechanistic insight into polyamine-induced inward rectification. The toxins are structurally similar to
spermine at one end and have a bulky hydrophobic aromatic group at the other (see Fig. 1 A). By studying competitive interactions between polyamines and these
polyamine toxins, we present evidence that polyamine
block is more complex than direct open channel block.
Our results suggest that direct open channel block accounts for low affinity block by spermine and spermidine, but high affinity block by these polyamines involves their binding to another region of the channel
(intrinsic gate) to form a blocking complex that occludes the pore.
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METHODS |
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Molecular Biology
In vitro T7-transcribed cRNA (Ambion Inc.) was injected (50-
5,000 ng) into stage V-VI Xenopus oocytes isolated by partial ovariectomy under tricaine anaesthesia. PCR mutagenesis was carried out by the overlap extension technique (Ho et al., 1989).
Electrophysiology
Patch clamp studies were performed at room temperature on giant excised inside-out patches, as described previously (Shieh et al.,
1996). The patch electrode contained (mM): 100 KOH, 1.8 CaCl2, and 5 HEPES, pH 7.4 with 100 MES. The standard bath solution contained (mM): 100-104 KCl + KOH, 5 EDTA, and 5 HEPES, adjusted to pH 7.2 with MES. Polyamines and polyamine
toxins were added directly to the bath solution. Rapid bath solution changes (<1 s) were accomplished using a rapid solution
exchange device (Shieh et al., 1996
). All current traces shown in
the figures were leak corrected by subtracting currents recorded
with 30 mM tetraethylammonium in the bath solution, which
blocked Kir2.1 currents completely (Shieh et al., 1996
). Curve-fitting and model simulations were performed using Sigmaplot version 2.0 (Jandel Scientific) and SCoP version 3.5 (Simulation Resources, Inc.).
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RESULTS |
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Polyamine Toxins Block Kir2.1 Channels
Kir2.1 channels were heterologously expressed in Xenopus oocytes. When applied to the cytoplasmic surface of
giant inside-out patches, both philanthotoxin and argiotoxin blocked outward currents through Kir2.1 channels in a voltage-dependent manner. At +40 mV, the
concentrations of philanthotoxin and argiotoxin producing half-maximal block (K0.5) were 47 nM (n = 4 patches), and 37 nM (n = 4), respectively (Fig. 1 C). Unlike N-methyl-D-aspartate receptors, spermine blocked
Kir2.1 channels with higher affinity (K0.5 of 5.9 nM at
+40 mV) than either toxin (Fig. 1 B). Since earlier mutagenesis studies have defined two key negatively
charged amino acids, D172 in the M2 region and E224
in the COOH terminus, as regulators of polyamine- and
Mg-induced inward rectification in Kir2.1 (Wible et al.,
1994; Taglialatela et al., 1995
; Yang et al., 1995
), we also
tested the effects of philanthotoxin on the mutants
D172N and E224G. Both mutants were considerably
less sensitive to philanthotoxin block than wild-type
Kir2.1, with K0.5 values of 1,220 nM for D172N and
7,090 nM for E224G (Fig. 1 C). These findings are generally consistent with the long-pore plugging model of
polyamine block (Lopatin et al., 1995
), in which polyamines such as spermine are proposed to insert
lengthwise into the channel pore. The polyamine end
of the toxin could also insert lengthwise, but would be
unable to insert crosswise because of the large hydrophobic end.
Interactions between Polyamines and Polyamine Toxins
We next investigated interactions between spermine and philanthotoxin block. A fixed concentration of philanthotoxin (30 nM) was chosen that partially blocked steady state outward current at +40 mV, by 44% (Fig. 2 A). In the presence of 30 nM philanthotoxin, the concentration of spermine required to block the remaining current by 50% (K0.5) increased from 5.9 to 78 nM (n = 5 patches; Fig. 2 B). The same effect was seen with the trivalent polyamine spermidine (Fig. 2 E), whose K0.5 increased from 31 nM in the absence of philanthoxin to 342 nM (n = 5 patches) when 30 nM philanthotoxin was present. Finally, argiotoxin (10 nM) also interfered with spermine block of outward Kir2.1 currents to a comparable extent as philanthotoxin, increasing the K0.5 for spermine from 5.1 to 53 nM (n = 5 patches) at +40 mV (Fig. 2 F).
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The ability of polyamine toxins to desensitize Kir2.1
channels to block by spermine or spermidine to this degree is difficult to reconcile with a direct open channel
block mechanism in which toxin and polyamine molecules compete independently for binding site(s) in the
pore. In the long-pore plugging model (Lopatin et al.,
1995; Fig. 3 A), it is hypothesized that two spermine molecules directly occupy the pore, producing either
shallow (B1) or deeply bound (B2 and B3) states. We
incorporated two additional blocked states to represent
direct block by a single toxin molecule occupying the
pore (the B4 state), and a toxin molecule capping a
deeply bound spermine molecule (the B5 state). 10 rate constants (five equilibrium constants) connect the
various states. At a concentration of toxin producing
44% block, this model does not produce a large shift in
the K0.5 for spermine (Fig. 3 B). A small increase in the
K0.5 for spermine (1.8-fold) can be produced if the B5
state is disabled (Fig. 3 C). As the affinity of the toxin for the B5 state is increased, however, the K0.5 for spermine progressively shifts to lower rather than higher
values. The same predictions were obtained with simpler models of direct open channel block, with totals of
two to four blocked states. These results indicate that
block of Kir2.1 channels by polyamines and polyamine toxins is more complex than a direct open channel
block mechanism (Lopatin et al., 1995
).
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Can the Intrinsic Gating Mechanism Account for the Interaction between Polyamines and Polyamine Toxins?
An intrinsic gating mechanism has previously been proposed to explain inward rectification of K channels
(Oliva et al., 1990). Recently we (Shieh et al., 1996
)
and others (Aleksandrov et al., 1996
) have characterized an intrinsic gating mechanism in the cloned Kir2.1
channels in which persistent inactivation of outward current occurs in the absence of polyamines or Mg. Fig.
4 shows the time course of inactivation of outward
Kir2.1 currents in giant inside-out patches excised into
a polyamine- and Mg-free solution. Over the first 5 min,
there was a time-dependent decrease in the rate of inactivation of outward current (measured at +60 mV),
previously attributed to the washout of endogenous
polyamines (Ficker et al., 1994
; Lopatin et al., 1994
;
Fakler et al., 1995
; Yang et al., 1995
), but thereafter no
further change occurred. During the polyamine- and
Mg-free washout period, if Kir2.1 currents were first inactivated by depolarizing the membrane to +60 mV, and then rapidly unblocked by hyperpolarizing to
30
mV, a subsequent depolarization to +60 mV still produced inactivation of outward current. This seems inconsistent with a blocking molecule lodged in the pore
of the channel, since it should diffuse away once released from its blocking site during hyperpolarization,
and not be available to reblock the channel upon the
next depolarization (unless a compartmentalized pool
of poorly diffusible polyamines is postulated). An alternative explanation is that polyamines bind to and enhance the effectiveness of an intrinsic gate. The polyamine molecule could remain tethered to the intrinsic gate when released from the pore by hyperpolarization, and thereby be available to reocclude the pore
during the subsequent depolarization.
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To determine whether this type of mechanism could account for the philanthotoxin-spermine interaction, we examined the model schematically illustrated in Figs. 4 C and 5 A. We hypothesize that, due to its linear structure with four symmetrically spaced positive charges (Fig. 1 A), spermine binds at one end to the intrinsic gate, and the other end to a docking site in the ion-conducting pathway (Fig. 4 C). This mechanism is assumed to account for the high affinity voltage-dependent block by spermine, whereas direct interaction of free, untethered spermine with the pore docking site produces lower affinity block. Philanthotoxin, however, can only block Kir2.1 channels when its spermine-like end lodges in the docking site in the pore (Fig. 4 C). If the spermine-like end of philanthotoxin binds to the intrinsic gate, the hydrophobic end is too bulky to reach and interact with the docking site in the pore. This hypothetical schema predicts an interaction between polyamine toxins and spermine: if, in the presence of a polyamine toxin, the putative site in the intrinsic gate to which spermine binds is occupied by the spermine-like end of the polyamine toxin molecule, then spermine will no longer have access to its high affinity blocking mechanism. Thus, spermine's ability to block Kir2.1 channels with high affinity should be reduced.
The simplest model of interaction between a
polyamine toxin (T), spermine (S), and an intrinsic
gate is shown in Fig. 5 A. Three open states reflect either nothing (O1), spermine (O2), or a toxin molecule
(O3) bound to the intrinsic gate site, with the pore
docking site empty. There are seven possible blocked states: the B1 state represents high affinity block by the
spermine-intrinsic gate complex occluding the pore.
The B2, B3, and B4 states represent an untethered spermine molecule bound to the docking site in the pore
(direct low affinity block) with either nothing, spermine, or a toxin molecule, respectively, bound to the intrinsic
gate. The B5, B6, and B7 states represent the analogous
conditions for a toxin molecule directly blocking the
pore. As in the long-pore plugging model (Fig. 3 A), 10 rate constants connect the various states, represented in
the diagram by their corresponding equilibrium constants K1-K5. Based on the difference in affinities for
spermine block between wild-type Kir2.1 and the E224G
mutation (Fig. 1 B), we chose K0.5 for high affinity block
by the spermine-intrinsic gate complex to be ~1,000×
higher than direct block by untethered spermine molecules. By altering the affinity of the toxin for the intrinsic
gate (i.e., adjusting the K3 equilibrium constant), the experimentally observed decreased affinity of spermine
block in the presence of toxin is readily reproduced by
the model, and yields an acceptable fit to the data (Fig.
5 B). The increase in K0.5 in the presence of toxin is explained by the O3 state being favored as the affinity of
the toxin for the intrinsic gate is increased, shifting the
equilibrium away from spermine's high affinity blocking
pathway (O1 O2
B1). In contrast, a direct open
channel block mechanism, in which both toxin and
spermine compete independently for a binding site in
the pore, provides no means for the toxin to shift the
equilibrium towards an open state, and so cannot account for the observed shift in the K0.5 for spermine in
the presence of philanthotoxin. Although we have not
tested for toxin-toxin interactions, we would predict
minimal interaction between different polyamine toxins,
since neither would have access to the high affinity
blocking route coupled to the intrinsic gate.
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The Polyamine-Polyamine Toxin Interaction in Kir2.1 Channels with Mutated Rectification Sites
The philanthotoxin-spermine competition experiments
strongly support the role of an intrinsic gate in high affinity polyamine block of Kir2.1 channels. Since D172
and E224 have both been shown to be important in
regulating the sensitivity of Kir2.1 to block by polyamines and Mg (Taglialatela et al., 1995; Yang et al., 1995
; Abrahms et al., 1996
), we also examined the
philanthotoxin-spermine interaction in the D172N
and E224G mutants. For D172N, the K0.5 for spermine
at +40 mV increased from 5.9 to 90 nM (n = 4), and for philanthotoxin, from 47 to 1,220 nM (n = 6), compared with wild-type Kir2.1 (Fig. 1, B and C). In the
presence of 700 nM philanthotoxin, which blocked
outward current at +40 mV by 40%, the K0.5 for spermine increased from 90 to 1,050 nM (Fig. 2 C). A possible interpretation of these findings is that D172 regulates binding of polyamines and toxin to the pore
docking site (Fig. 4 C). By collectively destabilizing the
binding to this site of the spermine-intrinsic gate complex, untethered spermine and philanthotoxin, D172N
should exhibit lower affinity block by both spermine
and philanthotoxin. Assuming that the highest affinity
block still occurs via the spermine-intrinsic gate complex, however, the ability of philanthotoxin to decrease
the affinity of spermine block should still be preserved.
All of these findings were obtained in the D172N mutant. By appropriately adjusting the equilibrium constants to reflect the lower affinities at the pore docking
site, these effects were readily simulated in the intrinsic
gating model (Fig. 5 C). In contrast, the direct block
model (Fig. 3 A) again failed to reproduce the philanthotoxin-spermine interaction (not shown).
For E224G, the K0.5s for block by spermine and
philanthotoxin were also both increased, from 5.9 to
6,030 nM (n = 4) and from 47 to 7,090 nM (n = 5), respectively (Fig. 1, B and C). However, in the presence
of 3,000 nM philanthotoxin, which blocked outward current at +40 mV by 31%, the increase in the K0.5 of
spermine block, as seen in both wild-type Kir2.1 and
the D172N mutant, was eliminated (6,030 nM in the absence of philanthotoxin versus 4,800 nM in its presence, n = 5). A possible interpretation of these findings
is that E224 primarily regulates polyamine binding to
the intrinsic gate. If the E224G mutation impairs binding of spermine and philanthotoxin to the intrinsic
gate, then block can occur only through direct interaction of untethered spermine or philanthotoxin with
the pore docking site (near D172). The state diagram
for the E224G mutant then reduces to a direct open channel block model containing only one open state
(O1 in Fig. 5 A) and two blocked states (B2 and B5);
i.e., a simplified version of Fig. 3 A. In this case, the
spermine-philanthotoxin interaction should be markedly reduced. Exclusive assignment of E224's role to
the polyamine binding site on the intrinsic gate, however, does not account for the increase in the K0.5 for
philanthotoxin block in the E224G mutant. Thus, E224
must also contribute to the stability of philanthotoxin
and, perhaps, spermine binding to the pore region.
This is consistent with previous observations showing
that the E224G mutation affects single channel properties through the Kir2.1 pore (Yang et al., 1995).
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DISCUSSION |
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These findings support the intrinsic gate model of spermine block hypothesized in Figs. 4 C and 5 A, in which
the D172 residue regulates the docking site in the pore,
and E224 regulates the polyamine binding site on the
intrinsic gate in addition to influencing binding of
polyamine toxins (and probably also polyamines) in the pore. The evidence can be summarized as follows:
(a) an intrinsic gating mechanism of inward rectification in Kir2.1 has been previously identified (Aleksandrov et al., 1996; Shieh et al., 1996
), which persists indefinitely after washout of endogenous internal
polyamines or Mg (Fig. 4 B). (b) Polyamines remain
available to reblock Kir2.1 channels after they are released from their blocking site in the pore by hyperpolarization (Fig. 4 A), suggesting that they remain tethered to another region of the channel from which they
dissociate slowly. (c) Polyamine toxins, such as philanthotoxin and argiotoxin, interfere with the ability of
spermine or spermidine to block Kir2.1 channels (Fig.
2). This effect cannot be explained by a direct open
channel block mechanism (Fig. 3 A), but is readily accounted for by an intrinsic gate model (Fig. 5 A). In the
latter, high affinity block is due to a spermine-intrinsic
gate complex, and low affinity block by direct open
channel block of untethered spermine molecules. (d)
The D172N mutation reduces the affinity of the channel for block by both spermine and philanthotoxin, but
does not eliminate the spermine-philanthotoxin interaction, suggesting its major effect is destabilization of
the pore docking site for all three blocking entities (the
spermine-intrinsic gate complex, untethered spermine,
and philanthotoxin). (e) The E224 mutation eliminates the spermine-philanthoxin interaction, consistent with destabilization of spermine's binding to the
intrinsic gate. The assignment of D172 to the pore
docking site, and E224 primarily to the intrinsic gate
binding site may explain why two such widely separated
amino acids play key roles in rectification.
Several limitations of this study should be recognized. We cannot absolutely exclude the possibility of a
compartmentalized pool of polyamines remaining after
extensive washing of excised patches. However, such a
pool would have to have multiple time constants with
fast and very slow (>>15 min) components (see Fig. 4
B), and cannot account for the philanthotoxin-spermine interaction by a pure open channel block mechanism (see Fig. 3). In the model proposed in Fig. 5 A,
the analysis was simplified by omitting the blocked state
corresponding to the intrinsic gate unoccupied by a
spermine molecule, even though outward current was
significantly attenuated at the end of the voltage clamp
pulse to +40 mV in the absence of polyamines or Mg
(Fig. 2 A). Long voltage clamp pulses (200 ms) were
therefore used to allow this process to reach quasi-steady state before measuring the effects of polyamines and polyamine toxins on the remaining current. Our
analysis was restricted to equilibrium conditions at +40
mV, and we have not yet evaluated whether the intrinsic
gate model can account for the complex kinetics and
voltage dependencies characterized extensively by Lopatin et al. (1995). The substantial block by the intrinsic gating mechanism itself (in the absence of
polyamines) complicates this analysis. Whether Mg or
other polyamines, such as putrescine, bind to or use
this putative intrinsic gate mechanism has also not yet
been determined. Although we think it likely based on earlier analysis of Kir2.1 mutants (Lee et al., 1997
), we
have not proven that the intrinsic gate that weakly
blocks outward current in the absence of polyamines is
the same structure to which polyamines bind to cause
high affinity block. The molecular configuration of the
putative intrinsic gate region and even number of intrinsic gates per channel (given the homotetrameric
structure of Kir2.1) remains purely speculative; it is
solely for clarity and convenience that the intrinsic gate
has been drawn as a single tethered gating particle in
Figs. 4 C and 5 A. In fact, the dual effect of the E224G
mutation on both the polyamine binding site on the intrinsic gate and its docking site in the pore is difficult to
reconcile physically with such a simple model. At first
glance, Fig. 5 A appears complicated. However, the
polyamine block mechanism (shaded area) has the
same number of rate constants and only one additional
state compared with the direct block model Fig. 3 A. Incorporating the toxin-spermine interaction adds four
additional states compared with the direct block
model, but retains the same number of rate constants.
Finally, whether the intrinsic gate mechanism also applies to other members of the strong inward rectifier K+ channel family is currently unknown.
In summary, these findings support the idea that a region of the Kir2.1 channel acts as an intrinsic gate that
binds a polyamine molecule such as spermine or spermidine to induce strong inward rectification. Multiple
positive charges on the polyamine molecule are likely
to facilitate the voltage-dependent interaction of the intrinsic gate-polyamine complex with a pore docking
site. Lower affinity block is produced by direct open
channel block by untethered polyamines. The intrinsic
gate mechanism retains the important feature of the
strong inward rectifiers, relief of inward rectification by
elevated K, hypothesized to result from destabilization
of the polyamine-intrinsic gate docking site in the pore
by external K ions. In view of recent structural information about pore dimensions of inward rectifier K channels (Doyle et al., 1998), the intrinsic gate is an appealing modification to the direct block long pore plugging
mechanism (Lopatin et al., 1995
), since it requires only
one spermine molecule, either tethered or untethered,
to block the pore. With the distance from the internal surface to the selectivity filter estimated at 3 nm (Doyle
et al., 1998
), a single spermine molecule (2 nm in
length; Lopatin et al., 1995
) occupying this section of
the pore is reasonable, but more problematic for a direct block mechanism requiring two spermine molecules stacked lengthwise on top of one another (4 nm
long) to fit into this region.
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FOOTNOTES |
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Address correspondence to James N. Weiss, M.D., Division of Cardiology, Rm 3641 MRL Building, UCLA School of Medicine, Los Angeles, CA 90095. Fax: 310-206-5777; E-mail: jweiss{at}mednet.ucla.edu
Original version received 25 November 1998 and accepted version received 1 March 1999.
Jong-Kook Lee and Scott A. John contributed equally to this work and should be considered co-first authors.We thank Drs. K. Philipson, F. Bezanilla, and C.G. Nichols for critically reading the manuscript and Dr. Yujuan Lu for expert technical support.
Supported by National Institutes of Health grants RO1 HL60025, RO1 HL36729, and P50 HL52319, by an American Heart Association, Greater Los Angeles Affiliate Grant-in-Aid (to S.A. John) and Research Fellowship (to J.-K. Lee), and by the Laubisch Fund and Kawata Endowment.
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