Membrane Transport Group, Department of Chemistry, The Faculties, The Australian National University, Canberra City, Australian Capital Territory 0200, Australia
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ABSTRACT |
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The lipid bilayer
technique was used to characterize the
Ca2+ dependence of a fast
K+ channel formed by a synthetic
17-amino acid segment [OaCNP-39-(1-17)] of
a 39-amino acid C-type natriuretic peptide (OaCNP-39) found in platypus (Ornithorhynchus
anatinus) venom (OaV). The
OaCNP-39-(1-17)-formed K+ channel was reversibly
dependent on
1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid-buffered cis (cytoplasmic)
Ca2+ concentration
([Ca2+]cis).
The channel was fully active when
[Ca2+]cis
was >104 M and
trans (luminal)
Ca2+ concentration was 1.0 mM, but
not at low
[Ca2+]cis.
The open probability of single channels increased from zero at
1 × 10
6 M
cis
Ca2+ to 0.73 ± 0.17 (n = 22) at
10
3 M
cis
Ca2+. Channel openings to the
maximum conductance of 38 pS were rapidly and reversibly activated when
[Ca2+]cis,
but not trans
Ca2+ concentration
(n = 5), was increased to >5 × 10
4 M
(n = 14). Channel openings to the
submaximal conductance of 10.5 pS were dominant at
5 × 10
4 M
Ca2+.
K+ channels did not open when
cis
Mg2+ or
Sr2+ concentrations were increased
from zero to 10
3 M or when
[Ca2+]cis
was maintained at 10
6 M
(n = 3 and 2). The Hill coefficient
and the inhibition constant were 1 and 0.8 × 10
4 M
cis
Ca2+, respectively. This
dependence of the channel on high
[Ca2+]cis
suggests that it may become active under
1) physiological conditions where
Ca2+ levels are high, e.g., during
cardiac and skeletal muscle contractions, and
2) pathological conditions that lead
to a Ca2+ overload, e.g., ischemic
heart and muscle fatigue. The channel could modify a cascade of
physiological functions that are dependent on the
Ca2+-activated
K+ channels, e.g., vasodilation
and salt secretion.
calcium-activated potassium; cytotoxic
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INTRODUCTION |
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USING THE LIPID BILAYER technique, we have demonstrated that, in addition to their interaction with ion transport mechanisms, C-type natriuretic peptides (CNP) can exert their effects on signal transduction by directly forming ion transport pathways (28, 31). CNP are present in platypus venom (12-16) and mammalian cells and are thought to be involved in several pathologies such as cytokine-associated disorders (52). OaCNP-39, isolated from platypus venom, was shown to cause sustained tonic relaxation of the rat uterus in vitro, an effect mimicked by synthetic OaCNP-39 (12-16), pointing to a role of CNPs as potent toxins. The formation of these ion channels modifies the membrane potential and second-messenger systems (e.g., Ca2+ homeostasis). We suspect that the channels can also be modified by changes in the second-messenger systems. These cyclic events would further augment the abnormal electrical activity and distortion of the signal transduction, causing loss of cellular compartmentation and cell dysfunction. Such a cyclic link appears to exist between the activities of natriuretic peptides and cytoplasmic Ca2+ (31). We were interested in cytosolic modulators of K+ channels, particularly those the concentration of which is modified under pathological conditions, e.g., ATP and Ca2+ overload; therefore, we examined the Ca2+ activation properties of the OaCNP-39-(1-17)-formed K+ channels.
The activity of the fast K+
channel formed by incorporating the platypus venom (OaV)
OaCNP-39 and OaCNP-39-(1-17) into bilayers exposed to a symmetrical Ca2+
concentration ([Ca2+])
of 103 M
was characterized previously (27). The properties of
the channel, e.g., that formed by OaV, include the
following. 1) "Bursts" occur
in the outward currents at voltages between
20 and +140 mV.
These bursts are separated by well-defined periods of channel inactivation or closure. 2) The
values of the maximal single-channel conductance (
) and the
concentration for half-maximal single-channel conductance
(KS) are 63.1 pS and 169 mM
K+ at +140 mV and 21.1 pS and 307 mM K+ at 0 mV, respectively.
3) The conductance values of 38.8 ± 4.6 and 60.7 ± 7.1 pS in 250/50 and 750/50 mM
cis/trans, respectively, are within
the range of 25-135 pS of the loosely defined group of
intermediate Ca2+-activated
K+
(IKCa) channels.
4) The OaV-,
OaCNP-39-, and OaCNP-39-(1-17)-formed channels are K+ selective. The
selectivity sequence and permeability
(P) ratio for monovalent
cations obtained for OaCNP-39-(1-17) are
K+ > Rb+ > Na+ > Cs+ > Li+ and 1:0.76:0.21:0.09:0.03
PK+:PRb+:PNa+:PCs+:PLi+, respectively (27). This selectivity sequence is different from that of
the IKCa channel in
Aplysia
(Tl+ > Rb+
NH+4 > Cs+ > Li+ and
Na+) (22) but similar to that of
the 265-pS big Ca2+-activated
K+
(BKCa) channel in secretory
bovine chromaffin cells (37). Similarly, ionic selectivity of the 11- to 30-pS cloned human IKCa
channel, estimated from bi-ionic reversal potentials, gave the
permeability (PK-Px)
sequence K+ = Rb+ (1.0) > Cs+ (9.3)
Na+,
Li+, and
N-methyl-D-glucamine
(>51) (25).
This study shows that the OaCNP-39-(1-17)-formed fast
K+ channel responds rapidly and
reversibly to changes in cis
[Ca2+]
([Ca2+]cis)
but not to trans
[Ca2+]
([Ca2+]trans),
being fully active at 5 × 10
4 M
cis
Ca2+.
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METHODS |
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Solutions
Unless otherwise stated, the initial experimental solution for incorporating OaCNP-39-(1-17) into the bilayers contained KCl (250/50 mM cis/trans) plus 1 mM CaCl2 and 10 mM HEPES buffer (adjusted to pH 7.4 with 4.8 mM KOH). Solutions contained 250/50 mM cis/trans KCl or CsCl and 10 mM cis/trans HEPES (pH adjusted to 7.4 with KOH). [Ca2+] was adjusted by perfusion of the cis chambers with solutions containing 2 mM 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (BAPTA) and titrated with CaCl2 to the desired [Ca2+], as described previously (29).Lipid Bilayer Membranes and Incorporation of OaCNP-39-(1-17)
Bilayers were formed across a 150-µm hole in the wall of a 1-ml Delrin cup with use of a mixture of palmitoyloleoyl phosphatidylethanolamine, palmitoyloleoyl phosphatidylserine, and palmitoyloleoyl phosphatidylcholine (5:3:2, by volume) (26, 30, 41) obtained in chloroform from Avanti Polar Lipids (Alabaster, AL). The lipid mixture was dried under a stream of N2 and redissolved in n-decane at a final concentration of 50 mg/ml. Our stringent precautions against contamination of the lipid by other peptides included ascertaining that 1) no channels formed in the absence of the experimental peptide OaCNP-39-(1-17), 2) unlike bilayers containing peptides, bilayers devoid of peptides can withstand a large voltage range, e.g., ±200 mV, and 3) no ion channel was formed in the presence of an excess of phospholipid mixture in the cis chamber (26). OaCNP-39-(1-17) was then incorporated into the lipid bilayer by addition to the cis chamber up to a final peptide concentration of 0.1-1 µg/ml. The concentrations of OaCNP-39-(1-17) used to obtain channel activity were comparable to concentrations of several cytotoxic channel-forming peptides (2, 3, 34, 42). The side of the bilayer to which the OaCNP-39-(1-17) was added was defined as cis and the other side as trans. The experiments were conducted at room temperature (20-25°C).Recording Single-Channel Activity
The pCLAMP6 program (Axon Instruments) was used for voltage command and acquisition of K+ current families with an Axopatch 200 amplifier (Axon Instruments). The current was monitored on an oscilloscope and stored on a compact disk recorder. The cis and trans chambers were connected to the amplifier head stage by Ag-AgCl electrodes in agar-salt bridges containing the solutions present in each chamber. Voltages and currents were expressed relative to the trans chamber. Data were filtered at 1 kHz (4-pole Bessel,Data Analysis
CHANNEL 2 (developed by P. W. Gage and M. Smith, The John Curtin School of Medical Research), an in-house analysis program, was used to measure the parameters of single-channel activity (10, 30). These parameters include 1) mean open time (To, i.e., the total time that the channel was not closed and including openings to all conductance levels divided by the number of events), 2) mean closed time (Tc, i.e., the total time that the channel was closed divided by the number of events), 3) frequency of the channel opening (Fo), and 4) the open probability (Po, i.e., the sum of all open times as a fraction of the total time). The value of the current amplitude was obtained by measuring the distance (in pA) between two lines: one set on the maximum baseline noise of the closed level and the other on the noise of the majority of distinct open events >0.5 ms. The threshold for channel detection was set at 50% of the current (46). The reversal potential needed to calculate the conductance of the channel was corrected for the liquid junction potential by using JPCalc software (5).Statistics
Unless otherwise stated, each ion channel was used as its own control, and comparison was made between biophysical parameters of the channel before and after the cis or trans solutions were changed by any applied treatment. Values are means ± SE of channels, and the difference in means was analyzed by Student's t-test. Data were considered statistically significant when P < 0.05. ![]() |
RESULTS |
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Ca2+ Dependence of Conductance of the OaCNP-39-(1-17)-Formed Channel
To characterize the Ca2+ sensitivity of single currents through the fast K+ channel, we incorporated the OaCNP-39-(1-17) into lipid bilayer membranes. Families of ionic currents were measured at different voltages (Fig. 1, A and B). The effects of lowering [Ca2+]cis to 5 × 10
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Table 1 shows detailed analysis of the
number of bursts and intrabursts and their durations for six fast
IKCa channels at 104 and
10
3 M
cis
Ca2+ and at membrane potentials of
+120 and +140 mV. The number of events per episode decreased fourfold
from 7.33 ± 0.88 and 4.66 ± 0.66 at
10
3 M
cis
Ca2+ to 1.66 ± 0.33 and 1.50 ± 0.5 at 10
4 M
cis
Ca2+ and +140 and +120 mV,
respectively. The mean duration of the channel burst was not affected
significantly: 2.39 ± 0.40 and 2.87 ± 0.59 s at
10
3 M
cis
Ca2+ and 1.29 ± 0.48 and 2.15 ± 0.88 s at 10
4 M
cis
Ca2+ and +140 and +120 mV,
respectively. On the other hand, the duration of the intrabursts under
these conditions increased three- to sixfold, from 1.98 ± 0.28 and
3.75 ± 0.81 s at 10
3 M
cis
Ca2+ to 13.64 ± 3.21 and 11.35 ± 3.01 s at 10
4 M
cis
Ca2+ and +140 and +120 mV,
respectively. The increase in the duration of the intrabursts at
10
4 M
cis
Ca2+ is due to the significant
decline (P < 0.01) in the bursts'
frequency from 0.98 ± 0.18 and 0.62 ± 0.09 burst/s at
10
3 M
cis
Ca2+ to 0.22 ± 0.04 and 0.20 ± 0.04 burst/s at 10
4 M
cis
Ca2+ and +140 and +120 mV,
respectively.
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At <5 × 104 M
cis
Ca2+, the burst activity gave way
to isolated channel openings to the 36.31- and 10.7-pS maximal and
submaximal levels (Fig. 2,
B and
C). At
10
6 M
cis
Ca2+, all fast
K+ channels
(n = 22) that were active at
10
3 M
cis
Ca2+ became silent, and no channel
openings to any conductance level were observed (Fig.
2D). These effects of lowering
[Ca2+]cis
to 10
6 and
10
5 M on the channel
activity were reversed when
[Ca2+] was increased
to 10
3 M (Fig.
2E). The all-points histograms in
Fig. 2 (right) were obtained from
longer burst segments of channel activity. These histograms confirm
that the fast IKCa channels were
maximally open at 10
3 M
Ca2+ (Fig.
2A), partially open at
10
4 and
10
5 M
Ca2+ (Fig. 2,
B and
C), closed at
10
6 M
Ca2+ (Fig.
2E), and reactivated when
Ca2+ was increased to
10
3 M (Fig.
2E). The amplitude histograms show
that the high conductance levels had a low probability and that the
broad peak at ~36 pS disappeared between
10
4 and
10
6 M
Ca2+, with most activity in peaks
near 0 pS, the closed state. To ascertain that the loss of the channel
activity was due to the low
[Ca2+] per se, rather
than to the buffer ions, the free cis
BAPTA concentration was increased 10-fold from 2 to 20 mM and
calculated to the same [Ca2+]. This increase
in BAPTA concentration did not affect the channel activity
(n = 2), indicating that BAPTA did not
have an independent blocking action on the channel. The reduction in
channel activity was therefore due entirely to lowering
[Ca2+]. Lowering
[Ca2+]trans
from 10
3 to
10
6 M had no effect on the
biophysical properties of the channel (n = 5).
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The
[Ca2+]cis
dependence of the fast intermediate
K+ channel was also examined at
voltages between 160 and +140 mV. The time course of the
single-channel activity (data not shown), like those shown in Fig. 1,
indicates that changes in the bursting characteristics of the channel
activity occurred at all voltages between
20 and +140 mV. The
current amplitude obtained for
10
4 M
cis
Ca2+ indicates that the amplitude
of the maximum single currents remained unaffected at
10
4 M
cis
Ca2+ (Fig. 2), whereas at
<10
5 M
cis
Ca2+, the current amplitudes were
actually reduced to the current level of the subconductance state 10.75 pS, which is also seen at
10
3 M
cis
Ca2+.
Ca2+ Dependence of Kinetics of the OaCNP-39(1-17)-Formed Channel
The Ca2+ dependence of the Po of the single fast K+ channels was determined at voltages between
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The To of the
channel within the burst
To was not
affected at 5 × 104 M
cis
Ca2+.
To at +140 mV was
3.2 and 3.24 ms at 10
3 and
5 × 10
4 M
cis
Ca2+, respectively. According to
McManus and Magleby (39),
To for the
BKCa channel were 5-1,000 s
in the normal mode, 1.5-150 s in the intermediate mode, 1-7 s
in the brief mode, and 0.01-1 s in the buzz mode. Furthermore,
McManus and Magleby proposed a model for the kinetics of the
BKCa channel that includes two to
five closed states and two to four open states. According to this
model, reducing the
[Ca2+]cis
leads to shorter times in the open state, longer times in the closed
state, and low channel frequency. These findings are in agreement with
the low values obtained for the Hill coefficient (see below).
The data show that the channel activity undergoes long periods of
inactivation, where the conformation of the channel protein is in a
nonconductive state. In 250/50 mM
cis/trans KCl, these long periods of
inactivation are seen at 20 to +140 mV, thereby indicating that
they are voltage independent. It appears that, within the burst,
Tc was not
affected at 5 × 10
4 M
cis
Ca2+.
Tc at +140 mV was
1.1 and 1.5 ms at 10
3 and 5 × 10
4 M
cis
Ca2+, respectively. However, low
Ca2+ induced long periods of
inactivation between bursts of channel activity (Fig. 1), suggesting
changes to the inactivation mechanism of the fast
K+ channel. It has been previously
reported that changes in the Po are mainly due
to alterations in the duration of the long closures (7, 35).
Hill Coefficient and Number of Ca2+-Binding Sites
The presence of long inactivated intervals may indicate that the channel requires at least one Ca2+ before it enters the burst mode of activity. The number of the Ca2+ ions that bind to the fast K+ channel protein was estimated from the Hill equation: Po = Pmax[Ca2+]n/(K + [Ca2+]n), where Po is the steady-state Po of the channel, Pmax is the maximum Po of the channel, n is the slope factor of the curve or the Hill coefficient, and K is the nth root of K that gives the approximate midpoint of the activation curve. The lowest value of [Ca2+] at which the channel opening could be detected was 10
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The calculated value of n of 1.0 indicates that at least one Ca2+ must bind to fully stabilize the K+ channel in the open state. It has been proposed that n > 2.0 indicates that more than two ions must bind to the channel protein to fully activate the channel (4, 43, 44, 50). Similar suggestions were reported for BKCa channels in smooth muscle cells isolated from rat cerebral arteries (55).
Fast IK Channel Is Not Activated by cis Mg2+ or ATP
Some KCa channels, e.g., those in hippocampal neurons, are modulated by divalent internal Mg2+ and Sr2+ (38). The specificity of channel activation for Ca2+ was examined by using Mg2+ and Sr2+. K+ channels that were activated at 10
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DISCUSSION |
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The pharmacological and electrophysiological properties of the
Ca2+-activated channels indicate
the presence of three major classes of
KCa channels:
1) voltage-dependent large outwardly
directed K+
(BKCa) channels,
2) voltage-independent,
small-conductance (SKCa) channels, and 3)
voltage-independent, intermediate-conductance (IKCa) channels. The
IKCa channel reported here is
voltage dependent with a conductance of 38.8 ± 4.6 pS (250/50 mM
cis/trans). The extended
NH2 terminus of
OaCNP-39 forms the conductive pathway of the channel. This
is confirmed by the finding that the synthetic peptide
OaCNP-39-(1-17) mimics the conductance, kinetics,
selectivity, and pharmacological properties of the OaV- and
OaCNP-39-formed fast K+
channel. The channel activity of OaCNP-39-(1-17) is
characterized by bursts in the outward current at sustained
depolarizing voltage steps between 20 and +140 mV. The
OaCNP-39-(1-17)-formed
K+ channel was fully active
at
5 × 10
4 M
cis
Ca2+ and at 1.0 mM
trans
Ca2+. The
Po of single fast
IKCa channels increased from zero
at 1 × 10
6 M
cis
Ca2+ to 0.73 ± 0.17 at 10
3 M
cis
Ca2+.
Diversity and Significance of Variations in Ca2+ Sensitivity
The diversity of these channels reflects their involvement in various cellular functions. The low Ca2+ sensitivity at 10-100 mM cis Ca2+ of the fast IKCa channel is reminiscent of another IKCa channel (also known as the 123-pS IcF channel), which is induced by the expression of slo complementary DNAs in oocytes (1). However, unlike the fast IKCa channel, IcF is blocked with 0.14 mM tetraethylammonium ion. There is a large variation between the sensitivity of the Ca2+ sensitivity of the BKCa channel and that of the IKCa and SKCa channels, which appears to be dependent on cell type. For example, the BKCa channel is most sensitive in secretory cells, shows an intermediate sensitivity in smooth muscle cells, and is least sensitive in skeletal muscle cells. This variation implies that the channel may come into operation at physiological and pathological levels of Ca2+. Intrinsic differences in the channel protein, lipid composition of the membrane, channel phosphorylation and dephosphorylation, and other modulatory factors such as pH and Mg2+ may contribute to these variations in the Ca2+ sensitivity of the channel (23).Binding Properties and Mechanisms of Ca2+ Dependence of the K+ Channel
Compared with BKCa channels, little is known about the kinetics of Ca2+ binding to the IKCa and SKCa channel proteins. The characteristics of the Ca2+-binding sites on the channel protein are deduced from the Hill coefficient, localization of the Ca2+-binding sites, divalent selectivity, blockage by Ca2+ and Ba2+, and kinetics of activation. The Hill coefficient for the BKCa channel is 1-6, indicating the need for one to six Ca2+ for the activation of the BKCa channel. The number of ions depends on various factors; e.g., in the presence of millimolar Mg2+, the number of Ca2+ needed for activation increases to four to six, indicating different binding sites for Mg2+, which may induce conformational changes that increase the affinity of the binding sites to Ca2+. In contrast to the abundance of data for Ca2+ binding to the BKCa channel (23), there is little information on the binding of Ca2+ to IKCa and SKCa channels.Ca2+-Binding Site and Inactivation Mechanism of the Fast K+ Channel
The exact molecular events by which low [Ca2+] induces changes in current transitions are not known. Our working hypothesis is that exposure of the channel-forming peptides to low [Ca2+]cis causes changes in the conformation of these peptides that lead to a change in current transitions to submaximal levels. However, it is difficult to ascertain whether such conformational changes take place at the Ca2+-activating binding sites and/or at binding sites for the assembly of the channel-forming peptides. The location of the Ca2+-binding domain for the activation of the KCa channel protein is not well understood. However, there is evidence to suggest that it may be located on the cis side of the membrane, since cis N-bromoacetamide blocks the BKCa channel and high cis Ca2+ induces a mode shift in the channel kinetics (45). The amino acids that constitute Ca2+-binding sites on KCa channels are not yet adequately identified. In the OaCNP-39-(1-17)-formed fast K+ channel, there is a negative site in the form of aspartic acid, D, at position 4. The close proximity of D to the end of the NH2 terminus is consistent with its being located on the cis side of the channel. In addition, the finding that low [Ca2+]cis initially alters the kinetics (Figs. 1 and 3) and transitions to the subconductances of the channel (Fig. 2) may be in agreement with the induction of conformational changes at the entrance of the channel protein rather than deep in its conductive pathway. If indeed the D site is involved in the fast K+ channel inhibition, then Ca2+ dependence of the channel activity could be explained as the neutralization of this site by Ca2+. We suggest that this inhibitory site is actually the inactivation site or part of the inactivation mechanism of the channel. Long periods of inactivation between the bursts of channel activity, induced by lowering [Ca2+]cis toFunctional Requirements of the IKCa Fast Channel
The lack of sensitivity to cis ATP concentration in the presence of cis Mg2+ suggests that activation and deactivation of the fast IKCa channel are not modulated by ATP. The immediate channel activation and the absence of channel deactivation kinetics are in agreement with the lack of such a modulatory mechanism. The low Ca2+ sensitivity of the fast K+ channel would normally indicate that this K+ channel contributes little to changes in the membrane conductance, since [Ca2+]cis in unstimulated vascular smooth muscle cells has been estimated at 4 × 10The fast K+ channel may be
partially activated by >5 × 106 M
cis
Ca2+ and fully activated at
>10
4 M
cis
Ca2+ (Figs. 1 and 2). However, it
is not activated by cytoplasmic Mg2+ concentration (Fig. 5). Under
pathological conditions, e.g., hypoxia-perfusion-induced
Ca2+ overload or near
Ca2+ release sites, where
[Ca2+] can increase to
10
4 M, e.g., as in muscles,
the K+ channel will be maximally
active during muscle contraction. The channel would not be fully
activated if bulk myoplasmic
[Ca2+] did not
increase to >5 × 10
5 M. Interestingly, it
has been reported that O2 causes
fetal pulmonary vasodilation through activation of a
KCa channel (11).
Pathological Role for a KCa Channel
The fact that OaCNP-39-(1-17) formed Ca2+-dependent channels implies that it interferes with mechanisms that link an intercellular second-messenger system to the electrical properties of the membrane. Many physiological functions are regulated by such a link, e.g., contraction and relaxation of muscles. Although a specific pathological role for the channel is difficult to predict from bilayer studies, we suggest that the OaCNP-39-(1-17)-formed Ca2+-activated fast K+ channel will modify those physiological functions that are dependent on the KCa channels. The cellular mechanisms that may be affected by CNP-formed channels include the following.Fluid and electrolyte homeostasis. Because natriuretic peptides have an important role in fluid and electrolyte homeostasis, they may mediate this role (which can be potentially pathological) in these tissues in their capacity as formers of Ca2+-dependent ion channels. The involvement of a modified Ca2+-dependent channel, which has a role in fluid and electrolyte homeostasis, in cell dysfunction is not surprising, given that, in erythrocytes from patients with myotonic muscular dystrophy, KCa channels experience and detect high [Ca2+] and can express an augmented Ca2+ sensitivity (47). It is also known that pathological activation of this channel is involved in the development of crises in sickle cell anemia (8) and arteriolar relaxation during endotoxic shock (48). The implication is that those natriuretic peptides that were involved in endotoxic shock could have mediated their effect via the formation of IKCa channels of the type shown in Fig. 1.
Excitation. The KCa channels involved in depolarization modify the resting membrane potential and the action potential by altering the depolarization phase of the action potential (32). It has been suggested that in the mammalian nerve terminal this channel causes frequency-dependent failure of action potentials, which underlies neurosecretion fatigue (7). Because the conductance and kinetic properties of the channel are important in excitable cells that respond to a train of action potentials where Ca2+ levels are modified or enhanced, this increase in Ca2+ modulates the KCa channel, which in turn lowers excitability of the cell.
Vasodilation and envenomization. The KCa channel has been invoked in pulmonary vasodilation (11). However, the fact that the fast IK channel does not inactivate suggests that it may affect the frequency of the action potential and the subsequent refractory period. The implication for muscle fibers is that they would remain in the relaxed state, which explains the vasodilation induced by natriuretic peptides. The OaV-formed IKCa channels are part of mechanism underlying severe local effects induced by OaV envenomization, such as intense pain, hyperalgesia and plasma extravasation (edema), hypotension, and peripheral vasodilation (12-16, 18).
Amyloidoses.
It was recently suggested that CNPs may be new members of the class of
cytotoxic amyloid-forming peptides that form channels and may be
responsible for disease pathology in various amyloidoses (28). Other
established cytotoxic peptides that belong to this class include
1) amyloid -protein
[A
P-(1-40)]-formed
Ca2+ and giant multilevel cation
channels (2, 3), 2) prion peptide [PrP-(106-126)]-formed voltage-independent
nonselective ion channels (34), and
3) amylin-formed voltage-dependent
nonselective ion-permeable channels (42). The molecular structures for
some of these important endogenous peptides have been investigated (17,
49).
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ACKNOWLEDGEMENTS |
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I thank Drs. P. J. Milburn and G. M. de Plater for encouragement and the generous gift of platypus venom, OaCNP-39, and OaCNP-39-(1-17); R. McCart for numerous discussions and suggestions; H. Wood for critical reading of the manuscript; and A. Culverson, C. Horan, and E. Sturgiss (Commonwealth Scientific and Industrial Research Organization Research Scheme) for laboratory assistance.
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FOOTNOTES |
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This research work is supported by National Health and Medical Research Council Project Grant 970122 and a small grant from The Australian Research Council (F99123).
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.
Address for reprint requests and other correspondence: J. I. Kourie, Membrane Transport Group, Dept. of Chemistry, The Faculties, The Australian National University, Canberra City, ACT 0200, Australia (E-mail: joseph.kourie{at}anu.edu.au).
Received 4 January 1999; accepted in final form 22 March 1999.
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