Calcium dependence of C-type natriuretic peptide-formed fast K+ channel

Joseph I. Kourie

Membrane Transport Group, Department of Chemistry, The Faculties, The Australian National University, Canberra City, Australian Capital Territory 0200, Australia


    ABSTRACT
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
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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 >10-4 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


    INTRODUCTION
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ABSTRACT
INTRODUCTION
METHODS
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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 10-3 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 (gamma ) 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+.


    METHODS
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INTRODUCTION
METHODS
RESULTS
DISCUSSION
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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, -3 dB) and digitized via a TL-1 DMA interface (Axon Instruments) at 2 kHz. The formed optimal bilayers had specific capacitance values >0.42 µF/cm2, because the bilayer area included some of the thick film of the annulus with much lower capacitance than that of biological membranes, which had a capacitance of ~1.0 µF/cm2.

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.


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INTRODUCTION
METHODS
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DISCUSSION
REFERENCES

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 × 10-4 M on the channel activity are characterized by the appearance of long periods of between-channel bursts (intrabursts), with no apparent change in current amplitude or transitions within the bursts (Fig. 1B).


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Fig. 1.   Ca2+ dependence of fast K+ channels 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. Representative families of current traces illustrate activity of fast K+ channels recorded from a voltage-clamped bilayer in 250/50 mM cytosolic/luminal (cis/trans) KCl. Following convention, upward deflections denote activation of outward K+ current, i.e., K+ moving from cis chamber to trans chamber. Luminal Ca2+ concentration was kept at 10-3 M. For a better display, data are filtered at 1 kHz, digitized at 2 kHz, and reduced by a factor of 5. Current traces are separated by a 10-pA offset. Vm, membrane potential; [Ca2+]cis, cytosolic Ca2+ concentration.

Table 1 shows detailed analysis of the number of bursts and intrabursts and their durations for six fast IKCa channels at 10-4 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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Table 1.   Effects of [Ca2+]cis on burst properties

At <5 × 10-4 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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Fig. 2.   Activity of OaCNP-39-(1-17)-formed fast K+ channel activity at +140 mV as a function of [Ca2+]cis adjusted by perfusion with solutions at 10-3 M (cis/trans) Ca2+ (A, control), 10-4 M cis Ca2+ (B), 10-5 M cis Ca2+ (C), 10-6 M cis Ca2+ (D), and recovery, 10-3 M cis Ca2+ (E). Solid lines, channel's closed state and openings to a submaximal level of 10.70 pS and a maximal level of 36.31 pS at 10-3 and 10-4 M cis Ca2+. All-points histograms were obtained from segments of data lasting 89 s (A), 92 s (B), 93 s (C), 88 s (D), and 107 s (E). Bin width of histograms is 0.2.

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 -160 and +140 mV and over a range of [Ca2+] from 10-6 to 10-1 M. Figure 3A shows the effect of [Ca2+]cis on the Po of the fast K+ channel at 0 to +140 mV. Po increased from 0.01 at 10-6 M Ca2+ to ~0.73 at 10-3 M Ca2+, but no further increases were noted up to 10-1 M Ca2+. Similarly, the frequency of the channel activation at -160 to +140 mV increased from 5-10 s-1 at 10-6 M Ca2+ to 180-330 s-1 at 10-3 M Ca2+, but no further increases were noted up to 10-1 M Ca2+. The frequency of the single KCa channel in the membrane of cow cardiac Purkinje cells was increased by increasing the cytoplasmic Ca2+ (9). The Ca2+ dependence of the kinetic parameters of the channel is in agreement with the early findings, where it was demonstrated that the opening frequency of the channel, but not of the open life times, was Ca2+ dependent (33).


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Fig. 3.   Effects of [Ca2+]cis on voltage dependence of kinetic parameters of formed fast K+ channel. A: open probability (Po). B: frequency (Fo). open circle , 10-1 M cis Ca2+; , 10-2 M cis Ca2+; , 10-3 M cis Ca2+; , 10-4 M cis Ca2+; triangle , 10-5 M cis Ca2+; black-triangle, 10-6 M cis Ca2+. Threshold for channel detection was set at 50% of current.

The To of the channel within the burst To was not affected at 5 × 10-4 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 10-5 M. The mean value of [Ca2+]cis at which the single channels were open for half of the time was 0.8 × 10-4 M. The polyexponentials were fitted to the mean Po values at 10-6-10-1 M cis Ca2+ (Fig. 4).


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Fig. 4.   Concentration-dependent effects of cis Ca2+ on mean of relative probability of formed fast K+ channels exposed to different [Ca2+]cis at bilayer potential of +140 mV. Solid line is fitted to Hill equation, and values for Hill coefficient and inhibition constant were 1 and 0.8 × 10-4 M cis Ca2+, respectively.

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 10-3 M cis Ca2+ did not open when cis Mg2+ or Sr2+ concentration was increased from zero to 10-3 M, with [Ca2+]cis maintained at 10-6 M (n = 3 and 2, respectively; Fig. 5). Similarly, these divalent ions, in the presence of 10-3 M cis Ca2+, had no modulatory effects on the conductance or the kinetic parameters of the burst activity of the fast K+ channel. These results indicated that 1) the activation site was specific for Ca2+ and was not a general divalent cation binding site on the channel protein, 2) screening of surface charge was probably not important in channel activation, and 3) the channels would not open at the physiological Mg2+ concentrations found in some tissues, e.g., muscles. NH4Cl quaternary ammonium ions (50 mM), which suppressed ion passage through the KCa channel in Paramecia (51), failed to affect the fast IKCa channel (n = 3). The activity of the fast IK channel reported in this study was also not affected by ATP (n = 3).


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Fig. 5.   Specificity of fast K+ channel activation for Ca2+. Representative current traces illustrate activity of fast K+ channels recorded from a voltage-clamped bilayer at +140 mV in 250/50 mM cis/trans CsCl. A: activation at 10-3 M cis Ca2+; B: inhibition at 10-6 M cis Ca2+; C: inhibition at 10-6 M cis Ca2+ + 10-3 M cis Mg2+; D: recovery at 10-3 M cis Ca2+; E: inhibition at 10-6 M cis Ca2+; F: inhibition at 10-6 M cis Ca2+ + 10-3 M cis Sr2+; G: recovery at 10-3 M cis Ca2+.


    DISCUSSION
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
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 to <= 5 × 10-4 M, indicate that low Ca2+ induces changes in the inactivation site of the fast K+ channel (Fig. 1). Such a suggestion has also been proposed for KCa channels in Paramecia, in which trypsin pronase or thermolysin, which removes or cleaves the Ca2+-binding site, causes the loss of the Ca2+ dependence of the channel (32). The presence of the Ca2+-binding site at the end of the NH2 terminus of OaCNP-39-(1-17) is also in agreement with previous findings. Moreover, it has been found that amino acid deletions from the NH2 terminus, which had been treated with trypsin or by substitution of single amino acids, remove channel inactivation (21, 24, 57). In addition, the ShakerB-inactivating ball peptide (NH2 terminus) modifies the KCa channel (20, 53). These findings indicate that 1) the Ca2+-binding site is located at the end of the NH2 terminus and 2) the Ca2+-binding site is involved in the inactivation mechanism of the K+ channels (21, 24, 57).

Functional 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 × 10-8 M (55). However, the channel contributes significantly to membrane conductance when it is activated by 1) a general or a localized increase in [Ca2+] in the cytoplasm and the cytoplasmic face of the membrane and/or 2) lowering Ca2+ sensitivity of the channel in the presence of intracellular modulators (36, 56).

The fast K+ channel may be partially activated by >5 × 10-6 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 beta -protein [Abeta 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).


    ACKNOWLEDGEMENTS

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.


    FOOTNOTES

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.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

1.   Adelman, J., K. Shen, M. Kavanaugh, R. Waren, W. Y. Lagtutta, C. Bond, and A. North. Calcium-activated potassium channels expressed from cloned complementary DNAs. Neuron 9: 209-216, 1992[Medline].

2.   Arispe, N., H. B. Pollard, and E. Rojas. Giant multilevel cation channels formed by Alzheimer disease amyloid beta -protein. Proc. Natl. Acad. Sci. USA 90: 10573-10577, 1993[Abstract].

3.   Arispe, N., H. B. Pollard, and E. Rojas. Zn2+ interaction with Alzheimer amyloid beta  protein calcium channels. Proc. Natl. Acad. Sci. USA 93: 1710-1715, 1996[Abstract/Free Full Text].

4.   Barrett, J., K. Magleby, and B. Pallotta. Properties of single calcium-activated potassium channels in cultured rat muscle. J. Physiol. (Lond.) 331: 211-230, 1982[Medline].

5.   Barry, P. H. JPCalc, a software package for calculating liquid junction potential corrections in patch-clamp, intracellular, epithelial and bilayer measurements and for correcting junction potential measurements. J. Neurosci. Methods 51: 107-116, 1994[Medline].

6.   Benham, C., T. Bolton, K. Lang, and T. Takewaki. Calcium-activated potassium channels in single smooth muscle cells of rabbit jejunum and guinea-pig mesenteric artery. J. Physiol. (Lond.) 371: 45-67, 1986[Abstract].

7.   Bielfeldt, K., and M. B. Jackson. A calcium-activated potassium channel causes frequency-dependent action potential failures in mammalian nerve terminal. J. Neurophysiol. 70: 284-298, 1993[Abstract/Free Full Text].

8.   Brugnara, C., B. Gee, C. C. Armsby, S. Kurth, M. Sakamoto, N. Rifai, S. L. Alper, and O. S. Platt. Therapy with oral clortimazole induces inhibition of the Gardos channel and reduction of erythrocyte dehydration in patients with sickle cell disease. J. Clin. Invest. 97: 1227-1234, 1996[Abstract/Free Full Text].

9.   Callewaert, G., J. Vereecke, and E. Carmelite. Existence of a calcium-dependent potassium channel in the membrane of cow cardiac Purkinje cells. Pflügers Arch. 406: 424-426, 1986[Medline].

10.   Colqhoun, D., and A. G. Hawkes. Fitting and statistical analysis of single-channel recording. In: Single Channel Recording, edited by B. Sakmann, and E. Neher. New York: Plenum, 1983, p. 135-175.

11.   Cornfield, D. N., H. L. Reeve, S. Tolarova, E. K. Weir, and S. Archer. Oxygen causes fetal pulmonary vasodilation through activation of a calcium-dependent potassium channel. Proc. Natl. Acad. Sci. USA 93: 8089-8094, 1996[Abstract/Free Full Text].

12.   De Plater, G. M. Fractionation, Primary Structural Characterisation and Biological Activities of Polypeptides From the Venom of the Platypus (Ornithorhynchus anatinus) (Ph.D. thesis). Canberra, Australia: Australian National University, 1998.

13.   De Plater, G. M. Platypus (Ornithorhynchus anatinus) venom activates a Ca2+-dependent inward current in rat dorsal root ganglion neurones (Abstract). J. Physiol. (Lond.) 506: 154P, 1998.

14.   De Plater, G. M., R. L. Martin, and P. J. Milburn. A pharmacological and biochemical investigation of the venom from the platypus (Ornithorhynchus anatinus). Toxicon 33: 157-169, 1995[Medline].

15.   De Plater, G. M., R. L. Martin, and P. J. Milburn. The natriuretic peptide (ovCNP) from platypus (Ornithorhynchus anatinus) venom relaxes the isolated rat uterus and promotes oedema and mast cell histamine release. Toxicon 36: 847-857, 1998[Medline].

16.   De Plater, G. M., R. L. Martin, and P. J. Milburn. A C-type natriuretic peptide from the venom of the platypus (Ornithorhynchus anatinus): structure and pharmacology. Comp. Biochem. Physiol. C Pharmacol. Toxicol. Endocrinol. 120: 99-110, 1998[Medline].

17.   Durell, S. R., H. R. Guy, and H. B. Pollard. Theoretical models of the ion structure of amyloid beta -protein. Biophys. J. 67: 2137-2145, 1994[Abstract].

18.   Fenner, P. J., J. A. Williamson, and D. Myers. Platypus envenom---a painful learning experience. Med. J. Aust. 157: 829-832, 1992[Medline].

19.   Findley, I., M. Dunne, and O. Petersen. High-conductance K+ channel in pancreatic islet cells can be activated and inactivated by internal calcium. J. Membr. Biol. 83: 169-175, 1985[Medline].

20.   Foster, C., S. Chung, W. Zagotta, R. Aldrich, and I. Levitan. A peptide derived from the ShakerB K+ channel produces short and long blocks of reconstituted Ca2+-dependent K+ channels. Neuron 9: 229-236, 1992[Medline].

21.   Heginbotham, I., and R. MacKinnon. The aromatic binding site for tetraethylammonium ion on potassium channels. Neuron 8: 483-491, 1992[Medline].

22.   Hermann, A., and C. Erxleben. Charybdotoxin selectivity blocks small Ca-activated K channels in Aplysia neurons. J. Gen. Physiol. 90: 27-47, 1987[Abstract].

23.   Hinrichsn, R. D. Calcium-Dependent Potassium Channels. Austin, TX: Landes, 1993.

24.   Hoshi, T., W. Zagotta, and R. Aldrich. Biophysical and molecular mechanisms of Shaker potassium channel inactivation. Science 250: 533-538, 1990[Medline].

25.   Jensen, B. S., D. Strøbæk, P. Christophersen, T. D. Jørgensen, C. Hansen, A. Silahtarogul, S.-P. Olsen, and P. K. Ahring. Characterization of cloned human intermediate-conductance Ca2+-activated K+ channel. Am. J. Physiol. 275 (Cell Physiol. 44): C848-C856, 1998[Abstract].

26.   Kourie, J. I. Vagaries of artificial bilayers and gating modes of the SCl channel from the sarcoplasmic reticulum of skeletal muscle. J. Membr. Sci. 116: 221-227, 1996.

27.  Kourie, J. I. Characterization of a C-type natriuretic peptide (CNP-39)-formed cation-selective channel from platypus (Ornithorhynchus anatinus) venom. J. Physiol. (Lond.). In press.

28.   Kourie, J. I. Synthetic mammalian C-type natriuretic peptide forms large cation channels. FEBS Lett. 445: 57-62, 1999[Medline].

29.   Kourie, J. I., D. R. Laver, G. P. Ahern, and A. F. Dulhunty. A calcium-activated chloride channel in sarcoplasmic reticulum vesicles from rabbit skeletal muscle. Am. J. Physiol. 270 (Cell Physiol. 39): C1675-C1686, 1996[Abstract/Free Full Text].

30.   Kourie, J. I., D. R. Laver, P. R. Junankar, P. W. Gage, and A. F. Dulhunty. Characteristics of two types of chloride channel in sarcoplasmic reticulum vesicles from rabbit skeletal muscle. Biophys. J. 70: 202-221, 1996[Abstract].

31.   Kourie, J. I., and M. J. Rive. Role of natriuretic peptides in ion transport mechanisms. Med. Res. Rev. 19: 75-94, 1999[Medline].

32.   Kubalski, A., B. Martinac, and Y. Saimi. Proteolytic activation of a hyperpolarization- and calcium-dependent potassium channel in Paramecium. J. Membr. Biol. 112: 91-96, 1989[Medline].

33.   Leinders, T., R. Van Kleef, and H. Vijverberg. Single Ca2+-activated K+ channels in human erythrocytes: Ca2+-dependence of opening frequency but not of open lifetimes. Biochim. Biophys. Acta 1112: 67-74, 1992[Medline].

34.   Lin, M.-C., T. Mirzabekov, and B. L. Kagan. Channel formation by a neurotoxic prion protein fragment. J. Biol. Chem. 272: 44-47, 1997[Abstract/Free Full Text].

35.   Magelby, K., and B. Pallotta. Calcium-dependence of open and shut interval distributions from calcium-activated potassium channels in cultured rat muscle. J. Physiol. (Lond.) 344: 585-604, 1983[Abstract].

36.   Marrion, N. V., and S. J. Tavalin. Selective activation of Ca2+-activated K+ channels by co-localized Ca2+ channels in hippocampal neurons. Science 395: 900-905, 1998.

37.   Marty, A. Ca-dependent potassium channel with large unitary conductance in chromaffin cell membranes. Nature 291: 497-500, 1981[Medline].

38.   McLarnon, J. G., and D. Sawyer. Effects of divalent cations on the activation of a calcium-dependent potassium channel in hippocampal neurons. Pflügers Arch. 424: 1-8, 1993[Medline].

39.   McManus, O., and K. Magleby. Kinetic states and modes of single large-conductance calcium-dependence of open and shut interval distributions from calcium-activated potassium channels in cultured rat muscle. J. Physiol. (Lond.) 402: 79-120, 1988[Abstract].

40.   Methfessel, C., and G. Boheim. The gating of single calcium-dependent potassium channels is described by an activation/blockade mechanism. Biophys. Struct. Mech. 9: 35-60, 1982[Medline].

41.   Miller, C., and E. Racker. Ca++-induced fusion of fragmented sarcoplasmic reticulum with artificial planar bilayers. J. Membr. Biol. 30: 283-300, 1976[Medline].

42.   Mirzabeckov, T. A., M. C. Lin, and B. L. Kagan. Pore formation by the cytotoxic islet amyloid peptide amylin. J. Biol. Chem. 271: 1988-1992, 1996[Abstract/Free Full Text].

43.   Moczydlowski, E., and R. Latorre. Gating kinetics of Ca2+-activated K+ channels from rat muscle incorporated into planar lipid bilayers. J. Gen. Physiol. 82: 511-542, 1983[Abstract].

44.   Oberhauser, A., O. Alvarez, and R. Latorre. Activation by divalent cations of a Ca2+-activated K+ channel from skeletal muscle membrane. J. Gen. Physiol. 92: 67-86, 1988[Abstract].

45.   Pallotta, B. N-bromoacetamide removes a calcium-dependent component of channel opening from calcium-activated potassium channels in rat skeletal muscle. J. Gen. Physiol. 86: 601-611, 1985[Abstract].

46.   Patlak, J. B. Measuring kinetics of complex single ion channel data using mean-variance histograms. Biophys. J. 65: 29-42, 1993[Abstract].

47.   Pellegrino, M., M. Pellegrini, P. Bigini, and A. Scimemi. Properties of Ca2+-activated K+ channels in erythrocytes from patients with myotonic muscular dystrophy. Muscle Nerve 21: 1465-1472, 1998[Medline].

48.   Price, J. M., C. H. Baker, and R. F. Bond. Calcium-activated potassium channel-mediated arteriolar relaxation during endotoxic shock. Shock 7: 294-299, 1997[Medline].

49.   Prusiner, S. B., M. R. Scott, S. J. DeArmond, and F. E. Cohen. Prion protein biology. Cell 93: 337-348, 1998[Medline].

50.   Reinhart, P., S. Chung, and I. Levitan. A family of calcium-dependent potassium channels from rat brain. Neuron 2: 1031-1041, 1989[Medline].

51.   Saimi, Y., and B. Martinac. Calcium-dependent potassium channels in Paramecium studied under patch clamp. J. Membr. Biol. 112: 79-89, 1989[Medline].

52.   Suga, S., H. Itoh, Y. Komatsu, Y. Ogawa, N. Hama, T. Yoshimasa, and K. Nakao. Cytokine-induced C-type natriuretic peptide (CNP) secretion from vascular endothelial cells: evidence for CNP as a novel autocrine/paracrine regulator from endothelial cells. Endocrinology 133: 3038-3041, 1993[Abstract].

53.   Toro, L., E. Stefani, and R. Latorre. Internal blockade of a Ca2+-activated K+ channel by ShakerB-inactivating "ball" peptide. Neuron 9: 237-245, 1992[Medline].

54.   Vergara, C., and R. Latorre. Kinetics of Ca2+-activated K+ channels from rabbit muscle incorporated into planar bilayers: evidence for a Ca2+ and Ba2+ blockade. J. Gen. Physiol. 82: 543-568, 1983[Abstract].

55.   Wang, Y., and D. A. Mathers. Ca2+-dependent K+ channels of high conductance in smooth muscle cells isolated from rat cerebral arteries. J. Physiol. (Lond.) 462: 529-545, 1993[Abstract].

56.   Yagi, S., P. C. Becker, and F. S. Fay. Relationship between force and Ca2+ concentration in smooth muscle as revealed by measurement on single cells. Proc. Natl. Acad. Sci. USA 85: 4109-4113, 1988[Abstract].

57.   Zagotta, W., T. Hoshi, and R. Aldrich. Restoration of inactivation in mutants of Shaker potassium channels by a peptide derived from ShB. Science 250: 568-571, 1990[Medline].


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