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Received 12 August 1997; accepted after revision 12 December 1997.
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ABSTRACT |
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INTRODUCTION |
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TopAbstractIntroductionMethodsResultsDiscussionReferences |
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In recent studies polyamines have been shown to modulate membrane ion channels. External polyamines interact with the N-methyl-D-aspartate (NMDA) receptor-ion channel complex (Scott, Sutton & Dolphin, 1993) and enhance L-type calcium channel activity (Herman, Reuveny & Narahashi, 1993), while intracellular polyamines appear to gate inwardly rectifying potassium channels. At micromolar levels cytoplasmic spermine restores strong inward rectification to cardiac muscarinic potassium channels (Yamada & Kurachi, 1995) and to Kir2.1, the cloned inward rectifier potassium channel (Ficker, Taglialatela, Wible, Caporaso & Brown, 1994; Lopatin, Makhina & Nichols, 1994).
At millimolar levels intracellular spermine appears to affect a wide range of different membrane channels. It inhibits potassium outward and calcium inward currents in Aplysia neurones (Drouin & Hermann, 1994) and calcium-activated potassium currents in rat pituitary tumour cells (Weiger & Hermann, 1994). In rabbit ventricular myocytes, 0·94 mM spermine produces 50 % inhibition of KATP channel activity (Fan & Makielski, 1997), a result consistent with the absence of an effect of 10 µM spermine on KATP channels in rabbit atrial myocytes (Yamada & Kurachi, 1995). In the present study, we show that the activity of native KATP channels in inside-out patches from guinea-pig ventricular myocytes is inhibited by micromolar levels of polyamines applied to the inside membrane surface. This contrasts with recent findings on cloned KATP channels (Shyng, Ferrigni & Nichols, 1997). A preliminary report has been communicated to The Physiological Society (Niu & Meech, 1997b).
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METHODS |
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Preparation
Adult guinea-pigs were killed by cervical dislocation. The heart was removed and perfused retrogradely via the aorta using a Langendorff apparatus. Single ventricular myocytes were prepared by enzymatic dissociation as described previously (Rodrigo & Chapman, 1990) except that 40 mM taurine was included in the Tyrode solution used for the final perfusion. Myocytes were kept at room temperature (18-23°C) and used within 12 h of isolation.
Recording conditions
Currents through KATP channels were recorded using the inside-out configuration of the patch clamp technique (Hamill, Marty, Neher, Sakmann & Sigworth, 1981). Pipettes were made from borosilicate glass capillaries (Clark Electromedical Ltd) and had tip resistances of 5-25 M
when filled with pipette solution. We used a modified technique to excise inside-out patches; after seal formation, instead of removing a patch of membrane from the myocyte by withdrawing the pipette, the cell was dislodged and washed away by rapid superfusion of standard internal solution through the perfusion system. One advantage of this approach was that the cell could be maintained in a normal bathing medium and yet the inside surface of the patch was never exposed to calcium- or magnesium-containing solution.
KATP channel currents were recorded using a Axopatch-1D patch clamp amplifier and digitized via a TL-1 interface. For multi-channel analysis, currents were filtered at 0·5-1 kHz and digitized at 2-10 kHz. For single channel analysis the currents were filtered at 2 kHz and sampled at 20 kHz. All experiments were conducted at room temperature (18-23°C).
Solutions
The standard internal solution used to perfuse the internal surface of the cell membrane contained (mM): KCl, 140; Hepes, 10; EGTA, 5; pH 7·25 (adjusted with KOH). The pipette contained either Tyrode solution with (mM): NaCl, 140; KCl, 4; Hepes, 5; CaCl2, 1; pH 7·25 (adjusted with NaOH) or contained (mM): KCl, 140; Hepes, 5; CaCl2, 1; pH 7·25 (adjusted with KOH). All chemicals were obtained from Sigma Chemical Co. Ltd.
Analysis
The open and closed times were determined by the 50 % threshold method (Colquhoun & Sigworth, 1983). Closed times were measured in patches containing single channels. At a cut-off frequency (-3 dB) of 2 kHz, the rise time from 10 to 90 % full-amplitude is about 170 µs and so we included only events longer than 250 µs in the open and closed time distributions. Because the closed time constants covered several orders of magnitude we displayed the distributions using a logarithmic histogram with a square root ordinate (Sigworth & Sine, 1987). The open and closed time distributions were fitted, using the maximum likelihood method, to the probability density function:
where t is the measured dwell time, m is the number of components, 
j is the area of component j and 
j is its time constant.
Channel activity was calculated as the open probability:
where N is the number of channels in the membrane patch, tj is the time spent at each current level (j = 1, 2.....N). The total duration of the recording T was at least 15 s. For patches with a large number of channels, channel activity was taken as the average current.
KATP channels frequently show a rapid run-down of activity (Trube & Hescheler, 1984) and may enter a run-down state. Recordings with less than 90 % of the original channel activity were excluded from analysis. All data are given as means ±
KATP channel currents were studied in over 200 excised inside-out membrane patches from guinea-pig ventricular myocytes. Over 95 % of successfully isolated patches exhibited currents whose activity was reduced when ATP was applied to the internal surface of the cell membrane. Most patches contained multiple channels but in eleven patches there was only a single active channel. The outward unitary currents recorded from excised inside-out patches at +40 mV had an amplitude of approximately 2 pA under symmetrical 140 mM K+ conditions (Horie, Irisawa & Noma, 1987). Once the patch was excised into ATP-free solution, run-down of KATP channel activity was somewhat variable although in about 40 % of the patches obtained by our technique (see Methods) the open probability was greater than 90 % after 3 min. Recordings with less than 90 % of the original channel activity were excluded from analysis.
Effect of intracellular spermine
Figure 1A is the first 2 min of a recording from a membrane patch with twelve simultaneously active KATP channels. It shows the effect of successive applications of ATP and spermine at the cytoplasmic surface. Activity in all of the KATP channels was rapidly suppressed by 2 mM ATP. With subsequent exposure to 30 µM and 300 µM spermine, the open probability decreased in a dose-dependent fashion, although the progress of the inhibition was comparatively slow, becoming steady after about 5 s. In some experiments (not shown) the activity of the inhibited channel remained steady for as long as 3 min. In Fig. 1A the level of inhibition was approximately 50 % in 30 µM spermine and over 95 % in 300 µM spermine. Channel activity returned to the control level once the spermine had been removed but full recovery took 10-15 s. Note that in other, apparently stable, preparations there was only partial recovery of channel activity following spermine exposure. We have excluded such data from our analysis because of the progressive apparent increase in spermine sensitivity exhibited by such preparations. Incomplete recovery occurred even in a preparation that showed little run-down during an initial control period of over 3 min and was particularly obvious in preparations exposed to millimolar concentrations of spermine. In such cases channel activity returned to control levels if heparin, a large negatively charged molecule, was used as a scavenger.
Figure 1B shows the collected data from twenty-one patches studied at a holding potential of +40 mV. The dose-response curve of channel activity in the presence of spermine could be fitted by a Hill plot with a slope coefficient of 1·2 and half-maximal inhibition (ki) of 29 µM (continuous line). KATP channels can be seen to be reduced to negligible activity when the internal spermine level was above 1 mM. The addition (via the patch pipette) of 2 mM spermine to the outside surface of the membrane patch, however, caused no significant inhibition (not shown).
Figure 1. Effect of spermine on KATP channel activity
In A, the internal surface of the patch was exposed briefly to 2 mM ATP; all channel activity was inhibited and the zero current level is indicated. After washing with standard internal solution, channel activity was ~50 % inhibited by exposure to 30 µM spermine. More complete inhibition followed exposure to 300 µM spermine and channel activity again recovered upon washing with standard internal solution. Solution changes are indicated by horizontal bars. Inside-out patch held at +40 mV with both internal (bath) and external (pipette) solutions containing 140 mM K+. Data were filtered at 500 Hz. B, dose-response relationship for the effect of spermine on KATP channel activity. Relative activity was calculated by normalizing NPopen observed in different concentrations of spermine, to NPopen measured in standard internal solution during the first 30 s after patch isolation. The continuous line shown is a fit to the data of the equation: Relative activity = 1/{1 + ([spermine]/ki)h}, where h, the Hill coefficient, is 1·2 and ki, the concentration of half-maximal inhibition, is 29 µM. Data from 21 patches; holding potential, +40 mV.
To test whether the effect of spermine was at all voltage-dependent we examined the action of spermine over a range of different membrane potentials. In Fig. 2A the membrane was first held at +40 mV (upper trace). The open probability was initially 0·98 but perfusion with 100 µM spermine reduced it to 0·37. Once the spermine had been removed and the open probability had returned to its control value the membrane potential was switched to -40 mV (lower trace). There were more flickery closings at this potential but the open probability was not greatly affected. Re-exposing the patch to 100 µM spermine produced a reduction in channel activity similar to that seen at +40 mV. The effect of 100 µM spermine on KATP channel activity in four patches at different holding potentials between -80 and +80 mV is summarized in Fig. 2B. The activity of the patch in 100 µM spermine compared with the control activity in the absence of spermine was almost constant at the different membrane potentials. It appeared, however, that the unitary channel amplitude was slightly reduced at positive potentials (Fig. 2C).
Figure 2. Effect of spermine at different membrane potentials
A, inside-out patch current record at +40 mV (upper trace) and -40 mV (lower trace). Exposure to 100 µM spermine (shown by horizontal bars) caused marked inhibition. The dotted lines indicate the level of zero current. B, relative activity in four patches (different symbols) exposed to 100 µM spermine at holding potentials between -80 and +80 mV. Activity in spermine relative to control activity at each membrane potential. C, the unitary current-voltage relationship in the absence (
Single channel kinetics
Patches with a single active KATP channel were studied at 0 mV with 4 mM K+ in the pipette solution (Niu & Meech, 1997a). Figure 3A shows an example of the activity recorded from a single KATP channel when the patch was exposed successively to solutions with 10 and 100 µM spermine. In Fig. 3B, data for an all points histogram collected under control conditions and in the presence of 100 µM spermine, is shown fitted to a Gaussian distribution. There appeared to be no significant change in the unitary current amplitude. In fact, the most noticeable effect of spermine on channel gating kinetics was to introduce the channel into long-lived closings, and also shorten the duration of burst openings. The dose-dependent relationship obtained from an extended recording from one single channel patch is shown in Fig. 3C (
Figure 3. The effect of spermine on single KATP channel activity
A, single KATP channel current record showing effect of spermine. Internal surface of the patch perfused first with standard internal solution and then with solutions containing 10 and 100 µM spermine (indicated by horizontal bars above record). The current level during channel opening (
Figure 4 shows the distributions of open and closed times in the absence and presence of spermine. Under control conditions the open time distribution consisted of two exponential components while the closed time distribution had three exponential components (Spruce, Standen & Stanfield, 1987; Niu & Meech, 1997a). In the presence of 100 µM spermine the frequency of the short openings increased and the longer time constant decreased from 9·19 ± 0·49 ms (n = 4) to 3·38 ± 0·65 ms (n = 3). Overall there was a significant decrease (P < 0·001) in the mean open time from 9·08 ± 0·50 ms (n = 4) to 2·76 ± 0·70 ms (n = 3). Examination of the closed time distribution revealed that the major effect of spermine was to introduce a fourth closed state which lasted several seconds. As spermine appears to block open KATP channels the reciprocal of the mean open time plotted against the spermine concentration can be used to provide an estimate of the binding rate constant (Ogden & Colquhoun, 1985; Davies, Spruce, Standen & Stanfield, 1989). The slope of the line shown in Fig. 5 gives an apparent binding rate constant of 2·6 × 106 M-1 s-1
Figure 4. The effect of 100 µM spermine on open and closed times
Logarithmic histograms of open and closed time distributions from a single channel patch held at 0 mV. The distributions were fitted to the equation:
where t is the measured dwell-time, m is the number of components (j). The values returned for the fits were time constants,
Figure 5. Reduction of mean open time by spermine
Reciprocal of mean open time plotted against spermine concentration, [Spm]. The line is fitted to the equation:
where to is the mean open time and kb is the binding rate constant. (Each point is an average of data from 3-4 patches.)
Effect of intracellular magnesium ions
The weak rectification exhibited by KATP channels is thought to depend on the interaction of the channel pore with intracellular ions such as Mg2+ (Horie et al. 1987). Although the spermine effect we observed was not voltage dependent it seemed possible that there might be some interaction between intracellular spermine and intracellular Mg2+. We therefore examined the effect of spermine in the presence of 0·5 mM Mg2+ at the intracellular surface of the channel. We selected this concentration because preliminary experiments using higher concentrations confirmed that they accelerated run-down (Kozlowski & Ashford, 1990).
As found in rat ventricular myocytes (Findlay, 1987), a consistent effect of Mg2+ was to reduce the relative activity of the KATP channels in the patch, even at negative membrane potentials. With 0·5 mM Mg2+ in the intracellular bathing solution the relative activity was reduced to 0·65 ± 0·24 (n = 11; holding potential, -40 mV). Following the addition of spermine the relative activity was reduced still further and in many cases this was reversible. Figure 6 shows a dose-response curve for the reversible effect of spermine under these conditions. The theoretical Hill plot drawn through the data assumes a slope coefficient (1·2) and concentration of half-maximal inhibition (20 µM) similar to those in Mg2+-free solution (see Fig. 1).
Figure 6. Dose-response curve for the effect of spermine in the presence of 0·5 mM Mg2+
Relative activity was calculated by normalizing NPopen observed in different concentrations of spermine, to NPopen measured in standard internal solution before and after exposure. Only fully reversible changes were plotted. The continuous line shown is a fit to the data of the equation:
Relative activity = 1/{1 + ([spermine]/ki)h},
where h, the Hill coefficient, is 1·2 and ki, the concentration of half-maximal inhibition, is 20 µM. Data from 8 patches; holding potential, -40 mV.
Effect of intracellular arginine, spermidine, putrescine and tetraethylammonium
We tested the effect of other internal cations associated with the cellular metabolism of spermine. Consistent with a previous report (Smith, Sakura, Coles, Gummerson, Proks & Ashcroft, 1997), Fig. 7A shows that arginine in concentrations as high as 20 mM had no effect on KATP channel activity. However the polyamine spermidine markedly inhibited the channel at 0·1 mM (Fig. 7B). The action of spermidine was similar to that of spermine in that there was little effect of changing the membrane potential from -40 to +40 mV (see lower trace). A similar experiment showing the effect of putrescine is shown in Fig. 7C. When applied at a holding potential of -40 mV, 10 mM putrescine produced a partial inhibition of activity but upon depolarizing the membrane to +40 mV the channel was fully blocked. Channel activity recovered slowly upon washing. In the presence of low levels of putrescine (not shown), the KATP channel showed many brief openings interrupted by short closures and the inhibition could be relieved by hyperpolarizing the membrane to -40 mV. Because of the resolution of our recording system, we were unable to quantify the effect of putrescine on gating kinetics but the flickery block by putrescine appeared qualitatively similar to that caused by tetraethylammonium ions (TEA) shown in Fig. 7D. At a holding potential of -40 mV, 10 mM TEA produced a pronounced inhibitory effect which was followed by a slow recovery on wash-off. An equivalent inhibition was seen with 1 mM TEA when tested at +40 mV and 10 mM TEA produced full inhibition. The inhibitory effects of spermine, spermidine, putrescine, TEA and arginine at different holding potentials are summarized in Fig. 7E. In other experiments (not shown) we found neomycin to block KATP channels fully at 100 µM.
Figure 7. The effect of different cations on KATP channel activity
Multichannel current records from inside-out patches exposed to: A, 20 mM arginine followed by 0·3 mM spermine both at +40 mV; B, 0·1 mM spermidine at -40 mV followed by 0·1 mM spermidine at +40 mV; C, 10 mM putrescine at -40 mV with a step to +40 mV; D, 1 mM TEA at +40 mV followed by 10 mM TEA and (below) 10 mM TEA applied at -40 mV. Application of cations shown by horizontal bars. The dotted lines show the zero current levels. E, histogram of normalized KATP channel activity in the presence of cations at different membrane potentials. The number of patches and standard deviations are indicated.
Bath application of the polycation spermine to inside-out membrane patches from guinea-pig ventricular myocytes caused a dose-dependent inhibition of KATP channel activity associated with a decrease in mean open time and the introduction of long closures. Both the onset of inhibition and recovery upon washing was somewhat delayed when compared with the rapid suppression of activity that followed the application of ATP (see Fig. 1A). After isolation, KATP channels are particularly susceptible to the gradual loss of activity known as run-down (Trube & Hescheler, 1984), and although the method of producing detached patches used here appeared to delay run-down for some minutes, recovery of activity following spermine application was incomplete in some preparations. We have excluded such data from our analysis.
Deutsch, Matsuoka & Weiss (1994) have examined the effect of different polycations on rabbit ventricular myocytes. In addition to increasing the ATP sensitivity the polycations protamine, poly-L-lysine and poly-L-arginine caused an overall irreversible reduction in KATP current. The observed decrease in outward unitary current amplitude was consistent with fast open-channel block but to account for the reduction in inward current. Deutsch et al. (1994) propose that polycations screen negative charges that interact with the channel gate. The action of polyamines in reducing KATP channel open probability reported here is largely consistent with these findings except that in most cases the polyamines acted reversibly, at least at low concentrations. Higher (millimolar) concentrations of spermine inhibited the channels irreversibly unless they were treated with heparin, a large negatively charged molecule. It may be that at high levels spermine binds strongly to negative charges on the cell membrane. At micromolar levels, spermine caused a small reduction in the outward unitary current amplitude (Fig. 2C) but the major effects were independent of membrane potential as if the primary binding site was on the membrane surface rather than part way across the potential field. Spermidine also acted in a voltage-independent fashion but inhibition by putrescine was relieved by hyperpolarizing the membrane, suggesting that its site was within the channel pore. Furthermore, putrescine was markedly less effective than the other polyamines tested (order of effectiveness: neomycin > spermine > spermidine > putrescine), which suggests that the size of the inhibitor is a significant factor. By far the most effective inhibitor was neomycin which, with its six positive charges, was the largest molecule tested. It is difficult to incorporate this observation into a simple screening hypothesis and we suppose that inhibition involves binding of the polycation to an exposed site on the internal surface of the cell membrane or on the channel itself.
Kinetic analysis of spermine block
A graph of the reciprocal of the mean open time against spermine concentration (Fig. 5) gave an apparent binding rate constant of 2·6 × 106 m-1 s-1. Assuming that the concentration of half-maximal inhibition (ki; 20 × 10-6 M in Fig. 3) corresponds to the equilibrium dissociation constant for the blocking reaction, the apparent unbinding rate constant is 52 s-1 (time constant, 19 ms). The closed time distribution in 100 µM spermine is well fitted by a function with four time constants (Fig. 4) but two are less than 1 ms while one is greater than 1 s. The third component has an appropriate time constant (12·3 ms) and might include transitions to a spermine-blocked state, together with the closings to a normal shut state (time constant, 3·36 ms) which are seen in the absence of spermine. However, there is no increase in the proportion of such closures in the presence of spermine and so another possibility is that the longest closed state (not observed in the absence of spermine) is in reality interrupted by unresolved brief openings. An alternative explanation is that the true unbinding rate constant is about 0·3 s-1 (equivalent to a time constant of 3·3 s) but that the mean open times used to construct Fig. 5 included unresolved brief closings that alter the slope of the plotted relationship.
Cloned KATP channel
Functional KATP channels may be reconstituted from two subunits; a sulphonylurea receptor, SUR1 or SUR2, with a weak inward rectifier, either Kir6.1 or Kir6.2, (Inagaki et al. 1995a; 1996). Recent work on the cloned channels suggests that they have a (SUR/Kir6.x)4 stoichiometry (Clement et al. 1997). Mutagenesis of the strong inwardly rectifying potassium channel Kir2.1 has indicated at least two binding sites for polyamines. One, a negatively charged residue (aspartate) which lies within the transmembrane M2 domain, is essential for strong rectification (Stanfield et al. 1994; Lopatin et al. 1994). A second negatively charged amino acid (glutamate) at a site in the hydrophilic C-terminal domain, contributes independently to high affinity binding of Mg2+ and polyamines (Taglialatela et al. 1994; Yang, Jan & Jan, 1995). Both Kir6.1, which is present in heart tissue, and Kir6.2 have neutral asparagines in place of aspartate at the M2 site and neutral serines in place of glutamate at the C-terminal site (Inagaki et al. 1995a, b). Mutation of the M2 site in SUR1/Kir 6.2 channels to a negatively charged aspartate or glutamate resulted in KATP channels with outward currents that were largely abolished by 20 µM spermine, 100 µM spermidine, 100 µM putrescine or 1 mM Mg2+ (Shyng et al. 1997). In marked contrast to the voltage-independent inhibition of the native channels described here the steady-state inward current through the SUR1/Kir 6.2 channels remained unaffected by any of these treatments. SUR1 mRNA is known to be absent (or expressed at low levels) in heart (Inagaki et al. 1995b) and an alternate subunit SUR2 is likely to contribute to the heart channel structure (Inagaki et al. 1996). It is possible that the interaction between SUR2 and Kir6.1 in heart tissue leads to the inhibitory effect of polyamines and Mg2+ that we describe. Alternatively the native KATP channels may have an as yet undiscovered accessory subunit.
The action of the smallest polyamine putrescine, though weak, indicates that other regions of the pore besides the M2 and C-terminal sites contribute to voltage-dependent polyamine binding. This is consistent with the observation that the substitution with neutral amino acids of the aspartate and glutamate residues at the critical M2 and C-terminal sites in Kir2.1 did not significantly alter the voltage dependence of Mg2+ block (Yang et al. 1995). Yang et al. have suggested that multiple distributed binding sites might favour interaction with the longer polyamines (with their dispersed positive charges) but presumably a single site deep within the pore could account for the putrescine effect. The H5 region with its negatively charged glutamate may be expected to contribute to this weak interaction which would account for the sensitivity of Kir1.1 to putrescine (Yang et al. 1995).
Spermine-membrane lipid interactions
In Kir6.2 two positively charged amino acid residues in the C-terminus are particularly important for KATP channel activity and interactions with anionic membrane phospholipids seems possible (Fan & Makielski, 1997). Polyamines may bind to or 'shield' negative charges on the cell membrane and in some way reduce the mobility of the channel subunits. Alternatively they may have a physical 'stabilizing' effect on the surrounding membrane lipid (Tabor et al. 1961). Polylysine for example penetrates about 2-3 Å into the head group region of an artificial fatty acid bilayer and changes the orientation of the hydrocarbon chains (McIntosh, Waldbillig & Robertson, 1976). Polyamines decrease the lateral mobility of erythrocyte membrane glycoproteins (Schindler, Koppel & Sheetz, 1979) and reduce erythrocyte membrane deformability (Ballas, Mohandas, Marton & Shohet, 1983). The characteristic polyamine effect on erythrocyte deformability, like that on ventricular KATP channels, was restricted to an intracellular site and had spermine > spermidine > putrescine as an order of effectiveness. These physical changes are reported to have developed over a period of hours rather than seconds, however, and so an electrostatic interaction seems the most likely explanation for our observations.
Spermine and the regulation of cardiac excitability during ischaemia
In the present study KATP channels were inhibited by micromolar concentrations of spermine and spermidine and so the possibility that they regulate cardiac excitability during ischaemia or hypoxia must be considered. The multiple sites and modes of action of different polyamines on cardiac ion channels may provide an approach to controlling cellular excitability. KATP channels are present at high density in cardiac myocytes but appear to be fully inhibited by the levels of ATP known to be present. Activation of less than 1 % of the available KATP current could account for the action potential shortening observed during hypoxia (Findlay, 1994) but the fall in intracellular ATP (at least during the early stages of hypoxia) is generally agreed to be small (Gibbs, 1978; Weiss & Lamp, 1989). Therefore to account for KATP channel activation during metabolic blockade (Noma & Shibasaki, 1985; Nichols & Lederer, 1990) it is necessary to propose the involvement of other factors (Trube & Hescheler, 1984) such as an increase in the ADP : ATP ratio and a decrease in intracellular pH (Lederer & Nichols, 1989).
The discovery that polyamines are primarily responsible for the voltage dependence of strong inward rectifier potassium channels has led to the suggestion that changes in their intracellular concentration might be another factor that modulates cardiac excitability (Nichols, Makhina, Pearson, Sha & Lopatin, 1996). Intracellular polyamine levels depend on metabolism (Tabor & Tabor, 1984; Shyng, Sha, Ferrigni, Lopatin & Nichols, 1996) and mechanisms for high affinity polyamine uptake may be present in the heart. In rat cerebral cortex (Harman & Shaw, 1981) there is a metabolically sensitive, 'very high'-affinity polyamine-uptake system which, if active in cardiac tissue, might mean that polyamine levels fall during metabolic inhibition. Also present is a sodium-dependent 'high'-affinity system which is less sensitive to metabolism and which might cause increased polyamine uptake upon sodium loading. Hence if KATP channel activity depends on polyamine levels (as well as other factors present naturally in the cell cytoplasm) any estimate of their role in the regulation of the heart must be based on a complex multifactorial analysis which is not feasible at present.
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Acknowledgements
This work on KATP channels was initiated by the late Professor R. A. Chapman who died in December 1995. It was funded by a grant to Professor Chapman from the British Heart Foundation. We thank Professor P. R. Stanfield for his comments on the manuscript. Mrs Valerie Buswell, Mr John Jordan and Mr Mike Rickard provided excellent technical assistance.
Corresponding author
R. W. Meech: Department of Physiology, School of Veterinary Science, University of Bristol, Bristol BS2 8EJ, UK.
Email: r.meech{at}bristol.ac.uk
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RESULTS
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Methods
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References
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) and presence (
) of 100 µM spermine.
) together with other less complete data assembled from other patches (). The continuous line which shows a theoretical Hill plot fitted to the most complete set of data is consistent with all data obtained. In spite of differences in external potassium, [K+]o, both the slope coefficient (1·13) and the concentration of half-maximal inhibition (20 µM) were similar to those in Fig. 1B .
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) is indicated at the left. Inside-out patch at 0 mV with 4 mM K+ in the external (pipette) solution and 140 mM K+ in the internal (bath) solution. B, the effect of internal spermine on the amplitude of unitary current. All points amplitude histograms taken from the data shown in A were fitted with Gaussian distributions. The values for open and closed currents were 2·24 ± 0·28 and 0·00 ± 0·29 pA under control conditions and 2·27 ± 0·28 and 0·00 ± 0·25 pA in the presence of 100 µM spermine. C, dose-response curve obtained by fitting data () from single channel patch shown in A. Hill coefficient, h, and the concentration of half-maximal inhibition (ki) are 1·13 and 20 µM, respectively. Data from other patches () were consistent with these values.
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(and relative areas, ) as follows. Control: open times, 1·57 ms (0·02), 9·60 ms (0·98); closed times, 0·11 ms (0·94), 0·34 ms (0·05), 3·36 ms (0·01). Spermine: open times, 0·51 ms (0·19), 3·64 ms (0·81); closed times, 0·15 ms (0·918), 0·60 ms (0·073), 12·31 ms (0·005), 3332 ms (0·004).
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DISCUSSION
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Abstract
Introduction
Methods
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References
REFERENCES
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Abstract
Introduction
Methods
Results
Discussion
References
-cells. The Journal of Physiology 499, 625-635.
S. H. Rolen, P. W. Sorensen, D. Mattson, and J. Caprio
Polyamines as olfactory stimuli in the goldfish Carassius auratus
J. Exp. Biol.,
May 15, 2003;
206(10):
1683 - 1696.
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