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Cardiovascular Research 2005 65(2):387-396; doi:10.1016/j.cardiores.2004.10.035
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Copyright © 2004, European Society of Cardiology

Modulation of the voltage-dependent K+ current by intracellular Mg2+ in rat aortic smooth muscle cells

Paolo Tammaro1, Amy L. Smith, Barry L. Crowley and Sergey V. Smirnov*

Department of Pharmacy and Pharmacology, University of Bath, Claverton Down, Bath, BA2 7AY, U.K.

* Corresponding author. Tel.: +44 1225 384471; fax: +44 1225 386114. Email address: S.V.Smirnov{at}bath.ac.uk

Received 19 May 2004; revised 18 October 2004; accepted 26 October 2004


   Abstract
Top
 Abstract
1. Introduction
 2. Methods
 3. Results
 4. Discussion
 References
 
Objective: Intracellular magnesium ions (Mg2+i) are important in the regulation of a wide range of cellular metabolic processes and modulation of a variety of ion channels. Mg2+ deficiency has been implicated in the aetiology of various cardiovascular diseases. However, potential targets and mechanisms of action of Mg2+i in the cardiovascular system remain poorly understood. We therefore investigated the effect of Mg2+i on the voltage-gated K+ (KV) channels in rat aortic myocytes (RAMs).

Methods: KV currents (IKv) were investigated in single RAMs isolated from adult Wistar rat thoracic aorta using the whole-cell patch clamp technique. Changes in the vascular reactivity were also assessed in endothelium-denuded rat aortic rings loaded with Mg2+.

Results: An increase in Mg2+i caused several significant effects on IKv: (1) slowed down kinetics of activation at high (10 mM) Mg2+; (2) caused inward rectification at positive membrane potentials; (3) shifted the voltage-dependent inactivation, but not steady-state IKv activation; (4) the effect of Mg2+i on IKv inactivation was enhanced in the presence of intracellular ATP. Selective changes in the voltage-dependent characteristics predict a significant inhibition of the whole-cell steady-state IKv ("window current"), resulting in membrane depolarisation and enhanced tissue excitability. An increased sensitivity to KCl and the inhibitors of the IKv, tetraethylammonium and 4-aminopyridine (4-AP), was observed in Mg2+-loaded aortas, confirming this hypothesis.

Conclusion: Our results demonstrate that intracellular magnesium can act as a potent modulator of the KV channel function in vascular smooth muscle cells in the physiological range of membrane potentials, representing a novel mechanism for the regulation of KV channel activity in the vasculature.

KEYWORDS Arteries; Ion channels; K-channels; Smooth muscle; Vasoconstriction


   1. Introduction
Top
 Abstract
 1. Introduction
2. Methods
 3. Results
 4. Discussion
 References
 
Magnesium (Mg2+) is the second most abundant intracellular cation which is involved, both directly (as a cofactor of numerous enzymes) or indirectly (via MgATP), in the regulation of intracellular metabolic processes. Hypomagnesia and dietary Mg2+ deficiency have been implicated in the aetiology of multiple cardiovascular diseases, while Mg2+ supplementation is beneficial in the reduction of blood pressure and stabilisation of cardiac arrhythmias and acute myocardial infarction [1]. Interestingly, a significant accumulation of intracellular Mg2+ (Mg2+i) in erythrocytes was observed in hypertensive patients in comparison to normal subjects, whereas no significant difference in serum ionised Mg2+ was found [2], suggesting a possible abnormal handling of Mg2+i in human hypertension. In addition, Mg2+i concentration can be significantly increased in response to endogenous vasoconstrictors in vascular smooth muscle cells (VSMCs) [3,4], indicating that Mg2+i might also be involved in the regulation of agonist-induced contraction of VSMCs. However, potential targets, which are affected by changes in Mg2+i and mechanisms of its action in the cardiovascular system, remain unclear.

Ion channels, which are essential for the function of the cardiovascular system, represent one possible target for Mg2+i. Indeed, an increase in Mg2+i suppresses voltage-dependent Ca2+ channels (VDCCs) [5–7], directly blocks inward rectifier K+ channels [7,8], and stimulates large conductance Ca2+-activated K+ (BKCa) channels [9]. The role of Mg2+i in the regulation of voltage-dependent K+ (KV) channels, which contribute to the control of both cardiac function and VSMC excitability, has been limited to a few descriptive reports. In the heart, dialysis of amphibian atrial myocytes with 1–10 mM Mg2+ caused voltage-independent block of KV currents [10,11]. Qualitatively similar suppression of KV currents was demonstrated in canine and rabbit arterial SMCs [12]. Although the mechanism of inhibition has not been clarified in these reports, it was apparently different to the voltage-dependent block of heterologously expressed KV channels by Mg2+i that occurs at positive membrane potentials (>+40 mV) [13–16].

The main aim of this study was to investigate the effect of Mg2+i on the whole-cell KV channel current (IKv) in adult rat aortic myocytes (RAMs). RAMs were chosen because they express a homogeneous population of KV channel currents (IKv) [17] which play a key role in the regulation of resting membrane potential and excitability of aortic smooth muscle [18], thus representing a useful model to study mechanisms of KV channel regulation in the vasculature. This paper describes a novel selective effect of Mg2+i on IKv voltage-dependent characteristics which leads to the inhibition of whole-cell steady-state IKv in the physiological range of membrane potentials and increased excitability of VSMCs.


   2. Methods
Top
 Abstract
 1. Introduction
 2. Methods
3. Results
 4. Discussion
 References
 
Male Wistar rats (225–300 g) were humanely killed and the thoracic aorta was removed, cleaned, and cut into rings which were either used for cell isolation or tension measurements. All procedures were performed in accordance with the Guide for the Care and Use of Laboratory Animals published by the U.S. National Institutes of Health (NIH Publication No. 85-23, revised 1996).

2.1. Composition of solutions
The composition of physiological salt solution (PSS) was (mM): 130 NaCl, 5 KCl, 1.5 CaCl2, 1.2 MgCl2, 10 HEPES, and 10 glucose, pH=7.2 (NaOH). The pipette solution contained (mM): 110 KCl, 10 NaCl, 0.5 MgCl2, 10 HEPES, 10 EGTA, and 0.5 CaCl2, pH=7.2 (KOH). Different Mg2+i concentrations were achieved by adding corresponding amounts of MgCl2 to the pipette solution. Corresponding changes in osmolarity were less than 10% and were neglected. Free [Ca2+] (8–10 nM) and [Mg2+] were calculated using Maxchelator software (Stanford University, USA).

The composition of Krebs solution was (mM): 118 NaCl, 25 NaHCO3, 4.9 KCl, 1.2 KH2PO4, 2.5 CaCl2, 1.2 MgSO4, 11.7 glucose. Since the Mg2+ loading procedure (modified from Ref. [19]) required the complete removal of Na+ to eliminate Na+-dependent Mg2+ efflux via the Na+–Mg2+ exchanger, one of the major mechanisms of Mg2+ extrusion from the cell [20], HEPES-based buffers were utilised. Although changes in Mg2+i have not been directly measured, an increase up to 300% from the basal level of ~0.5 mM Mg2+i could be anticipated [21]. The Na+-containing buffer had composition (mM): 140 NaCl, 6 KCl, 2 CaCl2, 1.2 MgCl2, 5 HEPES, 11.7 glucose, pH=7.2 (KOH). In the Na+-free buffer, NaCl was replaced with N-methyl-D-glucamine (NMDG; pH=7.2, HCl). In the Mg2+-loading buffer 30 mM MgCl2 and 110 mM NMDG were used.

Basic chemicals and reagents were purchased from BDH Merck, Fisher or Sigma (all UK).

2.2. Cell isolation and electrophysiological recordings
Single RAMs were isolated from aortic rings (width ~1–1.5 mm) using collagenase (Type XI) and papain (both at 1 mg/ml) as previously described in detail [17,18].

Cells were placed in a chamber (100–200 µl) and whole-cell IKv (sampled at 10 kHz and filtered at 2 kHz) were recorded using the standard patch-clamp technique at room temperature as previously described [17,22]. Cell membrane capacitance (Cm) was determined from the area under capacitance transients (filtered at 50 kHz and sampled at 200 kHz) elicited by a 10-mV hyperpolarising step. To allow for equilibration of the pipette solution with cell interior, all recordings were started 5 min after establishing the whole-cell configuration. In addition, 1 µM paxilline and 10 µM glybenclamide were added to the PSS to block BKCa and ATP-sensitive K+ channels, respectively. Holding potential was –80 mV.

2.3. Isometric tension measurements and Mg2+-loading protocol
Isolated endothelium-denuded aortic rings (~2–2.5 mm width) were mounted in an organ bath under 1g resting tension and equilibrated for 60 min in Krebs solution, bubbled with 95% O2/5% CO2 at 37 °C. Endothelium was removed by gentle rubbing of the vessel lumen with a horse hair (verified by the absence of relaxation to 10 µM acetylcholine). Before commencing the experimental protocol, each preparation was stimulated with three applications of 2.5 µM phenylephrine (PE) followed by wash out with Krebs. Tension (expressed in grams) was measured using a Biegestab K30 isometric force transducer (Hugo Sack Elektronics, Germany), MacLab/4s interface and Chart v3.6/s software (ADI Instruments, UK). Data were sampled at 40 Hz.

To compare the effect of increased Mg2+i on excitability of intact rat aortas, three cumulative concentration responses to high K+ (KCl), tetraethylammonium chloride (TEA), 4-aminopyridine (4-AP), and PE (performed in the Na+-free buffer unless stated otherwise) were measured before (Control), after a 30-min incubation in the Mg2+-loading buffer (Mg2+-loaded), and after a 30-min washout (Recovery) in the same aortic preparation. Since we found that the recovery following washout with a Na+-free solution was incomplete after maximal KCl-induced contraction (not shown), washouts between agent-induced concentration responses were therefore performed with the Na+-containing or Krebs solutions, as indicated, to restore the basal level. We also found that 100 µM imipramine, a selective inhibitor of Na+–Mg2+ exchanger [19,23], irreversibly blocked both aortic contraction and IKv in single RAMs (not shown) and therefore has not been used in this study. The KV channel inhibitors TEA and 4-AP were used since they selectively block the IKv in adult RAMs [17,18].

Data are presented as mean ± S.E.M. and statistically compared using Student's unpaired t test with P<0.05 considered to be significant unless otherwise stated.


   3. Results
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
4. Discussion
 References
 
3.1. Effect of intracellular Mg2+i on IKv
RAMs were dialysed with a pipette solution containing 0.5, 5, or 10 mM MgCl2 ([MgCl]P) and families of IKv were recorded in response to 200-ms step depolarisation (Fig. 1A). The peak amplitude of IKv at each membrane potential was derived from a single exponential fit of the current activation kinetics [17], expressed as a current density and plotted against the test potential (Fig. 1B). This comparison shows that an increase in Mg2+i caused no significant inhibition of the current in the negative voltage range, but induced a marked inward rectification at positive membrane potentials. Moreover, a significant slow down of the current activation kinetics was observed in cells dialysed with 10 mM MgCl2 compared to those in the presence of 0.5 and 5 mM MgCl2 (0.0001<P<0.05 between +10 and +100 mV, Fig. 1C). In addition, a significant decrease in the maximal conductance of IKv (calculated from the Boltzmann fit of the current–voltage (I–V) relationships) was also observed in the presence of 10 mM MgCl2 (0.049 ± 0.005 nS/pF, n=20) compared to that in 0.5 mM (0.079 ± 0.009 nS/pF, n=12) and 5 mM (0.073 ± 0.005 nS/pF, n=19; Fig. 1D).


Figure 1
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  		Fig. 1 Effect of Mg2+i on IKv. (A) Families of IKv in RAMs dialysed with 0.5 (Cm=18.6 pF), 5 (Cm=14.1 pF) and 10 (Cm=15.2 pF) mM MgCl2. Note that traces are only shown between –40 and +100 mV in 20-mV increments. (B) and (C) Mg2+-dependence of I–V relationships and kinetics of activation (time constants derived from single exponential fit) of IKv. (D) Mg2+-dependent changes in maximal conductance calculated from I–Vs shown in panel (B). The peak IKv at each test potential was corrected off-line for a leak current (calculated from the mean slope resistance between –90 and –60 mV) and fitted with Boltzmann function:

Figure 1
where gKv is IKv conductance at each membrane potential (Vm) derived as a ratio of the peak IKv over the difference between the test Vm and the K+ equilibrium potential equal to –83 mV. Gmax, Va, and ka are the maximal whole-cell IKv conductance, the half-activation potential, and the slope factor, respectively.

 
3.2. Mg2+i-dependent changes in the voltage-dependent characteristics of IKv
The effect of Mg2+i on the steady-state activation was compared using the analysis of the normalised conductance–voltage relationship with the standard Boltzmann function (Fig. 2). No significant difference in the half-activation potential (Va) or the slope factor (ka) was observed at different Mg2+i levels (Table 1).


Figure 2
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  		Fig. 2 Effect of Mg2+i on the steady-state activation of IKv. IKv conductance was normalised to the maximal conductance in each cell, averaged and plotted against membrane potential (Vm) at each pipette [MgCl2] (shown in mM near symbols). Normalised conductance was fitted with the Boltzmann function similar to that described in the legend to Fig. 1 with mean parameters given in Table 1 (solid lines). Dashed lines show Va values.

 

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  		Table 1 Effect of intracellular Mg2+, ATP and pH on voltage-dependent characteristics of IKv

 
The effect of Mg2+i on inactivation of IKv was investigated with the availability protocol described in Fig. 3A. As can be seen from the representative recordings, the IKv amplitude at the test potential measured following the same conditioning potential was progressively decreased when RAMs were dialysed with 5 and 10 mM MgCl2 in the pipette compared to that in 0.5 mM (Fig. 3A, arrows). Overall, an increase of Mg2+i from 0.5 mM to 5 and 10 mM caused a marked effect on IKv availability shifting it towards more negative membrane potentials by ~5 and 12 mV (P<0.023), respectively (Fig. 3B). No difference in the slope factor (kh) or the noninactivating current component was found (Table 1).


Figure 3
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  		Fig. 3 Effect of Mg2+i on the IKv availability. (A) Families of representative IKv traces recorded in 3 RAMs dialysed with 0.5 (Cm=13.4 pF), 5 (Cm=19.9 pF), and 10 (Cm=25.4 pF) mM MgCl2 (as indicated) using the voltage protocol shown in the top panel. Cells were stepped to conditioning potentials (Vc) between –100 and 0 mV for 10 s in 10-mV increments followed by a test pulse to +60 mV. Interpulse interval: 10 ms. (B) Comparison of mean IKv availabilities in different Mg2+i (as indicated near symbols). Solid lines were drawn according to the Boltzmann function:

Figure 3
where I/Imax is the ratio of the IKv amplitude at the end of the test pulse over the IKv measured after Vc=–100 mV. Mean values of the half-inactivation potential (Vh), the slope factor (kh), and the noninactivating component (A) are given in Table 1. Dashed lines indicate Vh values.

 
Changes in Mg2+i in the presence of 1 mM MgATP had practically no effect on the mid-point of IKv activation; however, ka values were significantly increased compared to those in ATP-free solutions (Table 1). The IKv availability measured in the presence of 4 mM MgCl2 was also significantly shifted to more negative membrane potentials by a similar degree to that observed in the presence of 10 mM MgCl2 alone (Table 1). It is noteworthy that, when RAMs were dialysed with 0.5 mM MgCl2 or with 1 mM MgATP/0.2 mM MgCl2 (giving similar free [Mg2+]i=0.33 and 0.32 mM respectively), mean Vh and Va values were also similar (Table 1), indicating that observed shifts in the IKv inactivation were determined by changes in Mg2+i. In addition, no significant difference in the current activation kinetics was observed under these conditions (not shown).

The Mg2+i-dependent effect on IKv inactivation was not mimicked by an increased concentration of protons achieved via adjusting the pH of the pipette solution containing 0.5 mM MgCl2 to 6.2 instead of 7.2 (Table 1).

3.3. Effect of Mg2i on the steady-state open probability of IKv
The selective effect of Mg2+i on IKv inactivation, but not activation, can potentially affect the number of functional channels opened at rest by altering the steady-state open probability of the whole-cell KV current [24]. To evaluate the effect of Mg2+i on the whole-cell steady-state IKv, the changes in Va and Vh were plotted against the calculated free [Mg2+]i (Fig. 4A). The nearly linear dependence of the shifts on [Mg2+]i allows the prediction of Va and Vh at any [Mg2+]i in order to calculate the whole-cell steady-state open probability of IKv (Popen). The Popen was calculated as


Figure 4
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  		Fig. 4 (A) Relationships between the voltage-dependent characteristics of IKv and free [Mg2+]i. (B) and (C) Mg2+i-dependent changes in the whole-cell open state probability (Popen) of IKv in the absence and presence of 1 mM MgATP, respectively. The Popen was calculated as described in the text.

 


Formula

where m and h represent theoretical fractions of the steady-state activated and inactivated IKv at each membrane potential derived from equations similar to those described in the legends to Figs. 1 and 3Go, respectively (see Ref. [24] for details). Since no significant changes in the slope factors were found (Table 1), ka and kh values in different Mg2+i were averaged and used in calculations. Fig. 4B illustrates a decrease in Popen of IKv with increased Mg2+i. Both the sensitivity of IKv and degree of inhibition of the steady-state IKv by Mg2+i were increased in the presence of 1 mM MgATP (Fig. 4C). It is noteworthy that Popen was affected by changes in Mg2+i mostly in the negative voltage range, while at potentials positive to 0 mV the Popen was relatively independent of Mg2+i approaching the level determined by the noninactivating fraction of IKv.

3.4. Effect of Mg2+i on membrane potential in single RAMs
The analysis shown in Fig. 4B suggests that an increase in Mg2+i should result in membrane depolarisation of RAMs. Therefore, the effect of 0.2 and 4 mM MgCl2 in the pipette solution containing 1 mM MgATP on the cell membrane potential (Vm) was investigated in single RAMs. Since cell dialysis with both pipette solutions caused a gradual decrease in the membrane potential in the current clamp mode (as shown in examples in Fig. 5A), changes in the rate of membrane depolarisation during cell dialysis with different Mg2+i were monitored. Fig. 5A compares time-dependent changes in Vm recorded 20 s after breakthrough into the cell interior in two RAMs dialysed with 0.2 or 4 mM MgCl2. Overall, cells dialysed with higher Mg2+i had a more positive membrane potential which decreased faster than that in RAMs dialysed with low Mg2+i. Irregular fluctuations of Vm were also observed under both conditions. To analyse quantitatively the difference in the time course of Vm, Vm values at 20, 60, 90, 120, 150, and 180 s were averaged in 16 (0.2 mM MgCl2) and 15 (4 mM MgCl2) RAMs and plotted in Fig. 5B. At 20 s, no significant difference in the mean Vm was observed (–31.9 ± 4.2 mV in 4 mM vs. –39.3 ± 4.1 mV in 0.2 mM MgCl2). However, at 60 and 90 s, cells dialysed with high Mg2+i were significantly more depolarised than RAMs dialysed with 0.2 mM. Additionally, the difference between mean Vm values after 120 s of cell dialysis was marginally significant (P<0.031, one-tail t test). It is noteworthy that another 6 RAMs (3 cells in each pipette solution) were not included in this analysis due to their low Vm immediately after breakthrough (–12.8 ± 4.6 mV and –9.4 ± 2.8 mV in 0.2 and 4 mM MgCl2, respectively).


Figure 5
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  		Fig. 5 Effect of Mg2+i on membrane potential (Vm) in single RAMs. (A) Representative recordings of Vm in current clamp configuration in 2 RAMs dialysed with 0.2 (shown by arrow) and 4 mM MgCl2 in the presence of 1 mM MgATP. Recordings started 20 s after breakthrough into the cell. (B) Comparison of the time course of Vm during cell dialysis with 0.2 ({circ}; n=16) and 4 (bullet; n=15) mM MgCl2. *Significant difference at P<0.012 (60 s) and P<0.037 (90 s).

 
3.5. Effect of Mg2+-loading on excitability of endothelium-denuded rat aortic preparations
The significant Mg2+i-dependent inhibition of the steady-state IKv at negative membrane potentials associated with membrane depolarisation described above should result in increased excitability of aortic smooth muscles. To evaluate this possibility, contractile responses to various concentrations of KCl were examined in endothelium-denuded rat aortic preparations before and after tissue loading with Mg2+ as described in Methods. Fig. 6A shows a representative example of the experiment performed on the same aortic ring. Before Mg2+-loading, an increase in KCl concentration caused a marked contraction at 20 mM (Fig. 6Aa), whereas after incubation in high MgCl2 solution, sensitivity to KCl was significantly increased, eliciting contraction at as low as 10 mM and reaching nearly maximal contraction at 20 mM KCl (Fig. 6Ab). The effect of Mg2+-loading was fully reversible (Fig. 6Ac). The comparison of the mean absolute and normalised values of tension (Fig. 6Ba and Bb, respectively) clearly shows a significantly enhanced responsiveness of aortic preparations to low concentrations of KCl (between 10 and 20 mM) after Mg2+-loading. It is noteworthy that no significant changes in the maximal contraction were observed after Mg2+-loading in the Na+-free buffer, although a significant increase in the maximal tension was seen upon returning to Na+-containing solution (Fig. 6B). Such enhancement was not found when KCl concentration responses were recorded in the Na+-free buffer during recovery (Fig. 6Bc and Bd), indicating a possible Na+-dependence of this increase in the maximal tension.


Figure 6
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  		Fig. 6 Effect of Mg2+-loading on KCl-induced contraction of rat aorta. (A) Contraction to different KCl concentrations (indicated in mM) measured in a representative preparation before (a) and after (b) Mg2+-loading and after washout with the Na+-containing buffer (c). (Ba) and (Bb) Tension–concentration responses to KCl expressed in grams and normalised to the maximal contraction at 100 mM KCl, respectively (n=8–12, 3 rats). (Bc) and (Bd) illustrate the full reversibility of the effect of Mg2+ loading on KCl responses in the Na+-free buffer recorded in a separate set of experiments (n=16, 4 rats). Note the lack of potentiation of contraction at high KCl during the recovery in the absence of external Na+. Washouts (30 min) between KCl concentration responses were performed in Krebs. *, **, and *** indicate 0.012<P<0.046, 0.007<P<0.005, and P<0.0001, respectively. Recovery in the Na+-containing buffer was significantly different from both control and Mg2+-loaded preparations between 15 and 100 mM (Ba) and between 15 and 40 mM (Bb) KCl (0.0001<P<0.006). All statistical comparisons in this and subsequent figures were performed using paired t test.

 
If increased excitability of the rat aorta in Mg2+-loaded preparations is due to decreased IKv, then KV channel inhibitors should mimic the effect of KCl. Therefore, the effect of different concentrations of TEA and 4-AP (KV channel inhibitors which block IKv in RAMs in millimolar range [18]) was compared using the experimental protocol described in Methods. Experiments were performed in the presence of 1 µM paxilline to block BKCa. Contraction caused by IKv inhibitors was reversibly enhanced after loading the tissue with Mg2+i (Fig. 7A and B) in a manner similar to that for KCl. To account for differences in the size and force in individual preparations, concentration responses to both inhibitors were expressed as a percentage of maximal contraction induced by 2.5 µM PE in Krebs. Notably, maximal contractions to 2.5 µM PE measured in Krebs between concentration responses to the IKv inhibitors were not significantly different being equal to 1.3 ± 0.07, 1.4 ± 0.09, and 1.3 ± 0.08 g (n=12), suggesting therefore that the Na+-removal and Mg2+-loading procedures did not significantly alter tissue contractility throughout the experiment. A comparison of TEA and 4-AP concentration responses demonstrates a significant increase in the tension in response to low concentrations of both inhibitors after the Mg2+-loading procedure (Fig. 7C and D). During recovery, the effects of TEA and 4-AP were significantly diminished at low concentrations, but increased at higher doses, although this effect was only significant at 5 mM 4-AP when compared to the control value.


Figure 7
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  		Fig. 7 Effect of Mg2+-loading on TEA- and 4-AP-induced contraction. (A) and (B) Representative recordings in different concentrations of TEA (A) and 4-AP (B; as indicated in mM by arrows, each applied for 10 min) performed as described in Methods. Experiments were carried out in the presence of 1 µM paxilline (10 min pretreatment) to block the BKCa conductance. (C) and (D) Comparison of TEA- and 4-AP-induced contraction (expressed as percentage of the 2.5 µM PE-induced contraction) measured in 8 rings from 4 rats. *,**, and *** indicate significant difference between control and Mg2+-loaded preparations at P<0.02, 0.001<P<0.005, and P<0.0002, respectively. Recovery was significantly different from Mg2+-loaded rings for TEA at 0.5–2 mM (P<0.0001) and for 4-AP at 0.5 mM (P<0.005).

 
To investigate whether increased contractility of SMCs is responsible for the enhanced contraction described above, the effect of {alpha}1-adrenoreceptor agonist PE was studied. Since PE also causes membrane depolarisation and activation of VDCCs [18], experiments were performed both in the absence and presence of the L-type VDCC inhibitor diltiazem (2 µM). The comparison shows that Mg2+-loading significantly enhanced contraction to low PE concentrations in the absence (Fig. 8A) but not in the presence of diltiazem (Fig. 8B). Notably, contraction to higher concentrations of PE in Mg2+-loaded preparations was reduced in the presence of the inhibitor (Fig. 8B) compared to no significant effect in the absence of diltiazem (Fig. 8A). In addition, following recovery, PE-induced contraction (recorded in the presence of diltiazem and probably caused by PE-induced Ca2+ release from intracellular stores) was significantly suppressed (Fig. 8B). Consecutive PE concentration responses performed without Mg2+ loading, but in the presence of diltiazem, demonstrated only a relatively small suppression of contraction during the 3rd application of the agonist (Fig. 8C). This effect was significant between 20 and 1000 nM when compared to the two previous PE concentration dependencies.


Figure 8
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  		Fig. 8 Effect of Mg2+-loading on PE-induced contraction. (A) and (B) Comparison of PE-induced contractions recorded in the absence and presence of 2 µM diltiazem, an L-type VDCC inhibitor, respectively, using the protocol described in Methods. (C) Comparison of three consecutive PE concentration responses performed in the Na+-free buffer containing 2 µM diltiazem and used as a negative control (n=6, 3 rats). Tension was expressed as a percentage of 2.5 µM PE-induced contraction recorded in Krebs at the beginning of the experiment. *Significant difference between control and Mg2+-loaded preparations (n=10, 4 rats) at 0.002<P<0.034. The recovery was also significantly different from Mg2+-loaded aortas between 5 and 100 nM (P<0.001) in panel A and between 100 nM and 10 µM (0.007<P<0.014) in panel B. No significant differences between control responses in panels A and B were found.

 

   4. Discussion
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
 4. Discussion
References
 
In this paper, we have characterised the modulation of KV channels by Mg2+i in VSMCs. An increase in Mg2+i produced several significant effects on the IKv: (i) slowed down kinetics of activation at high (10 mM) Mg2+; (ii) caused inward rectification at positive membrane potentials; (iii) shifted voltage-dependent inactivation, but not steady-state IKv activation, to more negative voltages; (iv) the effect of Mg2+i on IKv inactivation was enhanced in the presence of intracellular ATP; and (v) the cell membrane was depolarised more rapidly in high Mg2+i. In addition, we demonstrated the enhanced sensitivity of Mg2+-loaded rat aortic preparations to membrane depolarisation by KCl and to IKv inhibitors TEA and 4-AP.

A significant slow down of the kinetics of IKv activation was observed only when cells were dialysed with the pipette solution containing 10 mM MgCl2, when a significant inhibition of the current was seen (Fig. 1). The slow down of IKv activation kinetics mimicked the effect observed in bullfrog atrial myocytes but at much lower concentrations of Mg2+i (1 mM) [11]. Conversely, an acceleration of the current kinetics in frog atrial cells dialysed with 10 mM Mg2+i was found [10]. The effect of Mg2+i on IKv kinetics is unlikely to be a result of a direct block of the channel since no significant effect of blocking cations on the activation rate of expressed KV channels was previously observed [15]. Although the mechanism of this effect remains to be clarified, it is likely to be mediated by a Mg2+i-dependent process.

The inward rectification of IKv in high Mg2+i closely mimics the voltage-dependent block of cloned KV channels [13–16] and could share a similar mechanism. However, occurring at very positive voltages, this effect is unlikely to have a great impact on vascular contractility. It is noteworthy that increased Mg2+i caused a similar type of rectification of the cloned Kv2.1 channel [16], which was proposed to be a molecular correlate of the IKv in RAMs [18].

By contrast, the differential effect on the steady-state activation and inactivation of IKv in RAMs can affect the whole cell steady-state open probability in the physiological range of membrane potentials, and thus excitability of VSMCs [24]. A classical explanation of shifts in voltage-dependencies of an ion current, when the extra- or intracellular concentration of divalent cations is changed, is their interaction with negative charges in the close vicinity of the voltage sensor, leading to similar shifts in both activation and inactivation dependencies [25]. Indeed, similar leftward shifts in the activation and inactivation dependencies of the KV current in increased Mg2+i were demonstrated in the giant squid axon. The magnitude of the shifts was increased in the presence of ATP and explained by the addition of new phosphorylated charged sites which interact with Mg2+i [26]. Although the presence of ATP increased the shift in the IKv inactivation in RAMs, no significant changes in Va values were observed, arguing against similar interactions in these cells. In addition, an increase in intracellular proton concentration did not significantly affect the voltage-dependent characteristics of IKv in RAMs (Table 1), whilst similar changes in the extracellular pH caused a significant shift in the activation of both heterologously expressed KV channels and KV currents in ventricular myocytes [27–29]. Additionally, no significant effect of Mg2+i on the KV activation was found in amphibian atrial myocytes [10,11]. However, the effect of Mg2+i on KV inactivation has not been investigated in these reports. Nevertheless, all the evidence argues against direct interactions of Mg2+ with fixed negative surface charges. Although the molecular mechanism of Mg2+i-induced changes in the IKv inactivation in RAMs has yet to be identified, the ATP-dependence of the effect (this study) and modulation of IKv inactivation by the protein kinase C inhibitor bisindolylmaleimide I [30] are indicative of the involvement of intracellular signalling cascades. To verify this hypothesis, further experimentation is necessary.

To evaluate the effect of Mg2+i on IKv in the physiological range of membrane potentials in RAMs, changes in the whole-cell steady-state open probability of IKv (also called "window current") were assessed using a theoretical approach previously described [24]. This analysis predicts that an increase in Mg2+i progressively reduces IKv between –60 and 0 mV and this effect was markedly enhanced in the presence of physiological levels of intracellular MgATP (Fig. 4). Such sensitivity of IKv to changes in Mg2+i in the voltage range close to the cell resting potential can favour membrane depolarisation and increase tissue excitability, leading to increased contractility due to increased Ca2+ entry via VDCCs. The increased rate of membrane depolarisation during cell dialysis with high Mg2+i in single RAMs (Fig. 5) and increased sensitivity to KCl (which causes vasoconstriction entirely via the voltage-dependent pathway) and to the IKv inhibitors TEA and 4-AP in Mg2+-loaded intact aortas (Figs. 6 and 7Go) strongly supports this conclusion. Although the effect of Mg2+i on VDCCs has not been directly investigated, such enhanced sensitivity of Mg2+i-loaded tissue is unlikely to be mediated by changes in VDCC activity since no significant differences in the maximal contraction, which would be expected if Ca2+ entry is increased, were observed before and after tissue loading with Mg2+ (Fig. 6). In addition, Mg2+i generally inhibits VDCCs [5,6,31], which would have an opposite effect on the tissue excitability.

The elevated contraction caused by KCl or IKv blockers is unlikely to originate from increased Ca2+ release from intracellular stores or increased contractility since PE, in the presence of diltiazem, did not cause an increase in tension in Mg2+-loaded preparations (Fig. 8B). Moreover, the PE-induced contraction was significantly decreased under these conditions. Such a marked decrease in tension is likely to be the result of the Mg2+ loading and not due to time-dependent changes in tissue contractility because only a small suppression of PE-induced contraction was observed under control conditions (Fig. 8C). Although from our data it is difficult to speculate about the exact mechanism of this force reduction, the inhibitory effect of Mg2+i on both IP3 and ryanodine receptors, which may be at least partly responsible for the decrease in tension in the presence of the VDCC blocker diltiazem, has been previously described [32,33]. An involvement of Mg2+i-dependent enzyme-mediated modulation (e.g. phosphorylation or dephosphorylation) downstream to the activation of {alpha}1-adrenoreceptors, which may contribute to the long-lasting inhibition of contraction following the recovery from Mg2+ loading, cannot be excluded. Nevertheless, the enhanced PE-induced contraction in the absence of diltiazem (Fig. 8A) points towards increased tissue excitability which does not contradict the results obtained with KCl and the IKv inhibitors. It is noteworthy that, like KCl (Fig. 6Bc), no potentiation of PE-induced contraction upon recovery was found (Fig. 8A), indicating that the significant increase in the maximal KCl-induced tension observed in the Na+-containing buffer (Fig. 6Ba) may require the presence of extracellular Na+. Although further experiments are necessary to clarify this question, it is possible that intracellular Na+ accumulation, resulting from Mg2+ extrusion via Na+-Mg2+ exchanger [20], may cause elevation of Ca2+i via Na+–Ca2+ exchanger working in the reversed mode.

Finally, the involvement of BKCa currents is also unlikely since: (1) BKCa (recorded in the presence of 200 nM free Ca2+i) was not affected by an increase in Mg2+i to 5 mM in RAMs (not shown); that (2) increased contractility to TEA and 4-AP was observed in the presence of the BKCa inhibitor paxilline (Fig. 7); and (3) BKCa do not significantly contribute to the regulation of aortic excitability [18].

In conclusion, our results demonstrate that Mg2+i can act as a potent modulator of the KV channel function in VSMCs in the physiological range of membrane potentials, representing a novel mechanism for the regulation of KV channel activity in the vasculature.


   Acknowledgements
 
This work was supported by the British Heart Foundation (grant FS/2000013).


   Notes
 
1 Present address: University Laboratory of Physiology Parks Road, Oxford OX1 3PT. Back

Time for primary review 24 days


   References
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
 4. Discussion
 References
 

  1. Chakraborti S., Chakraborti T., Mandal M., Mandal A., Das S, Ghosh S. Protective role of magnesium in cardiovascular diseases: a review. Mol. Cell Biochem. (2002) 238:163–179.[CrossRef][Web of Science][Medline]
  2. Sasaki S., Oshima T., Matsuura H., Ozono R., Higashi Y., Sasaki N., et al. Abnormal magnesium status in patients with cardiovascular diseases. Clin. Sci. (2000) 98:175–181.[CrossRef][Web of Science][Medline]
  3. Touyz R.M., Schiffrin E.L. Angiotensin II and vasopressin modulate intracellular free magnesium in vascular smooth muscle cells through Na+-dependent protein kinase C pathways. J. Biol. Chem. (1996) 271:24353–24358.[Abstract/Free Full Text]
  4. Okada K., Ishikawa S., Saito T. Cellular mechanisms of vasopressin and endothelin to mobilize [Mg2+]i in vascular smooth muscle cells. Am. J. Physiol. (1992) 263:C873–C878.[Web of Science][Medline]
  5. Yamaoka K., Seyama I. Regulation of Ca channel by intracellular Ca2+ and Mg2+ in frog ventricular cells. Pflügers Arch. (1996) 431:305–317.[CrossRef][Web of Science][Medline]
  6. Pearson H.A., Dolphin A.C. Inhibition of omega-conotoxin-sensitive Ca2+ channel currents by internal Mg2+ in cultured rat cerebellar granule neurones. Pflügers Arch. (1993) 425:518–527.[CrossRef][Web of Science][Medline]
  7. Agus Z.S., Morad M. Modulation of cardiac ion channels by magnesium. Annu Rev. Physiol. (1991) 53:299–307.[CrossRef][Web of Science][Medline]
  8. Breitwieser G.E. Mechanisms of K+ channel regulation. J. Membr. Biol. (1996) 152:1–11.[CrossRef][Web of Science][Medline]
  9. Xia X.M., Zeng X., Lingle C.J. Multiple regulatory sites in large-conductance calcium-activated potassium channels. Nature (2002) 418:880–884.[CrossRef][Medline]
  10. Tarr M., Trank J.W., Goertz K.K. Intracellular magnesium affects IK in single frog atrial cells. Am. J. Physiol. (1989) 257:H1663–H1669.[Web of Science][Medline]
  11. Duchatelle-Gourdon I., Lagrutta A.A., Hartzell H.C. Effects of Mg2+ on basal and β-adrenergic-stimulated delayed rectifier potassium current in frog atrial myocytes. J. Physiol. (1991) 435:333–347.[Abstract/Free Full Text]
  12. Gelband C.H., Ishikawa T., Post J.M., Keef K.D., Hume J.R. Intracellular divalent cations block smooth muscle K+ channels. Circ. Res. (1993) 73:24–34.[Abstract]
  13. Ludewig U., Lorra C., Pongs O., Heinemann S.H. A site accessible to extracellular TEA+ and K+ influences intracellular Mg2+ block of cloned potassium channels. Eur. Biophys. J. (1993) 22:237–247.[Web of Science][Medline]
  14. Gòmez-Hernandez J.M., Lorra C., Pardo L.A., Stuhmer W., Pongs O., Heinemann S.H., et al. Molecular basis for different pore properties of potassium channels from the rat brain Kv1 gene family. Pflügers Arch. (1997) 434:661–668.[CrossRef][Web of Science][Medline]
  15. Lopatin A.N., Nichols C.G. Internal Na+ and Mg2+ blockade of DRK1 (Kv2.1) potassium channels expressed in Xenopus oocytes. Inward rectification of a delayed rectifier. J. Gen. Physiol. (1994) 103:203–216.[Abstract/Free Full Text]
  16. Tammaro P., Smirnov S.V., Moran O. Effects of intracellular magnesium on Kv1.5 and Kv2.1 potassium channels. Eur. Biophys. J. (2004) [in press]. PubMed link: http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=pubmed&dopt=Abstract&list_uids=15243721; Springer link: http://springerlink.metapress.com/app/home/contribution.asp?wasp=8000375qq88hn2g&referrer=parent&backto=issue,17,30;journal,1,62;linkingpublicationresults,1:100412,1;.
  17. Belevych A.E., Beck R., Tammaro P., Poston L., Smirnov S.V. Developmental changes in the functional characteristics and expression of voltage-gated K+ channel currents in rat aortic myocytes. Cardiovasc. Res. (2002) 54:152–161.[Abstract/Free Full Text]
  18. Tammaro P., Smith A.L., Hutchings S.R., Smirnov S.V. Pharmacological evidence for a key role of voltage-gated K+ channels in the function of rat aortic smooth muscle cells. Br. J. Pharmacol. (2004) 143:303–317.[CrossRef][Web of Science][Medline]
  19. Handy R.D., Gow I.F., Ellis D., Flatman P.W. Na-dependent regulation of intracellular free magnesium concentration in isolated rat ventricular myocytes. J. Mol. Cell. Cardiol. (1996) 28:1641–1651.[CrossRef][Web of Science][Medline]
  20. Romani A.M., Scarpa A. Regulation of cellular magnesium. Front. Biosci. (2000) 5:D720–D734.[Web of Science][Medline]
  21. Zhang A., Cheng T.P., Altura B.T., Altura B.M. Extracellular magnesium regulates intracellular free Mg2+ in vascular smooth muscle cells. Pflügers Arch. (1992) 421:391–393.[CrossRef][Web of Science][Medline]
  22. Smirnov S.V., Beck R., Tammaro P., Ishii T., Aaronson P.I. Electrophysiologically distinct smooth muscle cell subtypes in rat conduit and resistance pulmonary arteries. J. Physiol. (2002) 538:867–878.[Abstract/Free Full Text]
  23. Günther T. Mechanisms and regulation of Mg2+ efflux and Mg2+ influx. Miner. Electrolyte Metab. (1993) 19:259–265.[Web of Science][Medline]
  24. Nelson M.T., Quayle J.M. Physiological roles and properties of potassium channels in arterial smooth muscle. Am. J. Physiol. (1995) 268:C799–C822.[Web of Science][Medline]
  25. Hille B. Ionic Channels of Excitable Membranes, 3rd ed. (2001) Sunderland Massachusetts: Sinauer Associates.
  26. Perozo E., Bezanilla F. Phosphorylation affects voltage gating of the delayed rectifier K+ channel by electrostatic interactions. Neuron (1990) 5:685–690.[CrossRef][Web of Science][Medline]
  27. Steidl J.V., Yool A.J. Differential sensitivity of voltage-gated potassium channels Kv1.5 and Kv1.2 to acidic pH and molecular identification of pH sensor. Mol. Pharmacol. (1999) 55:812–820.[Abstract/Free Full Text]
  28. Trapani J.G., Korn S.J. Effect of external pH on activation of the Kv1.5 potassium channel. Biophys. J. (2003) 84:195–204.[Web of Science][Medline]
  29. Vereecke J., Carmeliet E. The effect of external pH on the delayed rectifying K+ current in cardiac ventricular myocytes. Pflügers Arch. (2000) 439:739–751.[CrossRef][Web of Science][Medline]
  30. Tammaro P., Smirnov S.V. Modulation of Kv channel currents by bisindolylmaleimide I (BIM), a selective PKC, inhibitor, in rat aortic myocytes. Biophys. J. (2002) 82:285A. [Abstract].[CrossRef]
  31. Agus Z.S., Kelepouris E., Dukes I., Morad M. Cytosolic magnesium modulates calcium channel activity in mammalian ventricular cells. Am. J. Physiol. (1989) 256:C452–C455.[Web of Science][Medline]
  32. Mak D.O., Foskett J.K. Effects of divalent cations on single-channel conduction properties of Xenopus IP3 receptor. Am. J. Physiol. (1998) 275:C179–C188.[Web of Science][Medline]
  33. Ogawa Y., Kurebayashi N., Murayama T. Putative roles of type 3 ryanodine receptor isoforms (RyR3). Trends Cardiovasc Med. (2000) 10:65–70.[CrossRef][Web of Science][Medline]

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