Magnesium ions and ionic channels: activation, inhibition or block – a hypothesis

ARTICLE

Auteur(s) : Andrée Guiet-Bara¹, Jean Durlach², Michel Bara¹

¹Laboratory of Physiology and Physiopathology, University P.M. Curie – Paris VI, Paris, France
²SDRM, 64 rue de Longchamp, 92200 Neuilly, France

• – Mg²⁺ ions open the ionic channels and activate the ionic currents and fluxes through the membranes,
• – Mg²⁺ ions block the ionic channels, the ionic currents are reduced and the effect seems reversible,
• – Mg²⁺ ions obstruct the ionic channels, the ionic currents are inhibited and the effect is irreversible.

Magnesium ions activator effects: open channels

1) It has been demonstrated that magnesium ions interfere with the calcium-activated potassium channels which are classified in two groups: large-conductance K_Ca channels inhibited by tetraethylammonium, charybotoxin and iberotoxin (usually denoted BK channels), encoded by the slo1 gene, and an additional class of K_Ca channels with much lower conductance, usually referred to as SK channels inhibited by apamin. Moreover, the activation of BK channels by voltage, intracellular Mg²⁺ and intracellular Ca²⁺ was initiated by three distinct structural components: Segment S4, the low- and high-affinity metal binding-sites. Mg²⁺ ions activated the channel by binding to low-affinity metal-binding sites that were distinct from the high-affinity sites for Ca²⁺-dependent activation, the Mg²⁺-binding site was located in the intracellular RCK domain of the channel protein and the binding of Mg²⁺ in the intracellular RCK domains opened the activation gate by an allosteric mechanism [5]. It is possible to specify this interaction.

In fact:

• – Mg ions [6] activated BK channels independently of Ca²⁺ by preferentially binding to their open configuration, and also bound to Ca²⁺ sites. Quantitative computation of these effects revealed that the overall effect of Mg²⁺ ions under physiological conditions was to enhance the BK channel function. Using a combined site-directed mutagenesis and structural analysis, the authors demonstrated that a structurally new Mg²⁺-binding site in the RCK/Rossman field domain, an intracellular structural motif that immediately followed the activation gate S6 helix, was responsible for Mg²⁺-dependent activation. These results indicated distinct structural pathways for Mg²⁺ and Ca²⁺-dependent activation and suggested a possible explanation for the coupling mechanism between Mg²⁺ binding and channel opening.
• – A similar result was observed [5] and it was demonstrated that the interactions between S4 and a cytoplasmic domain may be involved in Mg²⁺-dependent activation. These results indicated that the voltage sensor was critical for Mg²⁺-dependent activation and the coupling between the voltage sensor and channel gate was a converging point for voltage- and Mg²⁺-dependent activation pathways.
• – An important site for Mg²⁺ sensing between intracellular Mg concentrations of 0.1-10 mM was identified [7].
• – Intracellular Mg²⁺, at physiological concentrations, activated the BK channel by binding to a metal-binding site in the cytosolic domain [8]. In certain conditions (electrostatic interactions…), conformation of the Mg²⁺ binding site may be affected.

Also, Mg²⁺ ions activate the BK channels through distinct intramolecular pathways by affecting different sets of local conformational changes that eventually lead to channel opening. This effect is also called: binding effect: the hydration shell was preserved and Mg²⁺ ions stayed at a distance far from head polar membrane groups, the channels remained open (figure 2).

2) Mg²⁺ ions interfere with different ionic channels:

• – Mg²⁺ salts (MgCl₂ and MgSO₄) regulated the Ca²⁺ influx through the voltage-gated Ca²⁺ channels in vascular smooth muscle cells and vascular endothelial cells from human allantochorial placental vessels by a channel opening mechanism, activating the ionic transfer through this channel type [9] and dilated small arteries [10].
• – In rabbit coronary artery smooth muscle cells, it has been demonstrated [11] that at low [Mg²⁺]_i, activation of Cl, Ca currents (I_{(Cl, Ca)}) and inhibition of K voltage-gated currents (K_(V)) depolarized membrane potential; at high [Mg²⁺]_i, the activation of I_(K,Ca) predominated. In conclusion, [Mg²⁺]_i hyperpolarized the membrane through a selective facilitation of I_(K,Ca) and was able to contribute to the relaxation of the coronary artery.
• – Moreover, Mg²⁺ ions decreased inhibition of human uterine contractility by K⁺_ATP channel openers, in preterm and term labour [12]. A similar effect was observed on mitochondrial K_ATP channels: this major effect which enhanced cardioprotection through the modulation of mitochondrial matrix volume, calcium accumulation and respiration [13].
• – A large family of voltage-gated potassium channel genes have been isolated and expressed, specially the irK (inward rectifier potassium channel) family: ROMK1, irK1, irK2, irK3, GirK1. Voltage-dependent inward-rectifying channels were able to mediate K⁺ fluxes across the plasma membrane, for example. The Mg²⁺ ions dependencies of channel time-dependent showed that MgATP as well as MgADP function as channel activators. In addition to K⁺ channels, studies revealed the presence of another outward-rectifying channel type (MgC) in the plasma membrane that however gates in a nucleotide-independent manner. MgC represented a new channel type distinguished for K channels by fast activation kinetics [14].
• – The structure of KirBac1.01 channel has been described in an open state using as a starting point the structure of the channel [15]. Significant movements of the outer helices take place in going from the closed to the open model in agreement with structural and biochemical data in potassium channels, which suggested that gating was accomplished by a conformational change that took place in the transmembrane domain upon an external stimulus like addition of Mg²⁺ ions. The open conformer was stable, providing a plausible all-atom model that will enable the study of potential gating mechanisms in more detail.

Also, in the case of open channel, it may be suggested that the magnesium salts stored the hydrated molecules and remained far from out external membranes sites. This hypothesis was confirmed by the different effects observed with MgCl₂ (6 H₂O) and MgSO₄ (7 H₂O) [16].

There are intermediate hydration states of the magnesium salts: from all water molecules present to all water molecules lacking.

Magnesium ions – inhibitor effects: close channels

For example, the Mg²⁺-inhibited cation current (MIC) was inhibited by millimolar concentrations of intracellular Mg²⁺ in a voltage-dependent manner. The screening effect of Mg²⁺ ions on the head polar group of membrane phospholipids induced an inhibition of ionic fluxes [17, 18].

Magnesium ions reduces the ionic transfer: block effect

There are numerous studies which indicate a block effect of magnesium ions:

K⁺ channels

• – In bovine pulmonary artery endothelial cells, extracellular Mg²⁺ ions blocked the inwardly rectifying K⁺ currents. The block was voltage-dependent and increased slightly with hyperpolarization (Kd at -160 mV and 0 mV was 9.5 and 23.2 mM respectively). The apparent fractional electrical distance of the Mg²⁺ ions binding site was calculated to be 0.07 from the outer mouth of the channel pore [19].
• – Intracellular Mg²⁺ ions blocked outward currents in BK channels in a highly voltage-dependent manner. An equation that combined an empirical Hill function for block together with a Boltzmann function for the voltage dependence of the block for channels with and without the ring of charge, explained this effect. The Hill coefficient was < 1 for Mg²⁺. When the intracellular concentration of KCl was increased from 150 mM to 3M, the ring of charge no longer facilitated block, Mg²⁺ block was reduced, and the Hill coefficient became approximately 1.0; the ring of negative charge facilitated block through preferential electrostatic attractions of Mg²⁺ over K⁺[20].
• – Magnesium sulphate inhibited the potassium current, in freshly dissociated hippocampal neurons of rats, which might contribute to protect the central neuronal system against damages induced by ischemia and oxygen deprivation [21].
• – The strong inward rectification of Kir2.1 currents was due to blockade of the outward current by cytoplasmic magnesium [22, 23]. Mg²⁺ ions entry in competition with other ions, block the high-affinity channel and induce three conductance states [24]. The inward rectifier potassium channel Kir2.1 was more sensitive to the weakly voltage-dependent block by extracellular Mg²⁺ ions than Kir2.2 and Kir2.3.
• – The blockade effect of the voltage-gated channels was important. Indeed, using an experimental animal model of autoimmune encephalomyelitis, it has been shown [25] that K⁺ channels blockers displaying high selectivity were potent immunosuppressive agents with beneficial symptomatic effects in experimental autoimmune encephalomyelitis.

Ca²⁺ channels

• – Extracellular Mg²⁺ ions inhibited the ionic transfer through the epithelial Ca²⁺ channel from rabbit kidney, structurally related to the family of six transmembrane-spanning ion channels with a pore-forming region between S5 and S6. This effect was concentrated on a single aspartate residue which determined Ca²⁺ permeation of this channel [26].
• – In HEK 293 cells, alpha1GT-type calcium channels were blocked by Mg²⁺ and there was a stronger competition between Ca²⁺ and Mg²⁺ ions [27].
• – Intracellular Mg²⁺ ions, inhibited also the permeation through open L-type Ca²⁺ channels at positive potential in rat ventricular myocytes [28]. This result suggested that, around its physiological concentration range, cytosolic Mg²⁺ ions modulated the extent to which channel phosphorylation regulated I(Ca). This modulation did not necessarily involve changes in channel phosphorylation, but more generally appeared to depend on the kinetics of gating induced by channel phosphorylation [29].

Other channels

• – Mg²⁺ ions blocked the NMDA receptor by binding to the channel pore with an apparent affinity that depended on the membrane potential [30].
• – Magnesium inhibited Ca²⁺ influx by blocking the voltage-dependent calcium channels in airway smooth muscle [31].
• – MgSO₄ blocked Na⁺ currents in isolated hippocampal CA1 neurons of rat and induced neuroprotection against damages induced by ischemia and oxygen deprivation [32].
• – In a TRP superfamily of cations channels (TRPV6), Mg²⁺ ions blocked the channel by binding to a site within the transmembrane electrical field. The block was relieved at positive potentials, indicating that under determining factors Mg²⁺ was able to permeate the selectivity filter of the channel [33, 34].
• – Extracellular Mg²⁺ blocked endothelin-1-induced contraction through the inhibition of non-selective cation channels in coronary smooth muscle [35].

Discussion

The passage of the channel from a state to another may be controlled by the hydration state of the Mg molecule which is different for each Mg salts, showing the importance of the anion.

The hypothesis, published in 1988 [4], on the screening/binding effects gives an explanation of the Mg ions effects on ionic channels and the three channel states may be explained:

• – The screening effects show the existence of ions at a few angstroms from the membrane surface in the aqueous diffuse layer and this effect differs from the Mg specific absorption. In this case, the membrane polar groups (+/-), whatever their location and distribution, are masked by the hydration shell around the cation and that considerably reduces the repulsive or attractive forces between Mg²⁺ ions and the surface groups, but there are non H-bond interactions. The external sites on the membrane are not accessible to the ions present in the medium, the membrane electrical resistance increases and the ionic transfer is inhibited, the effect is irreversible and the channel is closed.
• – In the binding process, the cations have lost their hydration shell and the interactions between cations and surface polar groups are direct and possible. These interactions are a function of the location and the distribution of the surface binding sites. The binding effects may explain the opening and/or block of the channels. Indeed, the variation in the ionic fluxes is due to an increase of the inter-site distance, the Mg ions lost hydration shell and there is repulsion between adjacent positive surface sites and Mg ions. The binding between surface sites are weakened and the inter-site distance is increased, as a result, the membrane stability is decreased and the ionic fluxes increased: the channel is open.
• – In the case of channel block, the Mg²⁺ ions are near the surface sites and there are attractive forces between cations and negative surface head polar groups and repulsive forces between cations and positive surface head polar groups; the Mg²⁺ ions are drawn towards two negative sites with formation of strong covalent bonds, the number of attainable sites is reduced, the membrane stability is increased, the ionic fluxes decreased and the channel is blocked. This effect may be reversible by breaking the bonds and in function of the Mg²⁺ ions concentrations, for example, the channel may oscillate between two states: open-block (figure 3).

Conclusion

References

2 Ebel H, Günther T. Stimulation of choline/Mg²⁺ antiport in rat erythrocytes by mefloquine. Magnes Res 2006; 19: 7-11.

3 Bara M, Ibrahim B, Guiet-Bara A. Magnesium and ionic membrane channels. In: Rayssiguier Y, Mazur A, Durlach J, eds. Advances in Magnesium Research: Nutrition and Health. John Libbey, 2001: 65-72.

4 Bara M, Guiet-Bara A, Durlach J. Analysis of magnesium membraneous effects: binding and screening. Magnes Res 1988; 1: 29-34.

5 Hu L, Shi J, Ma Z, Krishnamoorthy G, Sieling F, Zhang G, Horrigan FT, Cui J. Participation of the S4 voltage sensor in the Mg²⁺-dependent activation of large conductance (BK)K⁺-channels. Proc Natl Acad Sci USA 2003; 100: 10488-93.

6 Shi J, Cui J. Intracellular Mg²⁺ enhances the function of BK-type Ca²⁺-activated K⁺ channels. J Gen Physiol 2001; 118: 583-7.

7 Hu L, Yang H, Shi J, Cui J. Effects of multiple metal binding sites on calcium and magnesium-dependent activation of BK channels. J Gen Physiol 2006; 127: 35-49.

8 Yang H, Hu L, Shi J, Cui J. Tuning magnesium sensitivity of BK channels by mutations. Biophys J 2006; 91: 2892-900.

9 Bara M, Guiet-Bara A. Magnesium regulation of Ca²⁺ channels in smooth muscle and endothelial cells of human allantochorial placental vessels. Magnes Res 2001; 14: 11-8.

10 Zhang J, Wier WG, Blaustein MP. Mg²⁺ blocks myogenic tone but not K⁺-induced constriction: role for SOCs in small arteries. Am J Physiol 2002; 283: H2692-H2705.

11 Bae M, Kim S, Ko E, Lee SH, Ho WK, Earm YE. Intracellular Mg²⁺ hyperpolarizes rabbit coronary smooth muscle cells by differential modulation of Ca²⁺(i)-dependent ion channels. Pflugers Arch 2002; 444: 523-31.

12 Longo M, Jain V, Vedernikov YP, Hankins GD, Garfield RE, Saade GR. Effects of L-type Ca²⁺-channel blockade, K⁺ (ATP)-channel opening and nitric oxide on human uterine contractility in relation to gestational age and labour. Mol Hum Reprod 2003; 9: 159-64.

13 Rousou AJ, Ericsson M, Federman M, Levitsky S, McCully JD. Opening of mitochondrial KATP channels enhances cardioprotection through the modulation of mitochondrial matrix volume, calcium accumulation, and respiration. Am J Physiol 2004; 287: H1967-H1976.

14 Wolf T, Guinot DR, Hedrich R, Dietrich P, Marten I. Nucleotides and Mg²⁺ ions differentially regulate K⁺ channels and non-selective cation channels present in cells forming the stomatal complex. Plant Cell Physiol 2005; 46: 1682-9.

15 Domene C, Doyle DA, Venien-Bryan C. Modeling of an ion channel in its open conformation. Biophys J 2005; 89; (L01–L03).

16 Durlach J, Guiet-Bara A, Pagès N, Bac P, Bara M. Magnesium chloride or magnesium sulphate: a genuine question. Magnes Res 2005; 18: 1-6.

17 Silverman WR. tang CY, Mock AF, Huh KB, Papazian DM. Mg²⁺ modulates voltage-dependent activation in ether-A-go-go potassium channels by binding between transmembrane segments S2 and S3. J Gen Physiol 2000; 116: 663-78.

18 Kozak JA, Matsushita M, Nairn AC, Cahalan MD. Charge screening by internal pH and polyvalent cations as a mechanism for activation, inhibition and rundown of TRPM7/MIC Channels. J Gen Physiol 2005; 126: 499-514.

19 Leung YM, Kwan CY, Daniel EE. Block of inwardly rectifying K⁺ currents by extracellular Mg²⁺ and Ba²⁺ in bovine pulmonary artery endothelial cells. Can J Physiol Pharmacol 2000; 78: 751-6.

20 Zhang Y, Niu X, Brelidze TI, Magleby KL. Ring of negative charge in BK channels facilitates block by intracellular Mg²⁺ and polyamines through electrostatics. J Gen Physiol 2006; 128: 185-202.

21 Sang N, Meng ZQ. Blockade of magnesium sulphate on transient outward K+ current and delayed rectifier K⁺ current in acutely isolated rat hippocampal neurons. Yao Xue Xue Bao 2002; 37: 510-5.

22 Fujiwara Y, Kubo Y. Ser165 in the second transmembrane region of the Kir2.1 channel determines its susceptibility to blockade by intracellular Mg²⁺. J Gen Physiol 2002; 120: 677-93.

23 Murata Y, Fujiwara Y, Kubo Y. Identification of a site involved in the block by extracellular Mg²⁺ and Ba²⁺ as well as permeation of K⁺ in the Kir2.1 K⁺ channel. J Physiol 2002; 544: 665-77.

24 Yan DH, Ishihara K. Two Kir2.1 channel populations with different sensitivities to Mg²⁺ and polyamine blocks: a model for the cardiac strong inward rectifier K⁺ channel. J Physiol 2005; 563: 725-44.

25 Devaux J, Beeton C, Béraud E, Crest M. Canaux ioniques et démyélinisation: les fondements d’un traitement de l’encéphalomyélite auto-immune expérimentale par des bloqueurs des canaux potassium. Rev Neurol (Paris) 2004; 160: 4S16-4S27.

26 Nilius B, Vennekens R, Prenen J, Hoenderop JG, Droogmans G, Bindels RJ. The single pore residue Asp542 determines Ca²⁺ permeation and Mg²⁺ block of the epithelial Ca²⁺ channel. J Biol Chem 2001; 276: 1020-5.

27 Serrano JR, Dashti SR, Perez-Reyes E, Jones SW. Mg²⁺ block unmasks Ca²⁺/Ba²⁺ selectivity of alpha1G T-type calcium channels. Biophys J 2000; 79: 3052-62.

28 Wang M, Berlin JR. Voltage-dependent modulation of L-type calcium currents by intracellular magnesium in rat ventricular myocytes. Arch Biochem Biophys 2007; 458: 65-72.

29 Wang M, Berlin JR. Channel phosphorylation and modulation of L-type Ca²⁺ currents by cytosolic Mg²⁺ concentration. Am J Physiol Cell Physiol 2006; 291: C83-C90.

30 Liu Y, Hill RH, Arhem P, Von Euler G. NMDA and glycine regulate the affinity of the Mg²⁺- block site in NR1-1A/NR2A NMDA receptor channels expressed in Xenopus oocytes. Life Sci 2001; 68: 1817-26.

31 Gourgoulianis KI, Chatziparasidis G, Chatziefthimiou A, Molyvdas PA. Magnesium as a relaxing factor of airway smooth muscles. J Aerosol Med 2001; 14: 301-7.

32 Sang N, Meng Z. Blockade by magnesium of sodium currents in acutely isolated hippocampal CA1 neurons of rat. Brain Res 2002; 952: 218-21.

33 Voets T, Janssens A, Prenen J, Droogmans G, Nilius B. Mg²⁺-dependent gating and strong inward rectification of the cation channel TRPV6. J Gen Physiol 2003; 121: 245-60.

34 Gwanyanya A, Amuzescu B, Zakharov SI, Macianskiene R, Sipido KR, Bolotina V, Vereecke J, Mubagwa K. Magnesium-inhibited TRPM6/7 like channel in cardiac myocytes: permeation of divalent cations and pH-mediated regulation. J Physiol 2004; 559: 761-6.

35 Ko EA, Park WS, Earm YE. Extracellular Mg²⁺ blocks endothelin-1-induced contraction through the inhibition of non-selective cation channels in coronary smooth muscle. Pflugers Arch 2004; 449: 195-204.

36 Mubagwa K, Gwanyanya A, Zakharov S, Macianskiene R. Regulation of cation channels in cardiac and smooth muscle cells by intracellular magnesium. Arch Biochem Biophys 2007; 458: 73-89.