Loading rat heart myocytes with Mg<sup>2+</sup> using low‐[Na<sup>+</sup>] solutions

Almulla, Hasan; Bush, Peter G.; Steele, M G; Ellis, David; Flatman, Peter W.

doi:10.1113/jphysiol.2006.109850

Cited by 12 publications

(5 citation statements)

References 46 publications

(89 reference statements)

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“…Our results are also consistent with numerous experimental observations including work by Flatman et al [35] and Kubota et al, [25,26] with fluorescent indicators indicating a persistent elevation in cytosolic free [Mg 2+ ] i under conditions in which extracellular Na + is not available to favor Mg 2+ extrusion. The specific inhibition of Mg 2+ extrusion by imipramine or quinidine, two agents reported to block the Na + -dependent Mg 2+ transport in a variety of cells [11,36], further indicates that the extrusion of Mg 2+ induced by the addition of cyanide occurs through a specific transport mechanism.…”

Section: Discussionsupporting

confidence: 93%

Adenosine triphosphate depletion by cyanide results in a Na+-dependent Mg2+ extrusion from liver cells

Dalal

Romani

2010

Metabolism

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Addition of NaCN to isolated hepatocytes results in a marked and rapid decrease in cellular ATP content, and in the extrusion of a sizable amount of cellular Mg2+. This extrusion starts after a 10 min lag phase and reaches a maximum of 35 to 40 nmol Mg2+/mg protein within 60 min from the addition of CN−. A quantitatively similar Mg2+ extrusion is also observed following the addition of the mitochondrial uncoupler carbonyl cyanide p-trifluoromethoxy-phenylhydrazone (FCCP) but not that of the glycolysis inhibitor iodoacetate. The Mg2+ extrusion is completely inhibited by the removal of extracellular Na+ or the addition of imipramine, quinidine or glibenclamide, while it persists following the removal of extracellular Ca2+ or K+, or the addition of amiloride. An acidic extracellular pH or the removal of extracellular HCO3− inhibits the cyanide-induced Mg2+ extrusion by ≥80%. Taken together, these data suggest that the decrease in cellular ATP content removes a major Mg2+ complexing agent from the hepatocyte, and results in an extrusion of hepatic Mg2+ exclusively through a Na+-dependent exchange mechanism modulated by acidic changes in extracellular pH.

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Section: Discussionsupporting

confidence: 93%

Adenosine triphosphate depletion by cyanide results in a Na+-dependent Mg2+ extrusion from liver cells

Dalal

Romani

2010

Metabolism

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“…Mg 2ϩ blocks these outward currents significantly at physiological concentrations with an IC 50 of 0.53 mM for HCN2 channels and 0.81 mM for HCN1 channels at ϩ50 mV. The concentration of free, unbound Mg 2ϩ has been estimated to be between 0.5 and 1.5 mM in the intracellular environment of mammalian cells (1,6,13,15,30), suggesting that the Mg 2ϩ block of HCN channels is physiological. Lyashchenko and Tibbs (22) recently (in an article that was published during the final preparation of our manuscript) concluded that intracellular Mg 2ϩ blocks outward HCN currents in symmetrical K solutions, as well as in more physiological solutions (22).…”

Section: Discussionmentioning

confidence: 99%

Intracellular Mg²⁺ is a voltage-dependent pore blocker of HCN channels

Vemana¹,

Pandey²,

Larsson³

2008

American Journal of Physiology-Cell Physiology

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Hyperpolarization-activated cyclic nucleotide-gated (HCN) channels are activated by membrane hyperpolarization that creates time-dependent, inward rectifying currents, gated by the movement of the intrinsic voltage sensor S4. However, inward rectification of the HCN currents is not only observed in the time-dependent HCN currents, but also in the instantaneous HCN tail currents. Inward rectification can also be seen in mutant HCN channels that have mainly time-independent currents (5). In the present study, we show that intracellular Mg 2ϩ functions as a voltagedependent blocker of HCN channels, acting to reduce the outward currents. The affinity of HCN channels for Mg 2ϩ is in the physiological range, with Mg 2ϩ binding with an IC50 of 0.53 mM in HCN2 channels and 0.82 mM in HCN1 channels at ϩ50 mV. The effective electrical distance for the Mg 2ϩ binding site was found to be 0.19 for HCN1 channels, suggesting that the binding site is in the pore. Removing a cysteine in the selectivity filter of HCN1 channels reduced the affinity for Mg 2ϩ , suggesting that this residue forms part of the binding site deep within the pore. Our results suggest that Mg 2ϩ acts as a voltage-dependent pore blocker and, therefore, reduces outward currents through HCN channels. The pore-blocking action of Mg 2ϩ may play an important physiological role, especially for the slowly gating HCN2 and HCN4 channels. Mg 2ϩ could potentially block outward hyperpolarizing HCN currents at the plateau of action potentials, thus preventing a premature termination of the action potential. inward rectifying; hyperpolarization-activated current; instantaneous currents; divalent block; cysteine; hyperpolarization-activated cyclic nucleotide RHYTHMIC ACTIVITY OF PACEMAKER cells in the heart and thalamic neurons in the brain are dependent on the inward I h current through hyperpolarization-activated cyclic nucleotide-gated (HCN) channels (29,36). Following an action potential in pacemaker cells, I h contributes to the currents that slowly depolarize the membrane potential to threshold, thereby initiating another action potential (7). Four mammalian HCN channels have been cloned: HCN1-HCN4 (11,21,35). The deletion of HCN channels, or the presence of naturally occurring mutations in HCN channels, has been shown to have significant physiological consequences (20,25,37,40). HCN2 knockout mice, for example, showed spontaneous absence seizures and cardiac sinus dysrhythmia (20).HCN channels are members of the superfamily of voltagegated ion channels, possessing features such as a tetrameric structure, with each subunit containing six transmembrane domains (S1-S6) (29,36). In addition, HCN channels have a pore domain that shows conservation with voltage-gated potassium (Kv) channels, including a GYG signature motif in the selectivity filter, even though HCN channels are only modestly more selective (3:1) for K ϩ over Na ϩ (11, 21). Similar to Kv channels, HCN channels have an intracellular gate at the base of S6 that prevents access of ions to the pore (32, 33)....

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“…Metabolic inhibition did not affect total cellular HDP content (in vitro assay of lysates of control and DOG-treated hearts: 1.09 ± 0.12 and 1.04 ± 0.15 mM HDPs per gram of protein, respectively) (25), but it reduced [ATP] i by up to 95% (Fig. 3 A, iii) ] from 1 to 30 mM and removing extracellular Na + (27). This maneuver increased MagFluo4 fluorescence fourfold with a time constant of 3 min (Fig.…”

mentioning

confidence: 99%

Coupled Ca ²⁺ /H ⁺ transport by cytoplasmic buffers regulates local Ca ²⁺ and H ⁺ ion signaling

Swietach

Youm

Saegusa

et al. 2013

Proc. Natl. Acad. Sci. U.S.A.

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Significance The concentration of Ca 2+ ions is kept low in cells by specialized ion-pumping proteins at the membrane. We show that in cardiac cells, cytoplasm also has an intrinsic ability to pump Ca 2+ . Histidyl dipeptides and ATP are diffusible cytoplasmic buffer molecules. By exchanging Ca 2+ for H + , they act like local “pumps,” producing uphill Ca 2+ movement within cytoplasm in response to H + ion gradients. Intracellular H + ions are generated locally by metabolism and competitively inhibit many Ca 2+ -activated biochemical processes. Recruiting Ca 2+ to acidic zones facilitates these processes. Cytoplasmic histidyl dipeptides and ATP thus act like a biological pump without a membrane.

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Loading rat heart myocytes with Mg²⁺ using low‐[Na⁺] solutions

Cited by 12 publications

References 46 publications

Adenosine triphosphate depletion by cyanide results in a Na+-dependent Mg2+ extrusion from liver cells

Adenosine triphosphate depletion by cyanide results in a Na+-dependent Mg2+ extrusion from liver cells

Intracellular Mg²⁺ is a voltage-dependent pore blocker of HCN channels

Coupled Ca ²⁺ /H ⁺ transport by cytoplasmic buffers regulates local Ca ²⁺ and H ⁺ ion signaling

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Loading rat heart myocytes with Mg2+ using low‐[Na+] solutions

Cited by 12 publications

References 46 publications

Adenosine triphosphate depletion by cyanide results in a Na+-dependent Mg2+ extrusion from liver cells

Adenosine triphosphate depletion by cyanide results in a Na+-dependent Mg2+ extrusion from liver cells

Intracellular Mg2+ is a voltage-dependent pore blocker of HCN channels

Coupled Ca 2+ /H + transport by cytoplasmic buffers regulates local Ca 2+ and H + ion signaling

Contact Info

Product

Resources

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Loading rat heart myocytes with Mg²⁺ using low‐[Na⁺] solutions

Intracellular Mg²⁺ is a voltage-dependent pore blocker of HCN channels

Coupled Ca ²⁺ /H ⁺ transport by cytoplasmic buffers regulates local Ca ²⁺ and H ⁺ ion signaling