Measurements of Intracellular Mg2+ Concentration in Mouse Skeletal Muscle Fibers with the Fluorescent Indicator Mag-Indo-1

Csernoch, László; Bernengo, Jean Claude; Szentesi, Péter; Jacquemond, Vincent

doi:10.1016/s0006-3495(98)77584-8

Cited by 52 publications

(51 citation statements)

References 29 publications

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“…Other sensors, such as magnesium, 19 calcium, 43 and zinc 24 sensors, which also rely on the binding of the metabolite the sensor is designed for, could potentially encounter the same problems. pH Measurements in Time and Space.…”

Section: Resultsmentioning

confidence: 99%

Evaluating Nanoparticle Sensor Design for Intracellular pH Measurements

Benjaminsen

Sun²,

Henriksen³

et al. 2011

ACS Nano

166

195

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Particle-based nanosensors have over the past decade been designed for optical fluorescent-based ratiometric measurements of pH in living cells. However, quantitative and time-resolved intracellular measurements of pH in endosomes and lysosomes using particle nanosensors are challenging, and there is a need to improve measurement methodology. In the present paper, we have successfully carried out time-resolved pH measurements in endosomes and lyosomes in living cells using nanoparticle sensors and show the importance of sensor choice for successful quantification. We have studied two nanoparticle-based sensor systems that are internalized by endocytosis and elucidated important factors in nanosensor design that should be considered in future development of new sensors. From our experiments it is clear that it is highly important to use sensors that have a broad measurement range, as erroneous quantification of pH is an unfortunate result when measuring pH too close to the limit of the sensitive range of the sensors. Triple-labeled nanosensors with a pH measurement range of 3.2–7.0, which was synthesized by adding two pH-sensitive fluorophores with different pK a to each sensor, seem to be a solution to some of the earlier problems found when measuring pH in the endosome–lysosome pathway.

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Section: Resultsmentioning

confidence: 99%

Evaluating Nanoparticle Sensor Design for Intracellular pH Measurements

Benjaminsen

Sun²,

Henriksen³

et al. 2011

ACS Nano

166

195

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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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“…Results were either expressed in terms of indo-1% saturation or in actual free Ca 2+ concentration (for details of calculation, see [10,19]). In vivo values for R min , R max and β were measured using procedures previously described [5,7].…”

Section: Calibration Of the Indo-1 Response And [Ca 2+ ] Intra Calculmentioning

confidence: 99%

Expression of the muscular dystrophy-associated caveolin-3P104L mutant in adult mouse skeletal muscle specifically alters the Ca2+ channel function of the dihydropyridine receptor

Weiss

Couchoux

Legrand

et al. 2008

Pflugers Arch - Eur J Physiol

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Caveolins are plasma-membrane-associated proteins potentially involved in a variety of signalling pathways. Different mutations in CAV3, the gene encoding for the muscle-specific isoform caveolin-3 (Cav-3), lead to muscle diseases, but the underlying molecular mechanisms remain largely unknown. Here, we explored the functional consequences of a Cav-3 mutation (P104L) inducing the 1C type limb-girdle muscular dystrophy (LGMD 1C) in human on intracellular Ca(2+) regulation of adult skeletal muscle fibres. A YFP-tagged human Cav-3(P104L) mutant was expressed in vivo in muscle fibres from mouse. Western blot analysis revealed that expression of this mutant led to an approximately 80% drop of the level of endogenous Cav-3. The L-type Ca(2+) current density was found largely reduced in fibres expressing the Cav-3(P104L) mutant, with no change in the voltage dependence of activation and inactivation. Interestingly, the maximal density of intramembrane charge movement was unaltered in the Cav-3(P104L)-expressing fibres, suggesting no change in the total amount of functional voltage-sensing dihydropyridine receptors (DHPRs). Also, there was no obvious alteration in the properties of voltage-activated Ca(2+) transients in the Cav-3(P104L)-expressing fibres. Although the actual role of the Ca(2+) channel function of the DHPR is not clearly established in adult skeletal muscle, its specific alteration by the Cav-3(P104L) mutant suggests that it may be involved in the physiopathology of LGMD 1C.

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Measurements of Intracellular Mg2+ Concentration in Mouse Skeletal Muscle Fibers with the Fluorescent Indicator Mag-Indo-1

Cited by 52 publications

References 29 publications

Evaluating Nanoparticle Sensor Design for Intracellular pH Measurements

Evaluating Nanoparticle Sensor Design for Intracellular pH Measurements

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

Expression of the muscular dystrophy-associated caveolin-3P104L mutant in adult mouse skeletal muscle specifically alters the Ca2+ channel function of the dihydropyridine receptor

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Measurements of Intracellular Mg2+ Concentration in Mouse Skeletal Muscle Fibers with the Fluorescent Indicator Mag-Indo-1

Cited by 52 publications

References 29 publications

Evaluating Nanoparticle Sensor Design for Intracellular pH Measurements

Evaluating Nanoparticle Sensor Design for Intracellular pH Measurements

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

Expression of the muscular dystrophy-associated caveolin-3P104L mutant in adult mouse skeletal muscle specifically alters the Ca2+ channel function of the dihydropyridine receptor

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