Mg2+ block and inward rectification of mechanosensitive channels in Xenopus oocytes

 The effects of Mg²⁺ on single mechanosensitive (MS) channel currents recorded from Xenopus oocytes were studied using cell-attached and inside-out patch configurations. Mg²⁺ both permeates and blocks MS channels. Under symmetrical ionic conditions, the blocking effects of Mg²⁺ can be described by a Hill coefficient of 0.9 at ±100 mV and IC₅₀s of 0.12 mM (–100 mV) and 0.60 mM at (+100 mV). Although block by intracellular Mg²⁺ may contribute to inward MS channel rectification, significant current rectification is retained even under symmetrical KCl concentrations and in the complete absence of Mg²⁺. The observed voltage dependencies of the IC₅₀ for Mg²⁺ block and the K_m for K⁺ current saturation indicate asymmetries in the MS channel pore. In addition, the absence of K⁺ self block and anomalous mole fraction effects with K⁺/Tl⁺ mixtures indicate a single site pore model. Received: 11 September 1997 / Received after revision: 21 November 1997 / Accepted: 24 November 1997     

Mg2+ and memantine block of rat recombinant NMDA receptors containing chimeric NR2A/2D subunits expressed in Xenopus laevis oocytes.

N-methyl-d-aspartate receptors (NMDARs) display differences in their sensitivity to the channel blockers Mg(2+) and memantine that are dependent on the identity of the NR2 subunit present in the receptor-channel complex. This study used two-electrode voltage-clamp recordings from Xenopus laevis oocytes expressing recombinant NMDARs to investigate the actions of Mg(2+) and memantine at the two NMDARs displaying the largest differences in sensitivity to these blockers, namely NR1/NR2A and NR1/NR2D NMDARs. In addition, NR2A/2D chimeric subunits have been employed to examine the effects of pore-forming elements and ligand-binding domains (LBD) on the potency of the block produced by each of these inhibitors. Our results show that, as previously documented, NR2D-containing NMDARs are less sensitive to voltage-dependent Mg(2+) block than their NR2A-containing counterparts. The reduced sensitivity is determined by the M1M2M3 membrane-associated regions, as replacing these regions in NR2A subunits with those found in NR2D subunits results in a approximately 10-fold reduction in Mg(2+) potency. Intriguingly, replacing the NR2A LBD with that from NR2D subunits results in a approximately 2-fold increase in Mg(2+) potency. Moreover, when responses mediated by NR1/NR2A NMDARs are evoked by the partial agonist homoquinolinate, rather than glutamate, Mg(2+) also displays an increased potency. Memantine block of glutamate-evoked currents is most potent at NR1/NR2D NMDARs, but no differences are observed in its ability to inhibit NR2A-containing or NR2A/2D chimeric NMDARs. We suggest that the potency of block of NMDARs by Mg(2+) is influenced not only by pore-forming regions but also the LBD and the resulting conformational changes that occur following agonist binding.     

Phosphorylation modulates L-type Ca channels in frog ventricular myocytes by changes in sensitivity to Mg2+ block

 Maitotoxin (MTX) may exert its toxic effect by activating ion conductances and has been shown to elicit a fertilization-like response in Xenopus laevis oocytes. In the present study we investigated the electrophysiological response of stage V–VI Xenopus oocytes to MTX using the two-microelectrode voltage-clamp technique. Membrane voltage (V _m) measurements demonstrated that MTX (50 pM to 1 nM) depolarized the oocytes from –49±7 to –14±1 mV. Subsequent replacement of bath Na⁺ by the impermeant cation NMDG (N-methyl-d-glucamine) shifted V _m from –14±1 to –53±5 mV (n=29). This indicates that MTX activates a cation conductance. Indeed, current measurements at a holding potential of –60 or –100 mV showed that within 10 s of MTX application an inward current component developed which was largely abolished by extracellular Na⁺ removal. After a 1-min application of 1 nM MTX the NMDG-sensitive current increased more than 100-fold from 0.14±0.03 μA to a peak value of 21±3 μA (n=11). The effect of MTX was concentration dependent with an EC₅₀ of about 250 pM but only slowly reversible. Ion substitution experiments indicated that the stimulated conductance was nonselective for monovalent cations with a slight preference for NH₄ ⁺ (2.1) > K⁺ (1.5) > Na⁺ (1.0) > Li⁺ (0.7). Regarding divalent cations, a complex biphasic response to extracellular Na⁺ replacement by Ca²⁺ was observed, which suggests that the stimulated channels may have a small Ca²⁺ permeability but that exposure to high extracellular Ca²⁺ enhances recovery from MTX stimulation. No significant conductance for Mn²⁺ was observed. Application of 1 mM benzamil, 1 mM amiloride, or 100 μM 1-(β-[3-(4-Methoxyphenyl)-propoxy]-4-methoxyphenethyl)-1H-imidazole hydrochloride (SK&F 96365) reduced the MTX-stimulated inward current by 81%, 62%, or 65%, respectively. Gd³⁺ had an inhibitory effect of 29% and 38% at concentrations of 10 μM or 100 μM, respectively. Flufenamic acid, niflumic acid, (RS)-(3,4-dihydro-6,7-dimethoxyisoquinoline-1-γ1)-2-phenyl-N,N-di-[2-(2,3,4-trimethoxyphenyl)-ethyl]-acetamide (LOE908), and 3′,5′-dichlorodiphenylamine-2-carboxylic acid (DCDPC), known blockers of other nonselective cation channels, had no significant effect. We conclude that MTX activates a nonselective cation conductance in Xenopus oocytes. The underlying channels may be involved in changes in V _m that occur during the early stages of fertilization. Received: 30 December 1997 / Received after revision and accepted: 17 March 1998     

Modulation of Ca2+-activated K+ channels by Mg2+ and ATP in frog oxyntic cells

Ca²⁺-activated K⁺ channels in the basolateral plasma membrane of bullfrog oxynticopeptic cells are intimately involved in the regulation of acid secretion. Patch-clamp techniques were applied to study the regulating mechanism of these channels. In the excised inside-out configuration, intracellular Mg²⁺ decreased channel activity in a dose-dependent manner. In the absence of Mg²⁺, administration of adenosine 5 triphosphate (ATP) to the cytoplasmic side also inhibited channel activity. On the other hand, in the presence of Mg²⁺, addition of ATP markedly increased channel activity. At a fixed concentration of free Mg²⁺ the Mg-ATP complex caused channel activation and shifted the dose response relationship between channel activity and the intracellular Ca²⁺ concentration to the left. A nonhydrolysable ATP analogue, adenosine 5-[,-imido]triphosphate (AMP-PNP) adenylyl [,-methylene]diphosphate (AMP-PCP), could not substitute for ATP in channel activation, but a hydrolysable ATP analogue, adenosine 5-O-(3-thiotriphosphate) (ATP[S]) could do so. Furthermore, application of alkaline phosphatase to the cytoplasmic side inhibited channel activity. These results demonstrate that Ca²⁺-activated K⁺ channels are regulated by Mg²⁺ and ATP, and suggest that a phosphorylation reaction may be involved in the regulation mechanism of these channels.     

Modulation of Ca2+ channels by intracellular Mg2+ ions and GTP in frog ventricular myocytes

Under conditions of low intracellular [Mg²⁺] ([Mg²⁺]_i), achieved by dialysis with pipette solutions containing ethylenediamine tetraacetic acid (EDTA), 1,2-bis(2-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid (BAPTA) and adenosine triphosphate (ATP) as chelator, calcium currents through the L-type calcium channels (I _Ca) were increased in frog ventricular myocytes. Total suppression of phosphorylation by depleting the cell of ATP with a cocktail of β,γ-methyleneadenosine 5′-triphosphate (AMP-PCP) 2-deoxyglucose and carboxylcyanide-M-chlorophenylhydrazone (CCCP) did not inhibit the increase in I _Ca in the Mg²⁺-deficient medium. Thus, the involvement of phosphorylation process in the increase in I _Ca was not likely. Effective suppression of this enhancement of I _Ca was achieved by the application of guanosine triphosphate (GTP). From the dose-response curve for GTP, the GTP concentration required for half-maximal inhibition (IC₅₀) was estimated to be 4.0 μM at pMg 6. This GTP-induced suppression of I _Ca is not due to the guanine nucleotide binding protein (G-protein) cascade, because both activators and inhibitors of G-protein, which are structural analogues of GTP, suppressed I _Ca similarly. Treatment with pertussis toxin (PTX) did not affect the inhibitory action of Mg²⁺ and GTP on I _Ca. GTP is therefore assumed to bind directly to the Ca²⁺ channel. Interaction of Mg²⁺ and GTP with the Ca²⁺ channel activated in the Mg²⁺-deficient medium was examined by comparing the dose/response curves for GTP at two different [Mg²⁺]. The IC₅₀ for GTP suppression was estimated to be 5.7 μM at pMg 6 and 6.9 μM at pMg 5. The results suggest strongly that Mg²⁺ and GTP independently bind and control Ca²⁺ channels. Received: 22 December 1995/Received after revision and accepted: 11 March 1996     

Viral delivery of NR2D subunits reduces Mg2+ block of NMDA receptor and restores NT-3-induced potentiation of AMPA-kainate responses in maturing rat motoneurons

N-methyl-D-aspartate (NMDA) responsiveness of motoneurons declines during the initial 2 postnatal weeks due to increasing Mg2+ block of NMDA receptors. Using gene chip analyses, RT-PCR, and immunochemistry, we have shown that the NR2D subunit of the NMDA receptor (NMDAR), known to confer resistance to Mg2+ block, also declines in motoneurons during this period. We injected a viral construct (HSVnr2d) into the lumbar spinal cord on postnatal day 2 in an attempt to restore NMDAR function in motoneurons during the second postnatal week. Following HSVnr2d injection, we detected elevated levels of NR2D mRNA in spinal cord samples and NR2D protein specifically in motoneurons. These molecular changes were associated with marked functional alterations whereby NMDAR-mediated responses in motoneurons associated with both dorsal root (DR) and ventrolateral funiculus (VLF) inputs returned to values observed at E18 due to decreased Mg2+ blockade. Viruses carrying the beta-galactosidase gene did not induce these effects. NT-3 is known to potentiate AMPA-kainate responses in motoneurons if the response has an NMDAR-mediated component and thus is normally ineffective during the second postnatal week. Restoration of NMDAR-mediated responsiveness in the second postnatal week was accompanied by a return of the ability of neurotrophin-3 (NT-3) to potentiate the AMPA-kainate responses produced by both DR and VLF synaptic inputs. We conclude that delivery of the gene for a specific NMDA subunit can restore properties characteristic of younger animals to spinal cord motoneurons. This approach might be useful for enhancing the function of fibers surviving in the damaged spinal cord.     

SK channels and NMDA receptors form a Ca2+-mediated feedback loop in dendritic spines

Small-conductance Ca(2+)-activated K(+) channels (SK channels) influence the induction of synaptic plasticity at hippocampal CA3-CA1 synapses. We find that in mice, SK channels are localized to dendritic spines, and their activity reduces the amplitude of evoked synaptic potentials in an NMDA receptor (NMDAR)-dependent manner. Using combined two-photon laser scanning microscopy and two-photon laser uncaging of glutamate, we show that SK channels regulate NMDAR-dependent Ca(2+) influx within individual spines. SK channels are tightly coupled to synaptically activated Ca(2+) sources, and their activity reduces the amplitude of NMDAR-dependent Ca(2+) transients. These effects are mediated by a feedback loop within the spine head; during an excitatory postsynaptic potential (EPSP), Ca(2+) influx opens SK channels that provide a local shunting current to reduce the EPSP and promote rapid Mg(2+) block of the NMDAR. Thus, blocking SK channels facilitates the induction of long-term potentiation by enhancing NMDAR-dependent Ca(2+) signals within dendritic spines.     

Removal of NMDA receptor Mg(2+) block extends the action of NT-3 on synaptic transmission in neonatal rat motoneurons.

NT-3 has previously been reported to enhance AMPA/kainate receptor-mediated synaptic responses in motoneurons via an effect on the N-methyl-D-aspartate (NMDA) receptor. To investigate neurotrophin-3 (NT-3) action further, we measured the NMDA receptor (NMDAR)-mediated synaptic response directly by intracellular recording in motoneurons after blocking AMPA/kainate, GABA(A), GABA(B) and glycine receptor-mediated responses pharmacologically. Two pathways were stimulated, the segmental dorsal root (DR) and the descending ventrolateral fasciculus (VLF). The DR-evoked NMDAR-mediated response in motoneurons of rats younger than 1 wk has two components, the initial one of which is generated monosynaptically. NT-3 strongly potentiated both NMDA components in a rapidly reversible manner. No NMDAR-mediated responses were present at VLF connections and at DR connections in older (1- to 2-wk-old) neonates. Bath-applied NT-3-induced potentiation of the AMPA/kainate receptor-mediated response occurred only at connections that exhibit a synaptic NMDA receptor-mediated response. Reducing Mg(2+) concentration in the bathing solution restored the NMDAR-mediated response elicited by DR stimulation in older neonates and by VLF throughout the neonatal period (0-2 wk). In low-Mg(2+), NT-3 enhanced AMPA/kainate receptor-mediated responses elicited by inputs normally not influenced by NT-3. Thus a major reason for the loss of NT-3 action on AMPA/kainate synaptic responses is the reduced activity of the NMDA receptor due to developing Mg(2+) block of NMDA receptor-channel complex as the animal matures, and both can be re-established by reducing Mg(2+) concentration in fluid bathing the spinal cord.     

Microelectrode study of voltage-dependent Ba2+ and Cs+ block of apical K+ channels in the skin of Rana temporaria

The blockage of the apical K⁺ channels in frog species Rana temporaria by Ba²⁺ and Cs⁺ is strongly voltage-dependent. The interaction of both blockers with the K⁺ channels was studied by recording relations between the K⁺ currents (I _K) and the transepithelial and intracellular potential. Mucosal Ba²⁺ and Cs⁺ depress I _K, hyperpolarize the cell and induce pronounced nonlinearities in the current/voltage (I/V) relations. The nonlinearities are caused by the voltage-dependent interaction of Ba²⁺ and Cs⁺ with the binding site. Consequently, the apical membrane resistance not only depends on the blocker concentration but also on the apical membrane potential. Also the fractional resistance, fR _a, and the voltage divider ratio, fV _a, will change with blocker concentration and voltage. Owing to this non-ohmic behaviour, measurements of fV _a in the presence of Ba²⁺ deviate markedly from the expected fR _a values. The inhibitory effect of Ba²⁺ and Cs⁺ was analysed at different transepithelial and apical membrane voltages. The relation between the Michaelis-Menten constants and the voltage could be fitted with equations based on Eyring rate theory with the assumption of a single binding site. With this model we calculated the relative electrical position of the binding site for the blocker (), referred to the extracellular side of the channel. We obtained for Ba²⁺, =0.34±0.05 and for Cs⁺, =0.81±0.01. Comparison of the results from apical and transepithelial I/V relations demonstrates that the analysis of the transepithelial data provides overestimated values of the Hill coefficient and results in an underestimation of .     

A use-dependent tyrosine dephosphorylation of NMDA receptors is independent of ion flux

Tyrosine phosphorylation can upregulate NMDA receptor activity during pathological and physiological alterations of synaptic strength. Here we describe downregulation of recombinant NR1/2A receptors by tyrosine dephosphorylation that requires agonist binding, but is independent of ion flux. The tyrosine residues involved in this new form of NMDA receptor modulation likely form a 'ring' adjacent to the last transmembrane domain. The downregulation was due to a reduction in the number of functional channels, and was blocked by co-expressing a dominant-negative mu2-subunit of the clathrin-adaptor protein AP-2. Our results provide a mechanism by which synaptic NMDA receptors can be modulated in a use-dependent manner even when the postsynaptic membrane is not sufficiently depolarized to relieve channel block by magnesium ions.     

The polyamine diaminodecane (DA-10) produces a voltage-dependent flickery block of single NMDA receptor channels.

Receptor binding assays have shown that diaminodecane (DA-10) reduced binding of open channel blockers to the N-methyl-D-aspartate (NMDA) subtype of postsynaptic glutamate receptor through an interaction with the polyamine regulatory site. Because the action of DA-10 was opposite to that of the polyamine agonist spermine and was reversed by polyamine antagonists, DA-10 has been classified as an inverse agonist at the polyamine site. Using whole-cell voltage-clamp and single-channel recordings from cultured rat cortical neurons, we show that at negative holding potentials DA-10 (1-300 microM) reduced NMDA receptor whole cell current (IC50 = 34 microM) and produced a flickery block of NMDA single-channel currents. The flickery block of NMDA single channels was voltage-dependent and not reversed by the polyamine antagonist diethylenetriamine (DET). Potential mechanisms for the flickery block of NMDA single channel currents are discussed.     

Cross-linking of the (Ca2+ + Mg2+)-ATPase protein.

The addition of cupric-1,10,-phenanthroline, a cross-linking catalyst, to sarcoplasmic reticulum membranes caused protein sulfhydryl groups to form disulfide bridges. Following a short exposure to the catalyst (15 s, 22 degrees C) most of the protein was in a dimeric form (Mr = 248,000). Longer exposure times resulted in the formation of trimers, tetramers and other oligomers too large to enter the gel. At low temperatures (4 degrees C) dimer formation predominates, even for exposure times as long as 5 min. Cross-linking in the presence of 7.5 mM Triton X-100 (a concentration that resulted in clearing of the membrane suspension and thus solubilization of the membrane components) showed the appearance of a considerable dimer fraction, however, most of the (Ca2+ + Mg2+)-ATPase protein appeared as a monomer. Following 1 min of cross-linking at 22 degrees C, freeze-etched membranes showed no alteration in the number or appearance of 80 A intramembranous particles. Thus extensive cross-linking of the (Ca2+ + Mg2+)-ATPase protein can occur without disruption of the normal position of the intramembrane portion of the molecule.     

Ca2+ ions block and permeate serotonin 5-HT3 receptor channels in rat hippocampal interneurons

The serotonin 5-HT(3) receptor native to rat hippocampal CA1 stratum radiatum interneurons is blocked by Ca(2+) ions in a dose- and voltage-dependent manner, which is reflected by a region of negative slope conductance in the I-V curve. The steep dependence on the extracellular Ca(2+) concentration suggests that the channel contains more than one binding site for Ca(2+). A three barrier-two site model, based on Eyring rate theory, was used to describe the I-V curves. When extra- and intracellular K(+) and Cs(+) were substituted with Na(+), the I-V curves were accurately fit by the model, unlike the I-V curves recorded under standard ionic conditions. This suggests that the K(+) and Cs(+) permeabilities are small compared with that of Na(+). The distribution of the energy barriers and binding sites for Ca(2+) and Na(+) showed that the binding sites are located at approximately the 13' and the -4' position in the ion channel. The model predicts that at large hyperpolarized membrane potentials (more negative than -120 mV), the fractional Ca(2+) current amounts to approximately 1% of the total ion current. However, at physiologically relevant membrane potentials, the fractional Ca(2+) current is smaller (<0.1%) and the relative Ca(2+) permeability (P(Ca)/P(Na)) is estimated to be 0.10 at -60 mV.     

Changes in ionised Mg2+ and Ca2+ in maternal serum during weaning in goats.

Ionised levels of serum magnesium (Mg2+) and calcium (Ca2+) were measured in HEPES-buffered serum from lactating goats before and during weaning. During this period, there was a significant rise in serum Ca2+, and a concomitant fall in Mg2+. These opposing changes can be explained by the known enhanced absorption of Ca relative to Mg by the gut during lactation coupled with a Ca-induced suppression of PTH-driven renal Ca and Mg reabsorption. A rise in the serum Ca2+/Mg2+ ratio during weaning suggests that this is a period of potential cardiovascular risk for the mother, and merits closer study.     

Modulation of sarcoplasmic reticulum Ca2+-release channels by caffeine,Ca2+, and Mg2+ in skinned myocardial fibers of fetal and adult rats

Ryanodine causes depression of the caffeine-induced tension transient (ryanodine depression) in skinned muscle fibers, because it blocks the sarcoplasmic reticulum (SR) Ca²⁺-release channels [Su, J. Y. (1988) Pflügers Arch 411:132–136, 371–377; (1992) Pflügers Arch 421:1–6]. This study was performed to examine the sensitivity of SR Ca²⁺-release channels to ryanodine in fetal compared to adult myocardium and to investigate the influence of Ca²⁺, caffeine, and Mg²⁺ on ryanodine depression in skinned fibers. Ryanodine (0.3 nM–1 M) caused a dose-dependent depression in skinned myocardial fibers of the rat, and the fetal fibers (IC₅₀74 nM) were 26-fold less sensitive than those of the adult (IC₅₀2.9 nM). The depression induced by 0.1 M or 1 M ryanodine was a function of [caffeine], or [Ca²⁺] (pCa<6.0), which was potentiated by caffeine, and an inverse function of [Mg²⁺]. At pCa>8.0 plus 25 mM caffeine, a 20% ryanodine depression was observed in both the fetal and adult fibers, indicating independence from Ca²⁺. Ryanodine depression in skinned fibers of the fetus was less affected than that seen in the adult by pCa_i, [caffeine]_i, or 25 mM caffeine plus pCa_i or plus pMg_i (IC₅₀pCa 4.5 versus 5.1; caffeine 12.7 mM versus 2 mM; pCa 6.7 versus 7.3; and pMg 3.9 versus 3.3 respectively). The results show that the SR Ca²⁺-release channel in both fetal and adult myocardium is modulated by Ca²⁺, caffeine, and Mg²⁺. It is concluded that less ryanodine depression seen in the skinned fibers of the fetus, indicating a relatively insensitive SR Ca²⁺-release channel, could contribute to the resistance of intact myocardium to ryanodine.     

Na+/Mg2+ antiport in erythrocytes of spontaneously hypertensive rats: role of Mg2+ in the pathogenesis of hypertension.

Total Mg2+ content in plasma and erythrocytes did not significantly differ between WKY and SHR. Mg2+ efflux via Na+/Mg2+ antiport was 10% lower in non Mg(2+)-loaded erythrocytes of SHR than in WKY, and 16% lower in Mg(2+)-loaded erythrocytes of SHR. The activation of Na+/Mg2+ antiport in erythrocytes by Cl-, as tested by substitution of Cl- with SCN-, and the regulation of Na+/Mg2+ antiport by protein kinases, as tested by PMA and staurosporine, showed no differences between WKY and SHR. The reduction of Na+/Mg2+ antiport was explained by a reduction in the number of Na+/Mg2+ antiporter molecules in SHR erythrocytes. Mg2+ efflux in KCl medium by K+/Mg2+ antiport via the unspecific choline exchanger was not significantly reduced in SHR and was equally affected by PMA and staurosporine in WKY and SHR. An explanation for some controversial results, unchanged or reduced concentration of Mg2+ in serum, total Mg2+ and free Mg2+ in erythrocytes of SHR and patients with essential hypertension was proposed. The role of Na+/Mg2+ antiport and [Mg2+]i in the pathogenesis of experimental and clinical hypertension was discussed.