Weakening of ion-channel interactions of Na+ and Li+ in acetylcholine-receptor channels of frog skeletal muscle with an increase in agonist concentration

 The possibility that increases in agonist concentration beyond threshold levels may force changes in the character of high-conductance open states of skeletal muscle nicotinic acetylcholine receptor channels (nAChR) was examined by seeing whether differences in several critical ionic properties of nAChR currents could be detected with changes in agonist level. Single- and bi-ionic whole-cell currents of Na⁺ and Li⁺ in voltage-clamped frog (Rana pipiens) muscle fibers were measured during local superfusion of endplates with carbamylcholine (carb) at concentra- tions of 54 μm (low-carb) and 270 μM (high-carb). Three ionic properties that would be affected by changes in the open-state configuration of channel subunits were tested. First, ion-saturation characteristics. Peak Na⁺ and Li⁺ currents in low-carb trials showed sublinear dependence on ion concentrations from 0 to 60 mM with K _m values of 78 (Na⁺) and 49 (Li⁺) mM and a power function slope of 0.75 on double-log plot. In contrast, the concentration dependence of Na⁺ and Li⁺ currents in high-carb tests was linear through the origin with a power function slope of 1.02. Second, Na⁺/Li⁺ selectivity. The ratio of peak Na⁺ and Li⁺ currents in low-carb tests varied from 1.86 to 2.28 for ion concentrations of from 20 to 60 mM [mean = 2.02 ± 0.06 (SEM)] whereas the ratio for high-carb trials ranged from only 1.29 to 1.52 [mean = 1.42 ± 0.40 (SEM)]. Third, com- petitive interactions of Na⁺ and Li⁺ currents. Equimolar mixtures of Na⁺ and Li⁺ in low-carb tests produced bi-ionic inward currents which were never larger than the single-ion Na⁺ current alone, but bi-ionic currents at the high-carb level were always greater than the single-ion Na⁺ current, approximating the sum of the single-ion Na⁺ and Li⁺ currents in most cases. The results are consistent with a decrease in ion-channel binding at the high-carb level and support the possibility of agonist-induced changes in the high-conductance open-state configuration of nAChR subunits which result in a weakening of constraints on cation movements through the channel. Received: 1 July 1997 / Received after revision: 15 December 1997 / Accepted: 24 December 1997     

Kinetic evidence that desensitized nAChR may promote transitions of active nAChR to desensitized states during sustained exposure to agonists in skeletal muscle

During prolonged exposure of postjunctional nicotinic acetylcholine receptors (nAChR) of skeletal muscle to acetylcholine (ACh), agonist-activated nAChR (nAChR_a) gradually fall into a refractory “desensitized” state (nAChR_d), which no longer supports the high-conductance channel openings characteristic of the initially active nAChR_a. In the present study, the possibility was examined that nAChR_d, rather than simply constituting a passive “trap” for nAChR_a, may actively promote further conversions of nAChR_a to nAChR_d in a formally autocatalytic manner. Single-ion whole-cell voltage-clamp currents (Na⁺ and Li⁺ in separate trials) were measured using two KCl-filled capillary electrodes (5–10 MΩ) implanted at the postjunctional locus of single frog skeletal muscle fibers (Rana pipiens) equilibrated in 30 mM K⁺ bath media to eliminate mechanical responses. Various nAChR agonists (carbamylcholine, acetylcholine, suberyldicholine) at different concentrations were delivered focally by positive pressure microjet. It was found that the decline of postmaximal agonist-induced currents under these different conditions (driven by the growth of the subpool of nAChR_d) consistently followed an autocatalytic logistic rule modified for population growth of fixed units in a planar array: (where y represents the remaining agonist-induced current at time t, A=initial maximum current, and n is a constant). Some further experimental features that might result from a self-promoting growth of nAChR_d were also tested, namely, (1) the effect of increased nAChR_a and (2) the effect of increased nAChR_d. Increase in agonist concentration of the superfusate, by increasing the planar density of active nAChR_a at the outset, should enhance the probability of autocatalytic interactions with emerging nAChR_d, hence, the rate of decline of agonist-induced current, and this was a consistent finding under all conditions tested. Raising the initial level of desensitized nAChR_d by pretreatment of fibers with very low concentrations of agonist would be another way to increase autocatalytic interactions with active nAChR_a, and this was also found to produce increased rates of decline of agonist-induced currents when tested in additional trials. It is concluded that several kinetic features of nAChR desensitization in skeletal muscle are consistent with an action of nAChR_d to promote further transitions of nAChR_a to desensitized forms. This could occur by a direct effect of nAChR_d on contiguous nAChR_a or perhaps through some intermediary membrane component or local intracellular pathway.     

Monoclonal antibody to β2 subunit of neuronal nicotinic receptor depresses the postjunctional non-contractile Ca mobilization in the mouse diaphragm muscle

The involvement of subtypes of nicotinic acetylcholine receptor (nAChR) in the postjunctional non-contractile Ca²⁺ mobilization was investigated in mouse diaphragm muscles treated with an anticholinesterase, using monoclonal antibodies (mAbs) to nAChR subunits. mAb 210 (specific for 1 subunit of muscle nAChR) depressed contractile Ca²⁺ transients without affecting non-contractile Ca²⁺ transients. mAb 270 (specific for β2 subunit of neuronal nAChR) depressed only non-contractile Ca²⁺ transients. mAb 210 did not completely block the ACh-activated channel currents in flexor digitorum brevis muscle cells. The present findings indicate that the anti-β2 mAb 270-related subtype of nAChR may postsynaptically operate the non-contractile Ca²⁺ mobilization at the neuromuscular junction, suggesting the involvement of a subtype different from the usual muscle-type nAChR.     

Calcium permeability of nicotinic acetylcholine receptor channels in bovine adrenal chromaffin cells

The fractional contribution of Ca to current flow through neuronal-type nicotinic acetylcholine receptor channels was determined by quantitative fluorescence microfluorimetry using fura-2. The method, which has been applied already to several types of cells and channels is described in detail here. At –70 mV and 2 mM external Ca concentration it was found that Ca contributes 2.5% to the net current. The fractional contribution was found to be voltage dependent, increasing at negative potentials e-fold for a 110 mV potential difference. Total non-specific cation current was found to have a bell-shaped dependence on external Ca concentration peaking at 2 mM.     

Intracellular free Mg2+ concentration in skeletal muscle fibres of frog and crayfish

The free intracellular Mg²⁺ concentration ([Mg²⁺]_i) was investigated in frog sartorius and crayfish phasic and tonic skeletal muscle fibres, using a new Mg²⁺-sensitive microelectrode based on the ionophore ETH 5214 [Hu et al. (1989) Anal Chem 61:574–576]. In Ringer solution containing 0.5 mmol/l MgCl₂, the mean [Mg²⁺]_i of the frog muscle fibres was 1.3 mmol/l. In phasic crayfish muscle fibres, [Mg²⁺]_i was about twice as high (mean 3.5 mmol/l) as in tonic fibres (mean 1.5 mmol/l), measured in van Harreveld solution containing 1.2 mmol/l MgCl₂. Long-lasting (3–12 h) incubation of frog skeletal muscle fibres in Na⁺-free solution produced a reversible increase of [Mg²⁺]_i by a factor of about 1.7. A tenfold rise of extracellular Mg²⁺ led to an increase in [Mg²⁺]_i in the presence as well as in the absence of Na⁺. In these experiments, mean [Mg²⁺]_i values of 3.2 mmol/l were never exceeded. Thus, [Mg²⁺]_i remained at least 60 times lower than predicted from a passive distribution across the cell membrane. The results suggest the existence of a Na⁺-dependent and a Na⁺-independent Mg²⁺ extrusion mechanism, which is regulated by actual Mg²⁺ concentrations.     

The voltage and temperature dependence of the end-plate current in frog skeletal muscle

The effect of membrane potential (V) on the half-time (t _1/2) of the falling phase of the end-plate current (e.p.c.) was found to obey the equationt _1/2=A·e ^BV+C, whereA,B andC are constants.The temperature dependence oft _1/2 was found to follow the Arrhenius equation. The activation energy (E _a) varied from about 50 kJ/mol to about 120 kJ/mol.At membrane potentials between about –40 mV and –140 mV, theE _a/V relation was similar in all end-plates investigated:E _a increased if membrane potential was made more negative. At membrane potentials between about +60 mV and –40mV, however, theE _a/V relation was different in different end-plates: If membrane potential was made more negative,E _a was either increased, or not affected, or decreased.It is concluded that at negative levels of membrane potential the decay of the e.p.c. depends on average life-time of ionic channels, opened up by the action of acetylcholine on junctional receptors. At strongly positive levels of membrane potential, however, the decay of the e.p.c. can be determined by the average life-time of ionic channels or by the clearance of transmitter from the synaptic cleft, or both. Either of these processes can be reflected in the value of constant C in the above equation.     

Infraterminal spreading and extrajunctional expression of nicotinic acetylcholine receptors in denervated rat skeletal muscle

 Through the use of biotinylated-bungarotoxin and monoclonal antibodies, the nicotinic acetylcholine receptor (nAChR) was localized in the subneural apparatus of mammalian motor end plates of the flexor digitorum brevis muscle of the adult rat at the light and electron microscopic levels. Under normal conditions, nAChR was located in the primary post-synaptic membrane of the neuromuscular junction, and the depths of the junctional folds constituting the secondary post-synaptic membrane did not contain any nAChR. Up to 75 days after repeated transection of the related motor nerve (sciatic), there was no major alteration in the light-microscopic localization of junctional nAChR in the subneural apparatus, except for a moderate shrinkage and increased immunocytochemical reactivity of the subneural apparatus. At the electron microscopic level, however, immunocytochemical reactivity gradually occupied the entire extent of the secondary post-synaptic membrane, including the depths of the junctional folds, which exhibited extensive branching. In non-innervated portions of the muscle fibers, nAChR receptor appeared in a linear localization on the surfaces of denervated muscle fibers. This linear reaction was not continuous with the nAChR reaction of the motor end plates. It is concluded that denervation supersensitivity might not be due to spreading of junctional nAChR from the end-plate area, but rather to expression of nAChR in non-innervated portions of the muscle fiber and to the infraterminal (subsynaptic) spreading of nAChR into the depths of junctional folds. Received: 29 May 1998 / Accepted: 8 December 1998     

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     

Effects of Na+, K+ and Mg2+ on45Ca2+ uptake by pancreatic islets

Microdissected pancreatic islets of noninbredob/ob-mice were used to study ionic effects on the lanthanum-nondisplaceable⁴⁵Ca²⁺ uptake by islet cells. Omission of Mg²⁺ from the incubation medium had no effect, but the⁴⁵Ca²⁺ uptake was increased by omission of Na⁺ and decreased by omission of K⁺. Excess Mg²⁺ (1.2–15 mM) inhibited and excess K⁺ (4.7–25 mM) stimulated the⁴⁵Ca²⁺ uptake in a concentration-dependent manner. Stimulation of⁴⁵Ca²⁺ uptake in Na⁺-deficient islets was associated with an enhancement of the basal insulin release. Total abolishment of glucose-stimulated⁴⁵Ca²⁺ uptake in K⁺-deficient islets did not preclude a significant secretory response to glucose. It is concluded that the lanthanum-nondisplaceable⁴⁵Ca²⁺ uptake shows a partial correlation to insulin release.     

Effects of K+, Mg2+ deficiency and adrenal steroids on Na+, K+-pump concentration in skeletal muscle

Animal studies have shown that deficiency of K⁺ is associated with a reduction in the concentration of Na⁺, K⁺ pumps in skeletal muscle, and that this reduction is closely correlated with the reduction in the muscle K⁺ concentration. Furthermore, animals deficient in Mg⁺ show a downregulation of the Na⁺, K⁺-pump concentration, but this seems to be secondary to the concomitant K⁺ deficiency, which often accompanies Mg²⁺ deficiency. Measurements on skeletal muscle biopsies from patients who had been in long-term treatment with diuretics showed that 55% had reduced concentrations of both K⁺ and Mg²⁺, and that this was associated with a reduction in the concentration of Na⁺, K⁺ pumps. Furthermore, the Na⁺, K⁺-pump concentration correlated significantly with both muscle K⁺ and Mg²⁺, suggesting that the downregulation of the Na⁺, K⁺ pumps was related to the loss of K⁺, as predicted from the animal experiments. In accordance with this, normalization of muscle K⁺ and Mg²⁺ in response to oral Mg²⁺ supplementation, resulted in a restoration of the Na⁺, K⁺ pumps. Apart from thyroid hormone, which is another major regulator for the Na⁺, K⁺-pump concentration, other hormones may be of importance. It is well known that adrenal steroids control the synthesis of Na⁺, K⁺ pumps in the kidney and heart. Recently, treatment with dexamethasone was found to increase the Na⁺, K⁺-pump concentration in rat skeletal muscle. The increase was found in EDL, soleus, gastrocnemius and diaphragm muscles, and amounted to 23–52%. In contrast, treatment with aldosterone induced a decrease in the Na⁺, K⁺-pump concentration, which was closely correlated to the reduced K⁺ content of the muscles. The results indicate that in skeletal muscle, high doses of glucocorticoids upregulate the concentration of Na⁺, K⁺ pumps, whereas mineralocorticoids induce a downregulation which is secondary to the concomitant K⁺ deficiency.     

A study on early post-denervation changes of non-quantal and quantal acetylcholine release in the rat diaphragm

The d-tubocurarine (dTC) induced hyperpolarization of antiesterase-treated muscles at the endplate zone, miniature endplate potentials (mepps), resting membrane potentials (RMPs) and the input resistances of single muscle fibres (R_in) were measured in rat diaphragm at various times after denervation. The dTC-induced hyperpolarization decreased in two phases: 2 h after denervation it decreased transiently to 25%, after 4 h it had partially recovered to 60% and from 6 h it progressively decreased up to 12 h after which time it changed to depolarization. The initial fall and recovery were also present in muscles from sham-operated animals. The frequency of mepps decreased by 25% and the amplitude diminished by 10% within the first 2–4 h. After 10 h the frequency had decreased by 35% and the amplitude by 65%. After 12 h no mepps were present. The RMP was not significantly changed during the first 16 h after denervation. From 16 to 24 h the membrane became depolarized at a rate of about 1 mV/h. The input resistance of a single muscle fibre was constant for 12 h after denervation and from 12 to 24 h it increased by 25%. It is concluded that the early decrease in the dTC-induced hyperpolarization is probably due to the desensitization of acetylcholine (ACh) receptors caused by stress-activated non-quantal ACh release. The later decrease of dTC-hyperpolarization reflects a fall in the non-quantal ACh release. The depolarization of the resting membrane after denervation is related to the decrease in passive membrane permeability which is a secondary consequence of transmission failure. The present results do not distinguish between non-quantally released and quantally released ACh as possible trophic agents, since both types of release disappear at the same time.     

Interaction of the antiepileptic drug lamotrigine with recombinant rat brain type IIA Na+ channels and with native Na+ channels in rat hippocampal neurones

Actions of the new antiepileptic drug lamotrigine (LTG, Lamictal) were characterised using recombinant rat brain type IIA Na⁺ channels expressed in Chinese hamster ovary (CHO) cells and native Na⁺ channels in rat hippocampal pyramidal neurones, using whole-cell recording and intracellular recording techniques. In CHO cells, LTG caused a tonic inhibition of Na⁺ currents in a concentration-dependent and voltage-dependent manner. The half-maximal inhibitory concentration (IC₅₀) of approximately 500 M was obtained at a holding potential (V _h) of –90 mV compared with an IC₅₀ of 100 M at a V _h of –60 mV. LTG (50 M) caused a 10–mV negative shift in the slow, steady-state inactivation curve and delayed considerably the recovery from inactivation, but had no significant effects on the voltage dependence of activation or fast inactivation, suggesting that LTG acts mainly on the slow inactivated state. The affinity for the inactivated channels was estimated at 12 M. The tonic inhibition was augmented by a use-dependent action in which a further inhibition by the drug developed during rapid repetitive stimulation using a train of 20-ms duration pulses (11 Hz). These results were consistent with the drug action being on firing properties of pyramidal neurones. Only in those epileptiform bursts which caused cumulative inactivation of Na⁺ spikes did LTG produce a potent inhibition. Our data suggest that the inactivated channel is a primary target for LTG action at therapeutic concentrations.     

原发性高血压患者红细胞Mg2+/Na+交换速率的研究

目的：探讨国人原发性高血压（EH）患者红细胞Mg2 ／Na 交换速率（VNDmax）、红细胞镁含量（EMC）及其与血压的关系。方法：原子吸收光谱火焰法。结果：（1）EH组的VNDmax显著高于正常对照组（分别为155．3±15．2和96．8±13．2μm·L-1,P＜0．05）,而EMC显著低于对照组（分别为2．03±0．06和2．16±0．04mmol／L,P＜0．05）;（2）EH组的VNDmax与舒张压显著相关（r＝0．4872,P＜0．05）。结论：EH患者VNDmax明显升高,可能是造成EMC低下的原因,在疾病的发生和发展中起一定作用。     

The action of Ca2+, Mg2+ and H+ on the contraction threshold of frog skeletal muscle

Summary The dependence of the threshold potential for contraction on pH and the concentration of Ca²⁺ and Mg²⁺ in the bathing solution was measured in frog skeletal muscle.Decreasing the pH from 10.3 to 4.65 resulted in a threshold shift to more positive potentials. Between pH 6.5 and 8.5 the contraction threshold was almost pH-independent.Increasing [Ca²⁺]_o (in the concentration range 0.5–50 mM) shifted the curves relating contraction threshold to pH to less negative potentials and diminished the overall pH-dependence.The contraction threshold exhibited a similar dependence on [Ca²⁺]_o and [Mg²⁺]_o, the two curves running parallel in the concentration range of 5–50 mM, but Mg^2– was onlyc. 0.6 as effective as Ca²⁺.To explain these results a surface charge model is proposed which assumes that two acidic groups, ₁ and ₂, and one basic group, ₃, reside at the outer surface of the membrane of the T-system. Alterations in the extracellular medium exert their influence on the electro-mechanical coupling process by changing the surface potential. The groups will be titrated by protons and their charges screened off by the divalent cations. In addition, Ca²⁺ was supposed to bind with a weak dissociation constant (23 M) to the two acidic groups.The chosen charge densities are: ₁ = –0.0085/Ų[= –1e/(10.8Å)²], ₂ = –0.0037/Ų[= –1e/(16.4Å)²], ₃ = 0.0028/Ų[= +1e/(18.9Å)²] with intrinsic dissociation constantsK _H1=10^–2.0 M,K _H2=10^–4.1 M, andK _H3=10^–8.5 M.The measured threshold values are satisfactorily described by this model except at extreme alkaline and acid pH values.Supported by Deutsche Forschungsgemeinschaft, Bad Godes-berg-SFB 114 Bionach.     

Regulation of Ca channel by intracellular Ca2+ and Mg2+ in frog ventricular cells

The effects of changing the intracellular concentrations of Ca²⁺ or Mg²⁺ ([Ca²⁺]_i, [Mg²⁺]_i) on Ca current (I _Ca) was studied in frog ventricular myocytes using the whole-cell and cell-attached patch clamp techniques. In the physiological range of [Mg²⁺]_i an increase in [Ca²⁺]_i enhancedI _Ca whereas at lower [Mg²⁺]_i I _Ca was suppressed. The increase inI _Ca caused by Ca²⁺ loading was not mediated by phosphorylation since the kinase inhibitors H-8 {N-[2-(methylamino)-ethyl]-5-isoquinolinesulphonamide dihydrochloride}, staurosporine and KN-62 {1-[N,O-bis(5-isoquinoline-sulphonyl)-N-methyl-1-tyrosyl]-4-phenylpiperazine} and a non-hydrolysable adenosine 5-triphosphate analogue ,-methyleneadenosine 5-triphosphate did not prevent the Ca²⁺-inducedI _Ca increase.I _Ca was dramatically increased from 10 ± 6 (n = 4) to 71 ± 7 nA/nF (n = 4) when [Mg²⁺]_i was lowered from 1.0 × 10^–3 to 1.0 × 10^–6 M at a [Ca²⁺]_i of 10^–8 M. The concentration response relation for inhibition of Ca channels by [Mg²⁺]_i is modulated by [Ca²⁺]_i. To account for the experimental results it is postulated that competitive binding of Ca²⁺ or Mg²⁺ to the Ca channel accelerates the transition of the channel from an active to a silent mode. Single-channel recordings support this hypothesis. The regulation may have clinical relevance in cytoprotection during cardiac ischaemia.     

Modulation of Ca2+ channels, charge movement and Ca2+ transients by heparin in frog skeletal muscle fibres

Summary This study is an investigation into the modulatory effects of heparin, a component of the extracellular matrix that binds to dihydropyridine receptors, on contraction and Ca²⁺ channels in frog skeletal muscle. Using tension and Ca²⁺ signal measurements in single intact skeletal muscle cells we have found that heparin (100–200 g ml^-1) substantially potentiates twitch and tetanic tension (55% and 28%, respectively). In contrast, heparin reduces the amplitude of K⁺ contractures. Heparin most likely potentiates twitch tension by prolonging action potentials. The ionic basis of this effect was investigated in voltage-clamp experiments. Membrane currents were monitored in voltage-clamped segments of single fibres using the triple Vaseline gap technique. We found that heparin partially blocks delayed rectifier potassium channels. The depressive effects of heparin on K⁺ contractures prompted us to investigate the effects of heparin on charge movement and Ca²⁺ currents (I _Ca) under voltage-clamp. Charge movement was measured using a subtraction procedure that employed a -20 mV control pulse from a holding potential of 100 mV. Heparin depresses the total charge by 25%. We propose that the reduction in the amplitude of potassium contractures is related to a partial blockade of charge movement. Extracellular heparin shifts the I _Ca-V relation toward more negative voltages and delays the deactivation of tail currents. Double pulse experiments revealed that conditioning depolarizations speed the activation of I _Ca during test depolarizations. Heparin does not affect this process. The primary action of heparin is to accelerate the activation of I _Ca during pulses not preceded by conditioning depolarizations. Overall, the kinetic effects of heparin on I _Ca would increase the Ca²⁺ influx associated with action potentials. However, mechanical and optical experiments performed in Ca²⁺-free solutions and in the presence of Ca²⁺ channel blockers revealed that twitch and tetanic potentiation occur even in the absence of Ca²⁺-influx.     

alpha-Dystrobrevin isoforms differ in their colocalization with and stabilization of agrin-induced acetylcholine receptor clusters

In skeletal muscle, alpha-dystrobrevin (alphaDB) is expressed throughout the sarcolemma with high concentrations at the neuromuscular junction. Mice lacking alphaDB display a mild muscular dystrophy and perturbations at the neuromuscular junction that include disruptions to acetylcholine receptor (AChR) cluster stability and patterning. In adult skeletal muscle, three alternatively spliced isoforms (alphaDB1, alphaDB2, alphaDB3) are expressed, while two other splice variants (alphaDB1(-) and alphaDB2(-)) are expressed only during early development. alphaDB is clearly important in AChR stabilization; however, the degree to which individual alphaDB isoforms and their specific functional domains contribute to AChR cluster stability is not fully understood. To investigate this, we established a primary muscle cell culture system from alphaDB knockout mice and stably expressed individual alphaDB isoforms using retroviral infection. A comparison between wild-type and alphaDB knockout muscle cells showed that in the absence of alphaDB, fewer AChR clusters formed in response to agrin treatment, and these AChR clusters were very unstable. Retroviral expression studies revealed that the largest isoforms (alphaDB1, alphaDB1(-), alphaDB2, alphaDB2(-)) colocalized with agrin-induced AChR clusters and rescued the AChR cluster formation defects back to wild-type levels, while only the first three isoforms fully rescued AChR cluster stability back to wild-type levels. alphaDB2(-) conferred an intermediate level of stability to the AChR clusters. In contrast, alphaDB3 showed no specific colocalization with AChR clusters and little effect on AChR cluster formation or stabilization. Twice as much syntrophin was found associated with alphaDB2 compared with alphaDB2(-) in myotubes suggesting that increased recruitment of syntrophin by alphaDB may enhance the stability of AChR clusters. Taken together, these data demonstrate that different alphaDB isoforms have different functional capabilities in the formation and maintenance of AChR clusters in muscle cells, and that these differences are likely due to the presence of different functional domains in each isoform.     

Na+-dependent regulation of the free Mg2+ concentration in neuropile glial cells and P neurones of the leech Hirudo medicinalis

 To investigate the Mg²⁺ regulation in neuropile glial (NG) cells and pressure (P) neurones of the leech Hirudo medicinalis the intracellular free Mg²⁺ ([Mg²⁺]_i) and Na⁺ ([Na⁺]_i) concentrations, as well as the membrane potential (E _m), were measured using Mg²⁺- and Na⁺-selective microelectrodes. The mean steady-state values of [Mg²⁺]_i were found to be 0.91 mM (mean E _m=–63.6 mV) in NG cells and 0.20 mM (mean E _m=–40.6 mV) in P neurones with a [Na⁺]_i of 6.92 mM (mean E _m=–61.6 mV) and 7.76 mM (mean E _m=–38.5 mV), respectively. When the extracellular Mg²⁺ concentration ([Mg²⁺]_o) was elevated, [Mg²⁺]_i in P neurones increased within 5–20 min whereas in NG cells a [Mg²⁺]_i increase occurred only after long-term exposure (6 h). After [Mg²⁺]_o was reduced back to 1 mM, a reduction of the extracellular Na⁺ concentration ([Na⁺]_o) decreased the inwardly directed Na⁺ gradient and reduced the rate of Mg²⁺ extrusion considerably in both NG cells and P neurones. In P neurones Mg²⁺ extrusion was reduced to 15.4% in Na⁺-free solutions and to 6.0% in the presence of 2 mM amiloride. Mg²⁺ extrusion from NG cells was reduced to 6.2% in Na⁺-free solutions. The results suggest that the major [Mg²⁺]_i-regulating mechanism in both cell types is Na⁺/ Mg²⁺ antiport. In P neurones a second, Na⁺-independent Mg²⁺ extrusion system may exist. Received: 11 August 1998 / Received after revision: 14 October 1998 / Accepted: 15 October 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.     

Effects of Mg2+ on Ca2+ handling by the sarcoplasmic reticulum in skinned skeletal and cardiac muscle fibres

The influence of myoplasmic Mg²⁺ (0.05–10 mM) on Ca²⁺ accumulation (net Ca²⁺ flux) and Ca²⁺ uptake (pump-driven Ca²⁺ influx) by the intact sarcoplasmic reticulum (SR) was studied in skinned fibres from the toad iliofibularis muscle (twitch portion), rat extensor digitorum longus (EDL) muscle (fast twitch), rat soleus muscle (slow twitch) and rat cardiac trabeculae. Ca²⁺ accumulation was optimal between 1 and 3 mM Mg²⁺ in toad fibres and reached a plateau between 1 and 10 mM Mg²⁺ in the rat EDL fibres and between 3 and 10 mM Mg²⁺ in the rat cardiac fibres. In soleus fibres, optimal Ca²⁺ accumulation occurred at 10 mM Mg²⁺. The same trend was obtained with all preparations at 0.3 and 1 M Ca²⁺. Experiments with 2,5-di-(tert-butyl)-1,4-benzohydroquinone, a specific inhibitor of the Ca²⁺ pump, revealed a marked Ca²⁺ efflux from the SR of toad iliofibularis fibres in the presence of 0.2 M Ca²⁺ and 1 mM Mg²⁺. Further experiments indicated that the SR Ca²⁺ leak could be blocked by 10 M ruthenium red without affecting the SR Ca²⁺ pump and this allowed separation between SR Ca²⁺ uptake and SR Ca²⁺ accumulation. At 0.3 M Ca²⁺, Ca²⁺ uptake was optimal with 1 mM Mg²⁺ in the toad iliofibularis and rat EDL fibres and between 1 and 10 mM Mg²⁺ in the rat soleus and trabeculae preparations. At higher [Ca²⁺] (1 M), Ca²⁺ uptake was optimal with 1 mM Mg²⁺ in the iliofibularis fibres and between 1 and 3 mM Mg²⁺ in the EDL fibres. In the soleus and cardiac preparations Ca²⁺ uptake was optimal between 1 and 10 mM Mg²⁺. The results of this study demonstrate that SR Ca²⁺ accumulation is different from SR Ca²⁺ uptake and that these two important determinants of muscle function are differently affected by Mg²⁺ in different muscle fibre types.