Calcium-dependent potassium conductance in the photoresponse of a nudibranch mollusk

• 1.1. The response to light of Hermissenda photoreceptors when recorded intracellularly without interference from synaptic and action potentials consisted of three phases: an early depolarization (ED) followed by hyperpolarization (dip) and subsequent depolarization (tail).
• 2.2. The ED and the dip were associated with increased membrane conductance while decreased membrane conductance was involved with the tail.
• 3.3. The dip reversal potential was − 82.1 ± 5.3 mV and its amplitude varied inversely with the log of [K⁺].
• 4.4. Perfusing with agents which block K⁺ current like 4AP, Quinine, Quinidine or injection of TEA eliminated the dip and its associated increased membrane conductance, thus further supporting the role of K⁺ conductance in producing the dip.
• 5.5. The dip was enhanced by increased [Ca²⁺]_o, reduced by decreased [Ca²⁺]_o and abolished together with its associated increased membrane conductance when perfused with either D600, Cd²⁺, Mg²⁺, Mn²⁺, or Co²⁺, which block transmembrane Ca²⁺ current.
• 6.6. The dip and its associated increased membrane conductance were abolished by intracellular injection of EGTA and enhanced by perfusion with Ruthenium red.
• 7.7. Intracellular injection of Ca²⁺ mimicked the dip: membrane conductance was increased and the cell hyperpolarized.
• 8.8. These results indicate that the increase in intracellular [Ca²⁺] is primarily responsible for the light-induced increase of K⁺ conductance during the dip. The possible source of the Ca²⁺ is, at least in part, extracellular due to activation of an inward Ca²⁺ current.

     

Ca-activated potassium conductance in metabolically exhausted skeletal muscle fibres

Frog skeletal muscle fibres, when stimulated at low frequencies (1–5 Hz) until contractile activity ceases, show a drastic increase in potassium conductance. This results in an increased resting potential and a nearly complete disappearance of the afterpotential which follows a spike [1,2]. The origin of this phenomenon and some implications have been investigated and discussed in several publications from our laboratory. We summarize the main results in this review.     

Calcium-dependent potassium current in barnacle photoreceptor

When barnacle lateral eye photoreceptors are depolarized to membrane potentials of 0 to +50 mV in the dark, the plot of outward current through the cell membrane against time has two distinct maxima. The first maximum occurs 5-10 ms after the depolarization began. The current then decays to a minimum at approximately 500 ms after the onset of depolarization, and then increases to a second maximum 4-6 s after the depolarization began. If depolarization is maintained, the current again decays to reach a steady value approximately 1 min after depolarization began. The increase in current to the maximum at 4-6s from the minimum at approximately 500 ms is termed the "late current." It is maximum for depolarizations to around +25 mV and is reduced in amplitude at more positive potentials. It is not observed when the membrane is depolarized to potentials more positive than +60 mV. The late current is inhibited by external cobaltous ion and external tetraethylammonium ion, and shows a requirement for external calcium ion. When the calcium-sequestering agent EGTA is injected, the late current is abolished. Illumination of a cell under voltage clamp reduces the amplitude of the late current recorded subsequently in the dark. On the basis of the voltage dependence and pharmacology of the late current, it is proposed that the current is a calcium-dependent potassium current.     

Neutrophils injure cultured skeletal myotubes

The purpose of the study was to test the hypothesis that neutrophils can injure cultured skeletal myotubes. Human myotubes were grown and then cultured with human blood neutrophils. Myotube injury was quantitatively and qualitatively determined using a cytotoxicity (51Cr) assay and electron microscopy, respectively. For the 51Cr assay, neutrophils, under non-in vitro-stimulated and N-formylmethionyl-leucyl-phenylalanine (FMLP)-stimulated conditions, were cultured with myotubes at effector-to-target cell (E:T) ratios of 10, 30, and 50 for 6 h. Statistical analyses revealed that myotube injury was proportional to the E:T ratio and was greater in FMLP-stimulated conditions relative to non-in vitro-stimulated conditions. Transmission electron microscopy, using lanthanum as an extracellular tracer, revealed in cocultures a diffuse appearance of lanthanum in the cytoplasm of myotubes and a localized appearance within cytoplasmic vacuoles of myotubes. These observations and their absence in control cultures (myotubes only) suggest that neutrophils caused membrane rupture and increased myotube endocytosis, respectively. Myotube membrane blebs were prevalent in scanning and transmission electron micrographs of cultures consisting of neutrophils and myotubes (E:T ratio of 5) and were absent in control cultures. These data support the hypothesis that neutrophils can injure skeletal myotubes in vitro and may indicate that neutrophils exacerbate muscle injury and/or delay muscle regeneration in vivo.     

After hyperpolarization conductance time-course and repetitive firing in a motoneurone model with early inactivation of the slow potassium conductance system

Early inactivation of the slow potassium conductance system (GK), responsible for the spike afterhyperpolarization (AHP) in spinal alpha motoneurones, has been introduced in a motoneurone model whose G _K kinetics give rise to an exponentially decaying AHP conductance. After this modification, the model displays a plateau shaped time-course of the AHP conductance and a faster shortening of the first interval during repetitive firing induced by current steps of increasing intensities. Both features increase the resemblance between the model and the motoneurone behaviour. Comparison with real motoneurones also suggests that G _K inactivation may be more developed in slow than in fast motoneurones.     

Phosphorylation of the cardiac isoform of calsequestrin in cultured rat myotubes and rat skeletal muscle.

Calsequestrin is a high-capacity Ca(2+)-binding protein and a major constituent of the sarcoplasmic reticulum (SR) of both skeletal and cardiac muscle. Two isoforms of calsequestrin, cardiac and skeletal muscle forms, have been described which are products of separate genes. Purified forms of the two prototypical calsequestrin isoforms, dog cardiac and rabbit fast-twitch skeletal muscle calsequestrins, serve as excellent substrates for casein kinase II and are phosphorylated on distinct sites (Cala, S.E. and Jones, L.R. (1991) J. Biol. Chem 266, 391-398). Dog cardiac calsequestrin is phosphorylated at a 50 to 100-fold greater rate than is rabbit skeletal muscle calsequestrin, and only the dog cardiac isoform contains endogenous Pi on casein kinase II phosphorylation sites. In this study, we identified and examined both calsequestrin isoforms in rat muscle cultures and homogenates to demonstrate that the cardiac isoform of calsequestrin in rat skeletal muscle was phosphorylated in vivo on sites which are phosphorylated by casein kinase II in vitro. Phosphorylation of rat skeletal muscle calsequestrin was not detected. In tissue homogenates, cardiac and skeletal muscle calsequestrin isoforms were both found to be prominent substrates for endogenous casein kinase II activity with cardiac calsequestrin the preferred substrate. In addition, these studies revealed that the cardiac isoform of calsequestrin was the predominant form expressed in skeletal muscle of fetal rats and cultured myotubes.     

Triadin (Trisk 95) overexpression blocks excitation-contraction coupling in rat skeletal myotubes

To identify the function of triadin in skeletal muscle, adenovirus-mediated overexpression of Trisk 95 or Trisk 51, the two major skeletal muscle isoforms, was induced in rat skeletal muscle primary cultures, and the physiological behavior of the modified cells was analyzed. Overexpression did not modify the expression level of their protein partners ryanodine receptor, dihydropyridine receptor, and the other triadin. Caffeine-induced calcium release was also unaffected by triadin overexpression. Nevertheless, in the absence of extracellular calcium, depolarization-induced calcium release was almost abolished in Trisk 95 overexpressing myotubes (T95 myotubes), and not modified in Trisk 51 overexpressing myotubes (T51 myotubes). This was not because of a modification of dihydropyridine receptors, as depolarization in presence of external calcium still induced a calcium release, and the activation curve of dihydropyridine receptor was unchanged, in both T95 and T51 myotubes. The calcium release complex was also maintained in T95 myotubes as Trisk 95, ryanodine receptor, dihydropyridine receptor, and Trisk 51 were still co-localized. The effect of Trisk 95 overexpression on depolarization-induced calcium release was reversed by a simultaneous infection with an antisense Trisk 95 adenovirus, indicating the specificity of this effect. Thus, the level of Trisk 95 and not Trisk 51 is important on regulating the calcium release complex, and an excess of this protein can lead to an inhibition of the physiological function of the complex.     

A slow voltage-activated potassium current in rat vagal neurons.

Potassium currents play a key role in controlling the excitability of neurons. In this paper we describe the properties of a novel voltage-activated potassium current in neurons of the rat dorsal motor nucleus of the vagus (DMV). Intracellular recordings were made from DMV neurons in transverse slices of the medulla. Under voltage clamp, depolarization of these neurons from hyperpolarized membrane potentials (more negative than -80 mV) activated two transient outward currents. One had fast kinetics and had properties similar to A-currents. The other current had an activation threshold of around -95 mV (from a holding potential -110 mV) and inactivated with a time constant of about 3s. It had a reversal potential close to the potassium equilibrium potential. This current was not calcium dependent and was not blocked by 4-aminopyridine (5 mM), catechol (5 mM) or tetraethylammonium (20 mM). It was completely inactivated at the resting membrane potential. This current therefore represents a new type of voltage-activated potassium current. It is suggested that this current might act as a brake to repetitive firing when the neuron is depolarized from membrane potentials negative to the resting potential.     

Mitochondria fine-tune the slow Ca(2+) transients induced by electrical stimulation of skeletal myotubes

Mitochondria sense cytoplasmic Ca(2+) signals in many cell types. In mammalian skeletal myotubes, depolarizing stimuli induce two independent cytoplasmic Ca(2+) signals: a fast signal associated with contraction and a slow signal that propagates to the nucleus and regulates gene expression. How mitochondria sense and possibly affect these cytoplasmic Ca(2+) signals has not been reported. We investigated here (a) the emergence of mitochondrial Ca(2+) signals in response to electrical stimulation of myotubes, (b) the contribution of mitochondrial Ca(2+) transients to ATP generation and (c) the influence of mitochondria as modulators of cytoplasmic and nuclear Ca(2+) signals. Rhod2 and Fluo3 fluorescence determinations revealed composite Ca(2+) signals associated to the mitochondrial compartment in electrically stimulated (400 pulses, 45 Hz) skeletal myotubes. Similar Ca(2+) signals were detected when using a mitochondria-targeted pericam. The fast mitochondrial Ca(2+) rise induced by stimulation was inhibited by pre-incubation with ryanodine, whereas the phospholipase C inhibitor U73122 blocked the slow mitochondrial Ca(2+) signal, showing that mitochondria sense the two cytoplasmic Ca(2+) signal components. The fast but not the slow Ca(2+) transient enhanced mitochondrial ATP production. Inhibition of the mitochondrial Ca(2+) uniporter prevented the emergence of mitochondrial Ca(2+) transients and significantly increased the magnitude of slow cytoplasmic Ca(2+) signals after stimulation. Precluding mitochondrial Ca(2+) extrusion with the Na(+)/Ca(2+) exchanger inhibitor CGP37157 decreased mitochondrial potential, increased the magnitude of the slow cytoplasmic Ca(2+) signal and decreased the rate of Ca(2+) signal propagation from one nucleus to the next. Over expression of the mitochondrial fission protein Drp-1 decreased mitochondrial size and the slow Ca(2+) transient in mitochondria, but enhanced cytoplasmic and nuclear slow transients. The present results indicate that mitochondria play a central role in the regulation of Ca(2+) signals involved in gene expression in myotubes.     

Calcium-dependent,swelling-activated K+ conductance in human neuroblastoma cells

In most mammalian cells, regulatory volume decrease (RVD) is mediated by swelling-activated Cl(-) and K(+) channels. Previous studies in the human neuroblastoma cell line CHP-100 have demonstrated that exposure to hypoosmotic solutions activates Cl(-) channels which are sensitive to Ca(2+). Whether a Ca(2+)-dependent K(+) conductance is activated after cell swelling was investigated in the present studies. Reducing the extracellular osmolarity from 290 to 190 mOsm/kg H(2)O rapidly activated 86Rb effluxes. Hypoosmotic stress also increased cytosolic Ca(2+) in fura-2 loaded cells. Pretreatment with 2.5 mM EGTA and nominally Ca(2+) free extracellular solution significantly decreased the hypoosmotically induced rise in cytosolic Ca(2+) and the swelling-activated 86Rb efflux. In cell-attached patch-clamp studies, decreasing the extracellular osmolarity activated a K(+) conductance that was blocked by Ba(2+). In addition, the swelling-activated K(+) channels were significantly inhibited in the presence of nominally free extracellular Ca(2+) and 2.5mM EGTA. These results suggest that in response to hypoosmotic stress, a Ca(2+)-dependent K(+) conductance is activated in the human neuroblastoma cell line CHP-100.     

Regulation of the sodium-potassium pump in cultured rat skeletal myotubes by intracellular sodium ions

The properties of the Na-K pump and some of the factors controlling its amount and function were studied in rat myotubes in culture. The number of Na-K pump sites was quantified by measuring the amount of [3H]ouabain bound to whole-cell preparations. Activity of the pump was determined by measurement of ouabain-sensitive 86Rb-uptake and component of membrane potential. Chronic treatment of myotubes with tetrodotoxin (TTX), which lowers [Na]i, decreased the number of Na-K pumps, the ouabain-sensitive 86Rb uptake, and the size of the electrogenic pump component of Em. In contrast, chronic treatment with either ouabain or veratridine, which increases [Na+]i, resulted in an elevated level of Na-K pump sites. This effect was blocked by inhibitors of protein synthesis. Neither rates of degradation nor affinity of pump sites in cells treated with TTX, veratridine, or ouabain differred from those in control cells. The number and activity of Na-K pump sites were unaffected by chronic elevation in [Ca]i or chronic depolarization. We conclude that alterations in the level in intracellular Na ions play the major role in regulation of Na-K pump synthesis in cultured mammalian skeletal muscle.     

Regulation of glucose uptake in rat slow and fast skeletal muscles

1. Regulation of glucose uptake was compared between extensor digitorum longus (EDL) and soleus (Sol) muscles in rats. 2. Insulin stimulated glucose uptake more in EDL than in Sol. 3. Under high concentrations of insulin, the glucose uptake was higher in EDL than Sol. 4. Inhibition of oxidative phosphorylation by anoxia or an uncoupler stimulated glucose uptake more in EDL than in Sol. 5. Anoxia abolished the effect of insulin on glucose uptake in both EDL and Sol. 6. The blocker to glucose transport system reduced glucose uptake more in Sol than in EDL.     

TASK-3 dominates the background potassium conductance in rat adrenal glomerulosa cells

In a preceding study we showed that the highly negative resting membrane potential of rat adrenal glomerulosa cells is related to background potassium channel(s), which belong to the two-pore domain channel family. TWIK-related acid-sensitive K+ channel (TASK-1) expression was found in glomerulosa tissue, and the currents elicited by injection of glomerulosa mRNA (I(glom)) or TASK-1 cRNA (I(TASK-1)) showed remarkable similarity in Xenopus laevis oocytes. However, based on the different sensitivity of these currents to acidification, we concluded that TASK-1 may be responsible for a maximum of 25% of the weakly pH-dependent glomerulosa background K+ current. Here we demonstrate that TASK-3, a close relative of TASK-1, is expressed abundantly in glomerulosa cells. Northern blot detected TASK-3 message in adrenal glomerulosa, but not in other tissues. Quantitative RT-PCR experiments indicated even higher mRNA expression of TASK-3 than TASK-1 in glomerulosa tissue. Similarly to the glomerulosa background current, the current expressed by injection of TASK-3 cRNA (I(TASK-3)) was less acid-sensitive than I(TASK-1). Ruthenium red in the micromolar range inhibited I(glom) and I(TASK-3), but not I(TASK-1). Like I(TASK-1), I(TASK-3) was inhibited by stimulation of AT1a angiotensin II receptor coexpressed with the potassium channel. The high level of expression and its pharmacological properties suggest that TASK-3 dominates the resting potassium conductance of glomerulosa cells.     

Calcium dyshomeostasis in beta-amyloid and tau-bearing skeletal myotubes

The relative scarcity of inclusion-affected muscle cells or markers of cell death in inclusion body myositis (IBM) is in distinction to the specific and early intracellular deposition of several Alzheimer's Disease (AD)-related proteins. The current study examined the possible correlation between myotube beta-amyloid and/or Tau accumulations and a widespread mishandling of intracellular muscle calcium concentration that could potentially account for the unrelenting weakness in affected patients. Cultured myogenic cells (C(2)C(12)) expressed beta-amyloid-42 (Abeta(42)) and fetal Tau peptides, as human transgenes encoded by herpes simplex virus, either individually or concurrently. Co-expression of Abeta(42) in C(2)C(12) myotubes resulted in hyperphosphorylation of Tau protein that was not observed when Tau was expressed alone. Resting calcium concentration and agonist-induced RyR-mediated Ca(2+) release were examined using calcium-specific microelectrodes and Fluo-4 epifluorescence, respectively. Co-expression of Abeta(42) and Tau cooperatively elevated basal levels of myoplasmic-free calcium, an effect that was accompanied by depolarization of the plasma membrane. Sarcoplasmic reticulum (SR) calcium release, induced by KCl depolarization, was not affected by Abeta(42) or Tau. In contrast, expression of Abeta(42), Tau, or Abeta(42) together with Tau resulted in enhanced sensitivity of ryanodine receptors to activation by caffeine. Notably, expression of beta-amyloid, alone, was sufficient to result in an increased sensitivity to direct activation by caffeine. Current results indicate that amyloid proteins cooperate to raise resting calcium levels and that these effects are associated with a passive SR Ca(2+) leak and Tau hyperphosphorylation in skeletal muscle.     

Slow components of potassium tail currents in rat skeletal muscle

The kinetics of potassium tail currents have been studied in the omohyoid muscle of the rat using the three-microelectrode voltage-clamp technique. The currents were elicited by a two-pulse protocol in which a conditioning pulse to open channels was followed by a test step to varying levels. The tail currents reversed at a single well-defined potential (VK). At hyperpolarized test potentials (-100 mV and below), tail currents were inward and exhibited two clearly distinguishable phases of decay, a fast tail with a time constant of 2-3 ms and a slow tail with a time constant of approximately 150 ms. At depolarized potentials (-60 mV and above), tail currents were outward and did not show two such easily separable phases of decay, although a slow kinetic component was present. The slow kinetic phase of outward tail currents appeared to be functionally distinct from the slow inward tail since the channels responsible for the latter did not allow significant outward current. Substitution of Rb for extracellular K abolished current through the anomalous (inward-going) rectifier and at the same time eliminated the slow inward tail, which suggests that the slow inward tail current flows through anomalous rectifier channels. The amplitude of the slow inward tail was increased and VK was shifted in the depolarizing direction by longer conditioning pulses. The shift in VK implies that during outward currents potassium accumulates in a restricted extracellular space, and it is suggested that this excess K causes the slow inward tail by increasing the inward current through the anomalous rectifier. By this hypothesis, the tail current slowly decays as K diffuses from the restricted space. Consistent with such a hypothesis, the decay of the slow inward tail was not strongly affected by changing temperature. It is concluded that a single delayed K channel is present in the omohyoid. Substitution of Rb for K has little effect on the magnitude or time course of outward current tails, but reduces the magnitude and slows the decay of the fast component of inward tails. Both effects are consistent with a mechanism proposed for squid giant axon (Swenson and Armstrong, 1981): that (a) the delayed potassium channel cannot close while Rb is inside it, and (b) that Rb remains in the channel longer than K.     

Apoptosis and atrophy in rat slow skeletal muscles in chronic heart failure

Congestive heart failure is characterized by a skeletal musclemyopathy with muscle bulk loss. The mechanisms responsible for thesechanges are not clear at present. We have investigated the role ofapoptosis in the rat "slow" soleus muscle during the developmentof heart failure, which was induced by injection of monocrotaline (30 mg/kg). We looked at the time course of apoptosis by studying sixanimals at each of the following time points: 0, 17, 24, and 30 days.We found a decreased expression of the antiapoptotic protein Bcl-2,which was accompanied by a rise of proapoptotic caspase-3. Ubiquitinlevels did not change. DNA nick-end labeling showed an increased numberof apoptotic nuclei both in myofibers and interstitial cells when heartfailure occurred. At variance with previous observations in thefast-twitch tibialis anterior muscle in the same animals, in whichtumor necrosis factor- (TNF-) increased at the time thatapoptosis occurred, the magnitude of apoptosis is lower in soleusmuscle and there is no appearance of muscle atrophy. In soleus muscle,apoptosis is accompanied by activation of the caspase-3 system. Thereis no activation of the TNF-- and ubiquitin-dependent protein waste.In conclusion, slow muscles are less prone to develop apoptosis thanfast muscles. Muscle atrophy appears earlier in these latter ones.

     

Role of Na-K ATPase in regulation of resting membrane potential of cultured rat skeletal myotubes

The role of Na-K ATPase in the determination of resting membrane potential (Em) as a function of extracellular K ion concentration was investigated in cultured rat myotubes. The Em of control myotubes at 37 degrees C varied as a function of (K+)0 with a slope of about 58-60 mV per ten-fold change in (K+)0. Inhibition of the Na-K pump with ouabain or by reduced temperature revealed that this relation consists of two components. One, between (K+)0 of 10 and 100 mM, remains unchanged by alterations in enzyme activity; The second, between (K+)0 of 1 and 10 mM, is related to the amount of Na-K pump activity, the slope decreasing as pump activity decreases. Indeed, with complete inhibition of the Na-K pump, Em does not change over the range of (K+)0 1 to 10 mM. Measurements of 86Rb efflux and input resistance of individual myotubes showed that membrane permeability does not change as (K+)0 increases from 1 to 10 mM but increases as (K+)0 increases further. Monensin, which increases Na ion permeability, increases Em at values of external K+ below 10 mM, and is without effect at higher values of K+ concentration. The effect of monensin is blocked by ouabain. Tetrodotoxin, which blocks voltage-dependent Na+ channels, decreases Em at low (2-10 mM) K+. We conclude that changes in Em as a function of extracellular K+ concentration in the physiological range are not adequately explained by the diffusion potential hypothesis of Em, and that other theories (electrogenic pump, surface-absorption) must be considered.