Activation of nicotinic acetylcholine receptors increases the rate of fusion of cultured human myoblasts.

Amyotrophic lateral sclerosis (ALS) is characterized by progressive degeneration of motor neurons leading to muscle paralysis. Research in transgenic mice suggests that the muscle actively contributes to the disease onset, but such studies are difficult to pursue in humans and in vitro models would represent a good starting point. In this work we show that tiny amounts of muscle from ALS or from control denervated muscle, obtained by needle biopsy, are amenable to functional characterization by two different technical approaches: "microtransplantation" of muscle membranes into Xenopus oocytes and culture of myogenic satellite cells. Acetylcholine (ACh)-evoked currents and unitary events were characterized in oocytes and multinucleated myotubes. We found that ALS acetylcholine receptors (AChRs) retain their native physiological characteristics, being activated by ACh and nicotine and blocked by α-bungarotoxin (α-BuTX), d-tubocurarine (dTC), and galantamine. The reversal potential of ACh-evoked currents and the unitary channel behavior were also typical of normal muscle AChRs. Interestingly, in oocytes injected with muscle membranes derived from ALS patients, the AChRs showed a significant decrease in ACh affinity, compared with denervated controls. Finally, riluzole, the only drug currently used against ALS, reduced, in a dose-dependent manner, the ACh-evoked currents, indicating that its action remains to be fully characterized. The two methods described here will be important tools for elucidating the role of muscle in ALS pathogenesis and for developing drugs to counter the effects of this disease.voltage-clamp | patch-clamp

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“…A modest rectification in current-voltage relation for human muscle γ-AChR channels has been previously observed in native preparations (27,28).…”

Section: Discussionmentioning

confidence: 56%

Physiological characterization of human muscle acetylcholine receptors from ALS patients

Palma

Inghilleri

Conti

et al. 2011

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

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“…These latter cells are myoblasts cultured at low density in the differentiation medium for 2-3 days. In these conditions, myoblasts are induced to proceed through the steps preceding fusion but, as they cannot contact neighboring cells, they remain mononucleated (16). ] i similar to that of undifferentiated myoblasts (65 Ϯ 14 nM, n ϭ 393); the remaining 20% have a significantly (P Ͻ 0.001) increased resting [Ca 2ϩ ] i (105 Ϯ 21 nM, n ϭ 98).…”

Section: Resultsmentioning

confidence: 99%

T-type α1H Ca ²⁺ channels are involved in Ca ²⁺ signaling during terminal differentiation (fusion) of human myoblasts

Bijlenga¹,

Liu²,

Espinos³

et al. 2000

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

177

194

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Mechanisms underlying Ca 2؉ signaling during human myoblast terminal differentiation were studied using cell cultures. We found that T-type Ca 2؉ channels (T-channels) are expressed in myoblasts just before fusion. Their inhibition by amiloride or Ni 2؉ suppresses fusion and prevents an intracellular Ca 2؉ concentration increase normally observed at the onset of fusion. The use of antisense oligonucleotides indicates that the functional T-channels are formed by ␣1H subunits. At hyperpolarized potentials, these channels allow a window current sufficient to increase [Ca 2؉ ]i. As hyperpolarization is a prerequisite to myoblast fusion, we conclude that the Ca 2؉ signal required for fusion is produced when the resting potential enters the T-channel window. A similar mechanism could operate in other cell types of which differentiation implicates membrane hyperpolarization.

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“…Krause et al (9) observed a large up-regulation of ACh receptor density in cultured human myoblasts when they were switched from proliferative to differentiating growth conditions, but before fusion to myotubes. Our results are comparable to theirs in that we recorded large ACh receptor currents from nonfused uninucleate cells which had been growing in conditions that induce differentiation, again indicating that ACh receptor induction, like IRK up-regulation, is well underway prior to fusion.…”

Section: Discussionmentioning

confidence: 99%

The Small Conductance Calcium-activated Potassium Channel Regulates Ion Channel Expression in C3H10T1/2 Cells Ectopically Expressing the Muscle Regulatory Factor MRF4

Peña

Rane

1997

Journal of Biological Chemistry

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We investigated small conductance (SK) potassium channel-mediated regulation of muscle-specific, ion channel functional expression in the C3H10T1/2-MRF4 cell model system, a stable fibroblast line ectopically overexpressing the myogenic regulatory transcription factor, MRF4. Mitogenic stimulation of C3H10T1/2-MRF4 cells with basic fibroblast growth factor negatively regulates MRF4 transcriptional activity, inhibiting myogenesis. Using patch clamp techniques we found that mitogenic stimulation of C3H10T1/2-MRF4 cells also up-regulated SK. SK is a charybdotoxin-sensitive, apamin-insensitive channel that exerts positive proliferative control in fibroblasts. Mitogen withdrawal, which removes negative regulation of MRF4 thus initiating myogenesis, also eliminated SK channel currents, coincident both with induction of acetylcholine receptor channels, and up-regulation of muscle inward rectifier potassium channels. Addition of the SK channel blocker charybdotoxin to growth factor-containing culture medium overcame basic fibroblast growth factorinduced negative regulation of MRF4, as evidenced by induction of inward rectifier potassium and acetylcholine receptor channel expression identical to that observed in mitogen-withdrawn cells. Thus, the SK channel can govern electrophysiological phenotype in C3H10T1/2-MRF4 cells, consistent with an ability of SK to affect MRF4-dependent transcriptional activity. SK appears to be a pivotal signaling component for growth factor regulation of both cell proliferation and differentiation.The regulated expression of ion channels is a key component of developmental processes in many cell types. In nonexcitable cells such as lymphocytes and fibroblasts, both potassium and voltage-independent cation channels contribute to proliferative and cell fate control mechanisms (1-6), while in excitable tissues, the expression of both voltage-dependent cation and ligand gated channels is a culminating event in cell differentiation. The latter scenario is prominent in mammalian skeletal muscle, for which cell maturation is marked by the up-regulation of voltage-dependent channels and conditional expression of different ACh 1 receptor channel subtypes (7). In turn, the progression of muscle fiber differentiation may be affected by the activity of these channels (8 -10).To understand ion channels as both causal and effector agents in cell growth and differentiation, we have looked at the regulation of channel functional expression in the C3H10T1/2-MRF4 myogenic model cell line. C3H10T1/2 (10T1/2) is a multipotent, fibroblast-like cell line that can be manipulated to express a phenotype characteristic of either chondrocytes, adipocytes, or myocytes (11). A muscle phenotype can be selectively produced in 10T1/2 cells via overexpression of the myogenic regulatory transcription factor MRF4. In the presence of bFGF or other mitogenic stimuli, MRF4 is unable to initiate muscle-specific gene expression, even though MRF4 protein levels remain unchanged. Upon bFGF withdrawal, negative regulation of MRF4 is re...

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Activation of nicotinic acetylcholine receptors increases the rate of fusion of cultured human myoblasts.

Cited by 55 publications

References 44 publications

Physiological characterization of human muscle acetylcholine receptors from ALS patients

Physiological characterization of human muscle acetylcholine receptors from ALS patients

T-type α1H Ca ²⁺ channels are involved in Ca ²⁺ signaling during terminal differentiation (fusion) of human myoblasts

The Small Conductance Calcium-activated Potassium Channel Regulates Ion Channel Expression in C3H10T1/2 Cells Ectopically Expressing the Muscle Regulatory Factor MRF4

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Activation of nicotinic acetylcholine receptors increases the rate of fusion of cultured human myoblasts.

Cited by 55 publications

References 44 publications

Physiological characterization of human muscle acetylcholine receptors from ALS patients

Physiological characterization of human muscle acetylcholine receptors from ALS patients

T-type α1H Ca 2+ channels are involved in Ca 2+ signaling during terminal differentiation (fusion) of human myoblasts

The Small Conductance Calcium-activated Potassium Channel Regulates Ion Channel Expression in C3H10T1/2 Cells Ectopically Expressing the Muscle Regulatory Factor MRF4

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T-type α1H Ca ²⁺ channels are involved in Ca ²⁺ signaling during terminal differentiation (fusion) of human myoblasts