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An insulin-stimulated cation channel in skeletal muscle. Inhibition by calcium causes oscillation
1Department of Biochemistry and Molecular Biology, Harvard University, Cambridge, Massachusetts 02138.
Abstract:
A cation channel has been identified in the plasma membrane of skeletal muscle that oscillates open and closed in a regular manner. In an experimental system of patch-clamped reconstituted plasma membrane in phospholipid bilayers, the oscillations are calcium-dependent and constitute regular closing events due to inhibition of the channel by calcium with a Ki of 2.2 +/- 1 x 10(-6) M, followed by reopening. There are 3.7 +/- 1 calcium binding sites/channel. With sodium as the current vehicle, conductance is increased by voltage, insulin (Km = 5 +/- 0.6 x 10(-9) M), and hydrolyzable guanine nucleotides. Cyclic GMP alone with increase the conductance with a Km of 3.7 +/- 0.6 x 10(-7) M. In the absence of calcium, the unitary conductance with insulin + GTP or cGMP at 150 mM NaCl is 153 picosiemens. Sodium current is insensitive to 10(-5) M tetrodotoxin but inhibited by mu-conotoxin (Ki = 5 x 10(-8) M). These findings in the reconstituted system were verified in patch-clamped whole muscle cells where an insulin and cGMP-dependent sodium current inhibited by mu-conotoxin could be demonstrated. In the whole cell experiments, slow calcium-dependent oscillations of the sodium current were also detected.
Insights
Skeletal muscle cation channels exhibit calcium-dependent oscillations, regulated by insulin and cyclic GMP. These channels control sodium current, offering potential therapeutic targets for muscle function.
Area of Science:
- Biophysics
- Molecular Biology
- Muscle Physiology
Background:
- Skeletal muscle plasma membranes contain cation channels crucial for cellular function.
- Understanding the regulation of these channels is key to muscle physiology and disease.
Purpose of the Study:
- To characterize the properties and regulation of a novel oscillating cation channel in skeletal muscle.
- To investigate the roles of calcium, insulin, and cyclic GMP in channel activity.
Main Methods:
- Utilized patch-clamp electrophysiology on reconstituted plasma membrane bilayers and whole muscle cells.
- Quantified channel conductance, binding sites, and inhibition constants (Ki) for various molecules.
- Assessed the effects of voltage, insulin, guanine nucleotides, and toxins on channel activity.
Main Results:
- Identified a calcium-dependent oscillating cation channel in skeletal muscle plasma membranes.
- Demonstrated channel inhibition by calcium (Ki = 2.2 ± 1 x 10⁻⁶ M) with 3.7 ± 1 binding sites.
- Showed increased sodium conductance modulated by voltage, insulin (Km = 5 ± 0.6 x 10⁻⁹ M), and cyclic GMP (Km = 3.7 ± 0.6 x 10⁻⁷ M).
- Confirmed insulin and cyclic GMP-dependent sodium current, inhibited by mu-conotoxin, in whole muscle cells.
Conclusions:
- The identified cation channel exhibits complex regulation by calcium, insulin, and cyclic GMP.
- These findings provide insights into the molecular mechanisms governing skeletal muscle excitability and function.
- The channel's modulation by insulin suggests a potential role in metabolic regulation of muscle activity.