ABSTRACT
We investigated the effects of nerve growth factor (NGF) on expression of K+ channels in cultured skeletal muscle. The channels studied were (1) charybdotoxin (ChTx)-sensitive channels by using a polyclonal antibody raised in rabbits against ChTx, (2) Kv1.5 voltage-sensitive channels, and (3) apamin-sensitive (afterhyperpolarization) channels. Crude homogenates were prepared from cultures made from limb muscles of 1-2-day-old rat pups for identification of ChTx-sensitive and Kv1.5 channels by Western blotting techniques. Apamin-sensitive K+ channels were studied by measurement of specific [125I]-apamin binding by whole cell preparations. ChTx-sensitive channels display a fusion-related increase in expression, and NGF downregulates these channels in both myoblasts and myotubes. Voltage-dependent Kv1.5 channel expression is low in myoblasts and increases dramatically with fusion; NGF induces early expression of these channels and causes expression after fusion to increase even further. NGF downregulates apamin-sensitive channels. NGF increases the rate of fall of the action potential recorded intracellularly from single myotubes with intracellular microelectrodes. The results confirm and extend those of previous studies in showing a functional role for NGF in the regulation of membrane properties of skeletal muscle. Moreover, the findings demonstrate that the different K+ channels in this preparation are regulated in a discoordinate manner. The divergent effects of NGF on expression of different K+ channels, however, do not appear sufficient to explain the NGF-induced increase in the rate of fall of the action potential. The changes during the falling phase may rather be due to increases in channel properties or may result from an increased driving force on the membrane potential secondary to the NGF-induced hyperpolarization.
Subject(s)
Muscle, Skeletal/drug effects , Nerve Growth Factors/pharmacology , Potassium Channels, Voltage-Gated , Potassium Channels/metabolism , Potassium Channels/physiology , Action Potentials/drug effects , Animals , Animals, Newborn , Apamin/metabolism , Binding Sites , Cell Fusion , Cell Membrane/drug effects , Cell Membrane/metabolism , Cells, Cultured , Charybdotoxin/metabolism , Charybdotoxin/pharmacology , Electrophysiology , Gene Expression/drug effects , Ion Channel Gating/drug effects , Kv1.5 Potassium Channel , Molecular Weight , Muscle Fibers, Skeletal/cytology , Muscle Fibers, Skeletal/drug effects , Muscle Fibers, Skeletal/metabolism , Muscle Fibers, Skeletal/physiology , Muscle, Skeletal/cytology , Muscle, Skeletal/metabolism , Muscle, Skeletal/physiology , RatsABSTRACT
The Pattern of development and regulation of the apamin receptor (afterhyperpolarization channel) was studied in cultures of skeletal muscle prepared from 1-2-day-old rat pups. Expression was measured by the specific binding of (125)I-apamin. Apamin binding was virtually undetectable until the time of fusion (3-4 days in culture) of single myoblasts into myotubes. Mature myotubes (5-7 days in vitro) displayed a Bmax of 7.4 fmol/mg protein and a Kd of 376 pmol/L. When studied in mature muscle cells apamin binding was found to increase twofold in response to tetrodotoxin (TTX) and elevated Ko, which resulted in decreased Na(i). In contrast, treatments causing an increase in Na(i), such as monensin and veratridine, caused a decrease in apamin binding. The increase in apamin binding following TTX treatment was due mainly to synthesis of new channels, as the effect was blocked by cycloheximide. Alterations in cytosolic Ca2+ by calcium ionophore or Ca-channel blockers were without effect on apamin-sensitive channel expression. We conclude that afterhyperpolarization channel expression is regulated by the level of intracellular Na+ ions.
