Excitatory and inhibitory responses mediated by extracellular ATP in developing chick skeletal muscle.

Abstract

Intracellular recordings revealed that extracellular adenosine triphosphate (ATP) activated both an excitatory conductance and a potassium conductance in cultured myotubes of embryonic chick skeletal muscle. A series of ATP analogs were tested for their ability to activate either the excitatory or the potassium conductance. Activation of the two conductances had very similar pharmacological profiles, suggesting that the conductances might be activated by the same type of ATP receptor. The receptor was highly specific for ATP, and activation depended on sites within the base, sugar, and phosphate domains of ATP. Two irreversible antagonists of the ATP response were discovered: oxidized ATP and DIDS. The response to ATP exhibited long-term desensitization. Recovery from desensitization had a time constant of 5-6 hours at 37$\sp\circ$C. Its sensitivity to temperature and block by tunicamycin suggested that recovery from desensitization occurs by insertion of newly-synthesized ATP receptors into the plasma membrane. This hypothesis is consistent with the similar time course found for recovery from block with either oxidized ATP or DIDS. Ionic substitution experiments demonstrated that both cations and anions permeate the membrane following activation of the excitatory conductance. Analysis of the current noise suggested that cations and anions permeate a single class of small conductance (0.3 pS) ion channels. Analysis of the potassium conductance activated by ATP yielded an estimate for the single channel conductance of 20 pS. Single potassium-selective channels of this conductance were activated by ATP in cell-attached membrane patches when a ATP was applied outside of the pepette, and in excised outside-out patches. These results suggested that ATP activates potassium channels through an intramembranous second messenger system. Interestingly, treatment with a variety of pharmacological agents indicated that this second messenger system was activated independent of a G protein. One hypothesis for the role of ATP sensitivity is that ATP mediates an early event in the development of skeletal muscle such as fusion of myoblasts. However, at concentrations that completely blocked the response to ATP, the addition of DIDS to muscle cultures had no apparent effect on development, suggesting that ATP does not play an important role in muscle fusion. An alternate hypothesis is that ATP might be important for synaptogenesis.