The relationship between depletion of intracellular Ca2+ stores and activation of Ca2+ current by muscarinic receptors in neuroblastoma cells.
The relationship between the depletion of IP3-releasable intracellular Ca2+ stores and the activation of Ca(2+)-selective membrane current was determined during the stimulation of M1 muscarinic receptors in N1E-115 neuroblastoma cells. External Ca2+ is required for refilling Ca2+ stores and the voltage-independent, receptor-regulated Ca2+ current represents a significant Ca2+ source for refilling. The time course of Ca2+ store depletion was measured with fura-2 fluorescence imaging, and it was compared with the time course of Ca2+ current activation measured with nystatin patch voltage clamp. At the time of maximum current density (0.18 + .03 pA/pF; n = 48), the Ca2+ content of the IP3-releasable Ca2+ pool is reduced to 39 + 3% (n = 10) of its resting value. Calcium stores deplete rapidly, reaching a minimum Ca2+ content in 15-30 s. The activation of Ca2+ current is delayed by 10-15 s after the beginning of Ca2+ release and continues to gradually increase for nearly 60 s, long after Ca2+ release has peaked and subsided. The delay in the appearance of the current is consistent with the idea that the production and accumulation of a second messenger is the rate-limiting step in current activation. The time course of Ca2+ store depletion was also measured after adding thapsigargin to block intracellular Ca2+ ATPase. After 15 min in thapsigargin, IP3-releasable Ca2+ stores are depleted by > 90% and the Ca2+ current is maximal (0.19 + 0.05 pA/pF; n = 6). Intracellular loading with the Ca2+ buffer EGTA/AM (10 microM; 30 min) depletes IP3-releasable Ca2+ stores by between 25 and 50%, and it activates a voltage-independent inward current with properties similar to the current activated by agonist or thapsigargin. The current density after EGTA/AM loading (0.61 + 0.32 pA/pF; n = 4) is three times greater than the current density in response to agonist or thapsigargin. This could result from partial removal of Ca(2+)-dependent inactivation.
Mathes
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Department of Biological Sciences, Stanford University, Pacific Grove, California 93950, USA.
Thompson
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D000818:Animals; D002118:Calcium / Q000172:deficiency* / Q000378:metabolism*; D015220:Calcium Channels / Q000187:drug effects / Q000378:metabolism; D002217:Carbachol / Q000494:pharmacology; D002614:Chelating Agents / Q000494:pharmacology; D004533:Egtazic Acid / Q000494:pharmacology; D004594:Electrophysiology; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005728:Ganglia, Sympathetic / Q000166:cytology; D015544:Inositol 1,4,5-Trisphosphate / Q000502:physiology; D051379:Mice; D018721:Muscarinic Agonists / Q000494:pharmacology; D009447:Neuroblastoma; D009474:Neurons / Q000737:chemistry / Q000378:metabolism; D011976:Receptors, Muscarinic / Q000502:physiology*; D015290:Second Messenger Systems / Q000502:physiology; D013729:Terpenes / Q000494:pharmacology; D019284:Thapsigargin; D014407:Tumor Cells, Cultured / Q000737:chemistry / Q000166:cytology / Q000378:metabolism
D015220:Calcium Channels; D002614:Chelating Agents; D004791:Enzyme Inhibitors; D018721:Muscarinic Agonists; D011976:Receptors, Muscarinic; D013729:Terpenes; D004533:Egtazic Acid; D019284:Thapsigargin; D015544:Inositol 1,4,5-Trisphosphate; D002217:Carbachol; D002118:Calcium