Rogers_1995_Biophys.J_68_501

NMDA receptors, ATP receptors, and nicotinic ACh receptors respond to agonist by undergoing conformational changes that open weakly selective cationic channels that are permeable to calcium. We determined the fraction of the current carried by calcium by simultaneously measuring membrane current using whole-cell patch-clamp techniques and intracellular Ca2+ using the fluorescent indicator Fura-2. The Fura-2 response to free Ca2+ was calibrated individually for each cell. Two different calibration methods are compared: one uses voltage-activated Ca2+ channels, and the other uses the same ligand-gated channels that are being tested but in a pure Ca2+ solution. The two methods give quantitatively different results. The method using pure Ca2+ currents through ligand-gated channels calibrates the Fura-2 signal through the same influx pathway that generates the test response, thus controlling for the distribution of channels and ensuring a similar interaction between the incoming Ca2+ and Fura-2. In a physiologic solution containing 2.5 mM Ca2+ at a holding potential of -50 mV, the percentage of inward current carried by Ca2+ through NMDA receptors in hippocampal neurons is 12.4%. By comparison, in sympathetic neurons the percentage of current carried by Ca2+ through neuronal nAChRs is 4.7%, and through ATP-activated purinergic receptors it is 6.5%. These percentages can be used to estimate the amount of Ca2+ entry through these receptors during synaptic activation, but care must be exercised in considering the many subtypes of each receptor.