A new approach was developed to determine quantitatively the fraction of current carried by Ca2+ through an ion channel under physiological conditions. This approach entails the simultaneous measurement of membrane current and intracellular Ca2+ for single cells. Whole-cell patch-clamp techniques were used to measure current, and intracellular Ca2+ was monitored with the fluorescent indicator fura-2. To obtain a quantitative measure of the fraction of current carried by Ca2+, a cell-by-cell calibration method was devised to account for differences among cells in such factors as cellular volume and Ca2+ buffering. The method was used to evaluate the Ca2+ flux through muscle and neuronal nicotinic ACh receptors (nAChRs). In a solution containing 2.5 mM Ca2+ at a holding potential of -50 mV, Ca2+ carries 2.0% of the inward current through muscle nAChRs from BC3H1 cells and 4.1% of the inward current through neuronal nAChRs from adrenal chromaffin cells. The Ca2+ flux through neuronal nAChRs of adrenal chromaffin cells is insensitive to alpha-bungarotoxin. The influx of Ca2+ is voltage dependent, and because of the Ca2+ concentration difference across the cellular membrane, there is Ca2+ influx into the cell even when there is a large net outward current. At both muscle and neuronal cholinergic synapses, activity-dependent Ca2+ influx through nicotinic receptors produces intracellular signals that may have important roles in synaptic development, maintenance, and plasticity.

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Supplemental Digital Content is Available in the Text. Human sodium channel Na V 1.7 in induced pluripotent stem cell–derived sensory neurons sets the action potential threshold but does not support subthreshold depolarizations.

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.

The human electrophysiological and pharmacological properties of XEN-D0101 were evaluated to assess its usefulness for treating atrial fibrillation (AF). XEN-D0101 inhibited Kv1.5 with an IC50 of 241 nM and is selective over non-target cardiac ion channels (IC50 Kv4.3, 4.2 μM; hERG, 13 μM; activated Nav1.5, >100 μM; inactivated Nav1.5, 34 μM; Kir3.1/3.4, 17 μM; Kir2.1, >>100 μM). In atrial myocytes from patients in sinus rhythm (SR) and chronic AF, XEN-D0101 inhibited non-inactivating outward currents (Ilate) with IC50 of 410 and 280 nM, respectively, and peak outward currents (Ipeak) with IC50 of 806 and 240 nM, respectively. Whereas Ilate is mainly composed of IKur, Ipeak consists of IKur and Ito. Therefore, the effects on Ito alone were estimated from a double-pulse protocol where IKur was inactivated (3.5 µM IC50 in SR and 1 µM in AF). Thus, inhibition of Ipeak is because of IKur reduction and not Ito. XEN-D0101 significantly prolonged the atrial action potential duration at 20%, 50%, and 90% of repolarization (AF tissue only) and significantly elevated the atrial action potential plateau phase and increased contractility (SR and AF tissues) while having no effect on human ventricular action potentials. In healthy volunteers, XEN-D0101 did not significantly increase baseline- and placebo-adjusted QTc up to a maximum oral dose of 300 mg. XEN-D0101 is a Kv1.5/IKur inhibitor with an attractive atrial-selective profile.