{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,10,2]],"date-time":"2026-10-02T01:14:19Z","timestamp":1790903659388,"version":"4.1.0"},"reference-count":46,"publisher":"Public Library of Science (PLoS)","issue":"11","license":[{"start":{"date-parts":[[2022,11,23]],"date-time":"2022-11-23T00:00:00Z","timestamp":1669161600000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/publicdomain\/zero\/1.0\/"}],"funder":[{"DOI":"10.13039\/100000038","name":"U.S. Food and Drug Administration","doi-asserted-by":"publisher","id":[{"id":"10.13039\/100000038","id-type":"DOI","asserted-by":"publisher"}]}],"content-domain":{"domain":["www.plosone.org"],"crossmark-restriction":false},"short-container-title":["PLoS ONE"],"abstract":"<jats:sec id=\"sec001\">\n<jats:title>Background and purpose<\/jats:title>\n<jats:p>Ca<jats:sub>V<\/jats:sub>1.2 channels contribute to action potential upstroke in pacemaker cells, plateau potential in working myocytes, and initiate excitation-contraction coupling. Understanding drug action on Ca<jats:sub>V<\/jats:sub>1.2 channels may inform potential impact on cardiac function. However, literature shows large degrees of variability between Ca<jats:sub>V<\/jats:sub>1.2 pharmacology generated by different laboratories, casting doubt regarding the utility of these data to predict or interpret clinical outcomes. This study examined experimental factors that may impact Ca<jats:sub>V<\/jats:sub>1.2 pharmacology.<\/jats:p>\n<\/jats:sec>\n<jats:sec id=\"sec002\">\n<jats:title>Experimental approach<\/jats:title>\n<jats:p>Whole cell recordings were made on Ca<jats:sub>V<\/jats:sub>1.2 overexpression cells. Current was evoked using a \u201cstep-step-ramp\u201d waveform that elicited a step and a ramp current. Experimental factors examined were: 1) near physiological vs. room temperature for recording, 2) drug inhibition of the step vs. the ramp current, and 3) Ca<jats:sup>2+<\/jats:sup> vs. Ba<jats:sup>2+<\/jats:sup> as the charge carrier. Eight drugs were studied.<\/jats:p>\n<\/jats:sec>\n<jats:sec id=\"sec003\">\n<jats:title>Key results<\/jats:title>\n<jats:p>Ca<jats:sub>V<\/jats:sub>1.2 current exhibited prominent rundown, exquisite temperature sensitivity, and required a high degree of series resistance compensation to optimize voltage control. Temperature-dependent effects were examined for verapamil and methadone. Verapamil\u2019s block potency shifted by up to 4X between room to near physiological temperature. Methadone exhibited facilitatory and inhibitory effects at near physiological temperature, and only inhibitory effect at room temperature. Most drugs inhibited the ramp current more potently than the step current\u2014a preference enhanced when Ba<jats:sup>2+<\/jats:sup> was the charge carrier. The slopes of the concentration-inhibition relationships for many drugs were shallow, temperature-dependent, and differed between the step and the ramp current.<\/jats:p>\n<\/jats:sec>\n<jats:sec id=\"sec004\">\n<jats:title>Conclusions and implications<\/jats:title>\n<jats:p>All experimental factors examined affected Ca<jats:sub>V<\/jats:sub>1.2 pharmacology. In addition, whole cell Ca<jats:sub>V<\/jats:sub>1.2 current characteristics\u2014rundown, temperature sensitivity, and impact of series resistance\u2014are also factors that can impact pharmacology. Drug effects on Ca<jats:sub>V<\/jats:sub>1.2 channels appear more complex than simple pore block mechanism. Normalizing laboratory-specific approaches is key to improve inter-laboratory data reproducibility. Releasing original electrophysiology records is essential to promote transparency and enable the independent evaluation of data quality.<\/jats:p>\n<\/jats:sec>","DOI":"10.1371\/journal.pone.0276995","type":"journal-article","created":{"date-parts":[[2022,11,24]],"date-time":"2022-11-24T14:35:39Z","timestamp":1669300539000},"page":"e0276995","update-policy":"https:\/\/doi.org\/10.1371\/journal.pone.corrections_policy","source":"Crossref","is-referenced-by-count":6,"title":["Experimental factors that impact CaV1.2 channel pharmacology\u2014Effects of recording temperature, charge carrier, and quantification of drug effects on the step and ramp currents elicited by the \u201cstep-step-ramp\u201d voltage protocol"],"prefix":"10.1371","volume":"17","author":[{"given":"Ming","family":"Ren","sequence":"first","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-1222-6181","authenticated-orcid":true,"given":"Aaron L.","family":"Randolph","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Claudia","family":"Alvarez-Baron","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Donglin","family":"Guo","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Phu N.","family":"Tran","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-4354-7423","authenticated-orcid":true,"given":"Nicolas","family":"Thiebaud","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Jiansong","family":"Sheng","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-3663-1595","authenticated-orcid":true,"given":"Jun","family":"Zhao","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-5055-4840","authenticated-orcid":true,"given":"Wendy W.","family":"Wu","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"340","published-online":{"date-parts":[[2022,11,23]]},"reference":[{"issue":"4","key":"pone.0276995.ref001","doi-asserted-by":"crossref","first-page":"821","DOI":"10.1124\/pr.114.009654","article-title":"The Physiology, Pathology, and Pharmacology of Voltage-Gated Calcium Channels and Their Future Therapeutic Potential","volume":"67","author":"GW Zamponi","year":"2015","journal-title":"Pharmacol Rev"},{"issue":"5","key":"pone.0276995.ref002","doi-asserted-by":"crossref","first-page":"638","DOI":"10.1016\/j.hrthm.2006.12.046","article-title":"Cellular basis for the electrocardiographic and arrhythmic manifestations of Timothy syndrome: effects of ranolazine","volume":"4","author":"S Sicouri","year":"2007","journal-title":"Heart Rhythm"},{"issue":"1","key":"pone.0276995.ref003","doi-asserted-by":"crossref","first-page":"981","DOI":"10.1038\/s41598-017-01056-0","article-title":"Inhibition of late sodium current suppresses calcium-related ventricular arrhythmias by reducing the phosphorylation of CaMK-II and sodium channel expressions","volume":"7","author":"XH Wei","year":"2017","journal-title":"Sci Rep"},{"key":"pone.0276995.ref004","doi-asserted-by":"crossref","first-page":"34","DOI":"10.1016\/j.vascn.2017.02.021","article-title":"Proarrhythmia liability assessment and the comprehensive in vitro Proarrhythmia Assay (CiPA): An industry survey on current practice","volume":"86","author":"S Authier","year":"2017","journal-title":"Journal of pharmacological and toxicological methods"},{"key":"pone.0276995.ref005","doi-asserted-by":"crossref","first-page":"251","DOI":"10.1016\/j.vascn.2016.03.009","article-title":"An evaluation of 30 clinical drugs against the comprehensive in vitro proarrhythmia assay (CiPA) proposed ion channel panel","volume":"81","author":"WJ Crumb","year":"2016","journal-title":"Journal of pharmacological and toxicological methods"},{"key":"pone.0276995.ref006","article-title":"Assessment of an In Silico Mechanistic Model for Proarrhythmia Risk Prediction Under the CiPA Initiative","author":"Z Li","year":"2018","journal-title":"Clin Pharmacol Ther"},{"issue":"3","key":"pone.0276995.ref007","doi-asserted-by":"crossref","first-page":"277","DOI":"10.1016\/S0014-2999(98)00606-2","article-title":"Functional interaction between benzothiazepine- and dihydropyridine binding sites of cardiac L-type Ca2+ channels","volume":"358","author":"S Kanda","year":"1998","journal-title":"Eur J Pharmacol"},{"issue":"3","key":"pone.0276995.ref008","doi-asserted-by":"crossref","first-page":"195","DOI":"10.1007\/s00210-007-0190-6","article-title":"Effects of flavoxate hydrochloride on voltage-dependent Ba2+ currents in human detrusor myocytes at different experimental temperatures","volume":"376","author":"T Tomoda","year":"2007","journal-title":"Naunyn Schmiedebergs Arch Pharmacol"},{"issue":"2","key":"pone.0276995.ref009","doi-asserted-by":"crossref","first-page":"491","DOI":"10.1124\/mol.64.2.491","article-title":"Molecular determinants of Ca2+ potentiation of diltiazem block and Ca2+-dependent inactivation in the pore region of cav1.2","volume":"64","author":"N Dilmac","year":"2003","journal-title":"Molecular pharmacology"},{"issue":"5","key":"pone.0276995.ref010","doi-asserted-by":"crossref","first-page":"1236","DOI":"10.1124\/mol.104.000893","article-title":"Molecular determinants of frequency dependence and Ca2+ potentiation of verapamil block in the pore region of Cav1.2","volume":"66","author":"N Dilmac","year":"2004","journal-title":"Molecular pharmacology"},{"issue":"5911","key":"pone.0276995.ref011","doi-asserted-by":"crossref","first-page":"790","DOI":"10.1038\/302790a0","article-title":"Mechanism of calcium channel blockade by verapamil, D600, diltiazem and nitrendipine in single dialysed heart cells","volume":"302","author":"KS Lee","year":"1983","journal-title":"Nature"},{"issue":"1","key":"pone.0276995.ref012","doi-asserted-by":"crossref","first-page":"79","DOI":"10.1007\/PL00004921","article-title":"Kinetics and state-dependent effects of verapamil on cardiac L-type calcium channels","volume":"355","author":"H Nawrath","year":"1997","journal-title":"Naunyn Schmiedebergs Arch Pharmacol"},{"issue":"20","key":"pone.0276995.ref013","doi-asserted-by":"crossref","first-page":"6388","DOI":"10.1073\/pnas.81.20.6388","article-title":"Nitrendipine block of cardiac calcium channels: high-affinity binding to the inactivated state","volume":"81","author":"BP Bean","year":"1984","journal-title":"Proc Natl Acad Sci U S A"},{"issue":"1","key":"pone.0276995.ref014","doi-asserted-by":"crossref","first-page":"1","DOI":"10.1016\/S0028-3908(99)00153-7","article-title":"Determinants of voltage-dependent inactivation affect Mibefradil block of calcium channels","volume":"39","author":"C Jimenez","year":"2000","journal-title":"Neuropharmacology"},{"issue":"5","key":"pone.0276995.ref015","first-page":"543","article-title":"Inhibition of the cardiac L-type calcium channel current by the TRPM8 agonist, (-)-menthol","volume":"61","author":"RL Baylie","year":"2010","journal-title":"J Physiol Pharmacol"},{"issue":"2","key":"pone.0276995.ref016","doi-asserted-by":"crossref","first-page":"110","DOI":"10.1089\/adt.2013.552","article-title":"Evaluating state dependence and subtype selectivity of calcium channel modulators in automated electrophysiology assays","volume":"12","author":"YA Kuryshev","year":"2014","journal-title":"Assay Drug Dev Technol"},{"issue":"3","key":"pone.0276995.ref017","doi-asserted-by":"crossref","first-page":"336","DOI":"10.1161\/01.RES.55.3.336","article-title":"Voltage-dependent block of calcium channel current in the calf cardiac Purkinje fiber by dihydropyridine calcium channel antagonists","volume":"55","author":"MC Sanguinetti","year":"1984","journal-title":"Circulation research"},{"issue":"11","key":"pone.0276995.ref018","doi-asserted-by":"crossref","first-page":"e0241362","DOI":"10.1371\/journal.pone.0241362","article-title":"Mechanisms of QT prolongation by buprenorphine cannot be explained by direct hERG channel block","volume":"15","author":"PN Tran","year":"2020","journal-title":"PLoS One"},{"key":"pone.0276995.ref019","doi-asserted-by":"crossref","first-page":"395","DOI":"10.1113\/jphysiol.1985.sp015752","article-title":"Inactivation of calcium channels in mammalian heart cells: joint dependence on membrane potential and intracellular calcium","volume":"364","author":"KS Lee","year":"1985","journal-title":"The Journal of physiology"},{"issue":"6","key":"pone.0276995.ref020","doi-asserted-by":"crossref","first-page":"1301","DOI":"10.1016\/0896-6273(94)90446-4","article-title":"Mechanism of Ca(2+)-sensitive inactivation of L-type Ca2+ channels","volume":"12","author":"JP Imredy","year":"1994","journal-title":"Neuron"},{"key":"pone.0276995.ref021","doi-asserted-by":"crossref","first-page":"673","DOI":"10.1113\/jphysiol.1992.sp019286","article-title":"Phosphorylation restores activity of L-type calcium channels after rundown in inside-out patches from rabbit cardiac cells","volume":"454","author":"K Ono","year":"1992","journal-title":"The Journal of physiology"},{"key":"pone.0276995.ref022","doi-asserted-by":"crossref","first-page":"321","DOI":"10.1113\/jphysiol.1983.sp014675","article-title":"Modulation by intracellular ATP and cyclic AMP of the slow inward current in isolated single ventricular cells of the guinea-pig","volume":"338","author":"H Irisawa","year":"1983","journal-title":"The Journal of physiology"},{"issue":"2","key":"pone.0276995.ref023","doi-asserted-by":"crossref","first-page":"238","DOI":"10.1007\/s004240050628","article-title":"Effects of temperature on human L-type cardiac Ca2+ channels expressed in Xenopus oocytes","volume":"436","author":"TJ Allen","year":"1998","journal-title":"Pflugers Archiv: European journal of physiology"},{"issue":"3","key":"pone.0276995.ref024","doi-asserted-by":"crossref","first-page":"294","DOI":"10.1007\/BF00582574","article-title":"Temperature-induced transitory and steady-state changes in the calcium current of guinea pig ventricular myocytes","volume":"405","author":"A Cavalie","year":"1985","journal-title":"Pflugers Archiv: European journal of physiology"},{"issue":"4","key":"pone.0276995.ref025","doi-asserted-by":"crossref","first-page":"307","DOI":"10.1111\/j.1540-8167.1996.tb00532.x","article-title":"Temperature dependence of macroscopic L-type calcium channel currents in single guinea pig ventricular myocytes","volume":"7","author":"TJ Allen","year":"1996","journal-title":"Journal of cardiovascular electrophysiology"},{"issue":"1","key":"pone.0276995.ref026","doi-asserted-by":"crossref","first-page":"3282","DOI":"10.1038\/s41598-021-82077-8","article-title":"Computer modeling of whole-cell voltage-clamp analyses to delineate guidelines for good practice of manual and automated patch-clamp","volume":"11","author":"J Montnach","year":"2021","journal-title":"Sci Rep"},{"issue":"4","key":"pone.0276995.ref027","doi-asserted-by":"crossref","first-page":"420","DOI":"10.1097\/FJC.0b013e3181f1d21b","article-title":"Increased cardiac risk in concomitant methadone and diazepam treatment: pharmacodynamic interactions in cardiac ion channels","volume":"56","author":"YA Kuryshev","year":"2010","journal-title":"J Cardiovasc Pharmacol"},{"issue":"2","key":"pone.0276995.ref028","doi-asserted-by":"crossref","first-page":"338","DOI":"10.1161\/01.RES.64.2.338","article-title":"Voltage- and use-dependent modulation of cardiac calcium channels by the dihydropyridine (+)-202-791","volume":"64","author":"TJ Kamp","year":"1989","journal-title":"Circulation research"},{"key":"pone.0276995.ref029","doi-asserted-by":"crossref","first-page":"495","DOI":"10.1113\/jphysiol.1986.sp016266","article-title":"Dual effects of dihydropyridines on whole cell and unitary calcium currents in single ventricular cells of guinea-pig","volume":"379","author":"AM Brown","year":"1986","journal-title":"The Journal of physiology"},{"issue":"1","key":"pone.0276995.ref030","doi-asserted-by":"crossref","first-page":"43","DOI":"10.1074\/jbc.M109.076448","article-title":"Roscovitine binds to novel L-channel (CaV1.2) sites that separately affect activation and inactivation","volume":"285","author":"V Yarotskyy","year":"2010","journal-title":"J Biol Chem"},{"issue":"2","key":"pone.0276995.ref031","doi-asserted-by":"crossref","first-page":"145","DOI":"10.1016\/0022-2828(83)90291-2","article-title":"Diltiazem and verapamil preferentially block inactivated cardiac calcium channels","volume":"15","author":"S Kanaya","year":"1983","journal-title":"J Mol Cell Cardiol"},{"issue":"4","key":"pone.0276995.ref032","doi-asserted-by":"crossref","first-page":"485","DOI":"10.1124\/mol.119.117531","article-title":"Structural Basis for Diltiazem Block of a Voltage-Gated Ca(2+) Channel","volume":"96","author":"L Tang","year":"2019","journal-title":"Molecular pharmacology"},{"key":"pone.0276995.ref033","doi-asserted-by":"crossref","first-page":"2100","DOI":"10.1038\/srep02100","article-title":"MICE models: superior to the HERG model in predicting Torsade de Pointes","volume":"3","author":"J Kramer","year":"2013","journal-title":"Sci Rep"},{"key":"pone.0276995.ref034","doi-asserted-by":"crossref","first-page":"917","DOI":"10.3389\/fphys.2017.00917","article-title":"Uncertainty Quantification Reveals the Importance of Data Variability and Experimental Design Considerations for in Silico Proarrhythmia Risk Assessment","volume":"8","author":"KC Chang","year":"2017","journal-title":"Frontiers in physiology"},{"key":"pone.0276995.ref035","unstructured":"ICH S7B: THE NON-CLINICAL EVALUATION OF THE POTENTIAL FOR DELAYED VENTRICULAR REPOLARIZATION (QT INTERVAL PROLONGATION) BY HUMAN PHARMACEUTICALS, (2005)."},{"key":"pone.0276995.ref036","unstructured":"ICH E14: THE CLINICAL EVALUATION OF QT\/QTC INTERVAL PROLONGATION AND PROARRHYTHMIC POTENTIAL FOR NONANTIARRHYTHMIC DRUGS, (2005)."},{"key":"pone.0276995.ref037","unstructured":"ICH E14\/S7B IWG: Questions and Answers: Clinical and Nonclinical Evaluation of QT\/QTc Interval Prolongation and Proarrhythmic Potential, (2022)."},{"issue":"5","key":"pone.0276995.ref038","doi-asserted-by":"crossref","first-page":"1074","DOI":"10.1111\/j.1476-5381.2010.01091.x","article-title":"Interaction of diltiazem with an intracellularly accessible binding site on Ca(V)1.2","volume":"162","author":"W Shabbir","year":"2011","journal-title":"British journal of pharmacology"},{"issue":"2","key":"pone.0276995.ref039","doi-asserted-by":"crossref","first-page":"62","DOI":"10.1016\/j.vascn.2009.02.002","article-title":"Optimization of Ca(v)1.2 screening with an automated planar patch clamp platform","volume":"59","author":"B Balasubramanian","year":"2009","journal-title":"Journal of pharmacological and toxicological methods"},{"issue":"2","key":"pone.0276995.ref040","doi-asserted-by":"crossref","first-page":"262","DOI":"10.1124\/mol.51.2.262","article-title":"1,5-benzothiazepine binding domain is located on the extracellular side of the cardiac L-type Ca2+ channel","volume":"51","author":"J Kurokawa","year":"1997","journal-title":"Molecular pharmacology"},{"issue":"2\u20133","key":"pone.0276995.ref041","doi-asserted-by":"crossref","first-page":"229","DOI":"10.1016\/S0014-2999(97)00119-2","article-title":"Effects of a novel, potent benzothiazepine Ca2+ channel antagonist, DTZ323, on guinea-pig ventricular myocytes","volume":"325","author":"J Kurokawa","year":"1997","journal-title":"Eur J Pharmacol"},{"issue":"7","key":"pone.0276995.ref042","doi-asserted-by":"crossref","first-page":"1631","DOI":"10.1111\/j.1476-5381.2010.00798.x","article-title":"Alternative splicing modulates diltiazem sensitivity of cardiac and vascular smooth muscle Ca(v)1.2 calcium channels","volume":"160","author":"HY Zhang","year":"2010","journal-title":"British journal of pharmacology"},{"issue":"1","key":"pone.0276995.ref043","doi-asserted-by":"crossref","first-page":"112","DOI":"10.1016\/j.vascn.2013.04.007","article-title":"Variability in high-throughput ion-channel screening data and consequences for cardiac safety assessment","volume":"68","author":"RC Elkins","year":"2013","journal-title":"Journal of pharmacological and toxicological methods"},{"issue":"7","key":"pone.0276995.ref044","doi-asserted-by":"crossref","first-page":"H1466","DOI":"10.1152\/ajpheart.00808.2011","article-title":"An in silico canine cardiac midmyocardial action potential duration model as a tool for early drug safety assessment","volume":"302","author":"MR Davies","year":"2012","journal-title":"American journal of physiology Heart and circulatory physiology"},{"issue":"7618","key":"pone.0276995.ref045","doi-asserted-by":"crossref","first-page":"117","DOI":"10.1038\/nature19102","article-title":"Structural basis for inhibition of a voltage-gated Ca(2+) channel by Ca(2+) antagonist drugs","volume":"537","author":"L Tang","year":"2016","journal-title":"Nature"},{"issue":"4","key":"pone.0276995.ref046","doi-asserted-by":"crossref","first-page":"1600","DOI":"10.1111\/cts.13039","article-title":"Comprehensive in vitro pro-arrhythmic assays demonstrate that omecamtiv mecarbil has low pro-arrhythmic risk","volume":"14","author":"Y Qu","year":"2021","journal-title":"Clin Transl Sci"}],"container-title":["PLOS ONE"],"original-title":[],"language":"en","link":[{"URL":"https:\/\/dx.plos.org\/10.1371\/journal.pone.0276995","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2022,11,24]],"date-time":"2022-11-24T14:36:27Z","timestamp":1669300587000},"score":1,"resource":{"primary":{"URL":"https:\/\/dx.plos.org\/10.1371\/journal.pone.0276995"}},"subtitle":[],"editor":[{"given":"Daniel M.","family":"Johnson","sequence":"first","affiliation":[],"role":[{"vocabulary":"crossref","role":"editor"}]}],"short-title":[],"issued":{"date-parts":[[2022,11,23]]},"references-count":46,"journal-issue":{"issue":"11","published-online":{"date-parts":[[2022,11,23]]}},"URL":"https:\/\/doi.org\/10.1371\/journal.pone.0276995","relation":{},"ISSN":["1932-6203"],"issn-type":[{"value":"1932-6203","type":"electronic"}],"subject":[],"published":{"date-parts":[[2022,11,23]]}}}