Experimental factors that impact CaV1.2 channel pharmacology—Effects of recording temperature, charge carrier, and quantification of drug effects on the step and ramp currents elicited by the “step-step-ramp” voltage protocol

Abstract

Background and purpose CaV1.2 channels contribute to action potential upstroke in pacemaker cells, plateau potential in working myocytes, and initiate excitation-contraction coupling. Understanding drug action on CaV1.2 channels may inform potential impact on cardiac function. However, literature shows large degrees of variability between CaV1.2 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 CaV1.2 pharmacology. Experimental approach Whole cell recordings were made on CaV1.2 overexpression cells. Current was evoked using a “step-step-ramp” 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) Ca2+ vs. Ba2+ as the charge carrier. Eight drugs were studied. Key results CaV1.2 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’s 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—a preference enhanced when Ba2+ 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. Conclusions and implications All experimental factors examined affected CaV1.2 pharmacology. In addition, whole cell CaV1.2 current characteristics—rundown, temperature sensitivity, and impact of series resistance—are also factors that can impact pharmacology. Drug effects on CaV1.2 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.

Development of automated patch clamp assays to overcome the burden of variants of uncertain significance in inheritable arrhythmia syndromes

“…At an experimental level, temperature control can also be an issue, which is important to study ion channel kinetics of temperature-sensitive variants ( Vandenberg et al, 2006 ; Abdelsayed et al, 2015 ; Lei et al, 2019 ; Jones, 2022 ; Ren et al, 2022 ; Kriegeskorte et al, 2023 ). Whilst temperature can be preset on most APC systems (e.g., Biolin Scientific, Fluxion and Nanion Technologies, Table 1 ), most APC assays for cardiac ion channels have been conducted at room temperature ( Vanoye et al, 2018 ; Glazer et al, 2020b ; Ng et al, 2021 ) due to an inherent instability of cell membranes at physiological temperatures leading to lower success rates.…”