An experimental investigation of rundown of the L-type calcium current [version 1; peer review: awaiting peer review]

Agrawal, Aditi; Clerx, Michael; Wang, Ken; Gissinger, Evgenia; J. Gavaghan, David; Polonchuk, Liudmila; R. Mirams, Gary

Abstract

Background

L-type calcium channels (LCCs) are multi-protein macro-molecular ion channel complexes that are involved in several critical functions in cardiac, skeletal, neuronal, smooth muscle, and endocrine cells. Like other ion channels, LCCs can be selectively over-expressed in a host cell line and studied using voltage-clamp patch-clamp experiments. However, L-type calcium current (ICaL) recordings commonly exhibit a reduction in current magnitude over time, commonly termed ‘rundown’. Previous studies have shown the effect of phosphorylation on rundown, here we provide evidence that accumulation of Ca2+ inside the cell also contributes towards ICaL rundown.

Methods

We generated experimental conditions that should promote the accumulation of sub-membrane Ca2+ in a CHO expression system, by increasing calcium import or decreasing export. These interventions took the form of: a decrease in inter-pulse duration between sweeps, block of the sodium-calcium exchanger, and increased temperature.

Results

On average, we found that current reduced to 63% of its initial value within 325 seconds. This reduction of current with time was found to follow two main patterns: linear or saturating decay. Additionally, current magnitude in some cells increased before stabilising or decaying.

Conclusions

This study shows that the rundown of ICaL in patch-clamp experiments can be reduced by modifying the experimental conditions, and implies that reduced accumulation of Ca2+ inside the cell membrane reduces calcium-dependent inactivation of ICaL.

Citation

Agrawal, A., Clerx, M., Wang, K., Gissinger, E., J. Gavaghan, D., Polonchuk, L., & R. Mirams, G. An experimental investigation of rundown of the L-type calcium current [version 1; peer review: awaiting peer review]

• Working Paper Type: Preprint
• Deposit Date: Jul 29, 2024
• Publicly Available Date: Jul 30, 2024
• DOI: https://doi.org/10.12688/wellcomeopenres.20374.1
• Public URL: https://nottingham-repository.worktribe.com/output/34874016
• Publisher URL: https://wellcomeopenresearch.org/articles/9-250/v1