Modelling the effect of experimental conditions that influence rundown of L-type calcium current

Background L-type calcium channels (LCCs) are macro-molecular complexes that conduct I_CaL and are involved in several critical functions in cardiac, skeletal, neuronal, smooth muscle, and endocrine cells. In common with other ionic channels they can be studied by isolating and overexpressing in a cell line, and the current through them can be measured using patch-clamp experiments. However, LCC current recordings are known to be contaminated with attenuation of current, known as ‘rundown’. Previous work has shown that increased accumulation of intracellular calcium is likely associated with increased rundown. Methods We built a mathematical model of I_CaL conducted by LCCs overexpressed in CHO cells and systematically investigated the qualitative impact of both user-defined as well as experimental parameters within the typical patch-clamp setup on I_CaL rundown. Results Simulations show that calcium-dependent inactivation (CDI) of LCCs modestly contributes towards experimentally observed rundown. The underlying reason for the experimental rundown due to CDI (RCDI) was found to be the non-instantaneous diffusion and reactions of calcium and the calcium-chelating buffer inside the cell. In this study we show that RCDI occurs when the buffer does not have sufficient time to diffuse into the cell; both after patching before the LCCs are activated, and also during the experiment progression. This finding was validated by showing that rundown due to accumulation of Ca²⁺ can be reduced by increasing the concentration of the calcium-chelating buffer in the intracellular solution. Conclusions To minimise rundown due to CDI, we suggest optimising independent experimental parameters such as buffer concentration and the time scales for diffusion to enable buffer equilibration into the cell. Additionally, we suggest that use of large cells should be avoided since they are more prone to RCDI.