Functional expression of L- and T-type Ca 2+ channels in murine HL-1 cells

Abstract

In the search for a readily available source of native cardiac cells, we investigated the molecular and pharmacological properties of the immortalized cardiac atrial myocyte cell line, HL-1 cells. This work focused on the expression pattern of voltage-gated Ca 2+ channels (VGCC). Reverse transcription-polymerase chain reaction analysis revealed that HL-1 cells have mRNA for several types of Ca 2+ channels including the L-types, α1C and α1D, as well as T-types, α1H and α1G, but are lacking N-type, α1B and the T-type, α1I. Western blot analysis demonstrated significant α1C protein subunit expression, with less α1D subunit apparent, while α1A, α1B and α1E subunit expression was undetectable. Immunocytochemical staining showed that the α1C protein subunit is expressed predominantly on the cell surface, whereas the α1D protein is expressed mostly intracellularly. Whole-cell patch-clamp measurements demonstrated the presence of low ( I Ca,T) and high ( I Ca,L) voltage-activated Ca 2+ currents, with preferential sensitivity to mibefradil and nimodipine, respectively. Addition of increasing external Ca 2+ concentrations, [Ca 2+] o, resulted in Ca 2+ influx measured by fluorometric imaging with an EC 50 of 0.8 mM [Ca 2+] o. At a fixed [Ca 2+] o of 0.125 mM, Ca 2+ influx was also triggered by increasing the extracellular K + concentration, [K +] o, with an EC 50 of 3.7 mM [K +] o. As increasing [K +] o depolarizes the cell, this latter result is consistent with Ca 2+ influx through a voltage-dependent mechanism. L-type (nimodipine and verapamil) and T-type (mibefradil and pimozide) Ca 2+ channel blockers inhibited Ca 2+ influx with IC 50s of 1, 2, 0.4 and 0.2 μM, respectively. Antagonists of N-type (ω-conotoxins GVIA) and P/Q-type (MVIIC or ω-agatoxin IVA) did not inhibit Ca 2+ influx, consistent with the lack of expression of N-, P-, or Q-type channels observed in the molecular studies. Taken together, these findings indicate that HL-1 cells express L- and T-subtypes of VGCC and are a unique in vitro model system for the study of native, mammalian cardiac Ca 2+ channels.