Functional expression of L- and T-type Ca2+ channels in murine HL-1 cells

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 Ca2+ channels (VGCC). Reverse transcription-polymerase chain reaction analysis revealed that HL-1 cells have mRNA for several types of Ca2+ channels including the L-types, alpha1C and alpha1D, as well as T-types, alpha1H and alpha1G, but are lacking N-type, alpha1B and the T-type, alpha1I. Western blot analysis demonstrated significant alpha1C protein subunit expression, with less alpha1D subunit apparent, while alpha1A, alpha1B and alpha1E subunit expression was undetectable. Immunocytochemical staining showed that the alpha1C protein subunit is expressed predominantly on the cell surface, whereas the alpha1D protein is expressed mostly intracellularly. Whole-cell patch-clamp measurements demonstrated the presence of low (ICa,T) and high (ICa,L) voltage-activated Ca2+ currents, with preferential sensitivity to mibefradil and nimodipine, respectively. Addition of increasing external Ca2+ concentrations, [Ca2+]o, resulted in Ca2+ influx measured by fluorometric imaging with an EC50 of 0.8 mM [Ca2+]o. At a fixed [Ca2+]o of 0.125 mM, Ca2+ influx was also triggered by increasing the extracellular K+ concentration, [K+]o, with an EC50 of 3.7 mM [K+]o. As increasing [K+]o depolarizes the cell, this latter result is consistent with Ca2+ influx through a voltage-dependent mechanism. L-type (nimodipine and verapamil) and T-type (mibefradil and pimozide) Ca2+ channel blockers inhibited Ca2+ influx with IC50s of 1, 2, 0.4 and 0.2 microM, respectively. Antagonists of N-type (omega-conotoxins GVIA) and P/Q-type (MVIIC or omega-agatoxin IVA) did not inhibit Ca2+ 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 Ca2+ channels.