Technical Reference #1684

1684.

Oestrogen Directly Inhibits the Cardiovascular L-type Ca2+ Channel Cav1.2 Nina D. Ullrich; Alexandra Koschak; Kenneth T. MacLeod, Imperial College London, Biochemical and Biophysical Research Communications, 361(1684), (2007)

Abstract:
Oestrogen can modify the contractile function of vascular smooth muscle and cardiomyocytes. The negative inotropic actions of oestrogen on the heart and coronary vasculature appear to be mediated by L-type Ca2+ channel (Cav1.2) inhibition but the underlying mechanisms remain elusive. We tested the hypothesis that oestrogen directly inhibits the cardiovascular L-type Ca2+ current ICaL. The effect of oestrogen on ICaL was measured in Cav1.2-transfected HEK-293 cells using the whole-cell patch-clamp technique. The current revealed typical activation and inactivation profiles of nifedipine- and cadmium-sensitive ICaL. Oestrogen (50 lM) rapidly reduced ICaL by 50% and shifted voltage-dependent activation and availability to more negative potentials. Furthermore oestrogen blocked the Ca2+ channel in a rate-dependent way exhibiting higher efficiency of block at higher stimulation frequencies. Our data suggest that oestrogen inhibits ICaL through direct interaction of the steroid with the channel protein.

Keywords:
Oestrogen; Hormones; L-type Ca2+ channel; Heterologous expression; Whole-cell patch-clamp

Materials & Methods:
Transient expression of Cav1.2 in mammalian cells. Human embryonic kidney cells (HEK-293) were maintained at 37 C in an atmosphere of 95% O2 and 5% CO2 in Dulbecco’s modified Eagle’s medium (Sigma–Aldrich Company UK) supplemented with 10% (v/v) foetal bovine serum (Gibco UK) 2 mM L-glutamine and 100 U/ml of penicillin–streptomycin. For transient Ca2+ channel expression cells were plated onto 10 cm tissue culture dishes 12 h before transfection with Ca2+ phosphate precipitation using standard protocols. HEK-293 cells were transiently transfected with human Cav1.2a1 subunit cDNA (3 lg) together with the auxiliary subunits b2a (2 lg) and a2d1 (2.5 lg) as well as 1.5 lg of pUC18 carrier DNA. For identification of positively transfected cells green fluorescence protein (GFP 1 lg cDNA) was added to the transfection solution. Cav1.2- transfected cells were incubated at 37 C in 5% CO2 until used in experiments. 24 h after transfection cells were transferred to 35 mm glass bottom microwell dishes (MatTek Corporation Ashland USA) which served as recording chambers. Currents were measured 2–5 days after transfection.

Microscopic Technique
NA

Cell Type(s)
HEK-293