Properties of BK Ca Channels Formed by Bicistronic Expression of hSlo ? and ?1-4 Subunits in HEK293 Cells

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Abstract
Large-conductance Ca2+-activated K+ (BKCa) channels are sensitive to both voltage and internal [Ca2+] and are found in many tissues. Their physiological roles range from causing relaxation of smooth muscle to regulating the frequency of action potential firing. There is considerable variation between different tissues in their Ca2+- and voltage-dependence. Much of this variation results from the association of the pore-forming α subunit (hSloα) with different β subunits leading to altered channel properties. Since hSloα alone produces functional BKCa channels, we have used a bicistronic expression method to ensure that both α and β subunits are expressed, with the β subunit being in excess. Using this method we have investigated the effect of four β subunits (β1 to β4) on cloned BKCa channels. The four β subunits were individually cloned into a vector that had hSloα cDNA inserted downstream of an internal ribosome entry site. The constructs were transiently transfected into HEK293 cells together with a construct that expresses green fluorescent protein, as a marker for transfection. Fluorescent cells expressed BKCa channels whose currents were recorded from inside-out or outside-out patches. The currents we measured using this expression system were similar to those expressed in Xenopus oocytes by Brenner et al. (Brenner, R., Jegla, T.J., Wickenden, A., Liu, Y., Aldrich, R.W. 2000. Cloning and functional expression of novel large-conductance calcium-activated potassium channel β subunits, hKCNMB3 and hKCNMB4. J. Biol. Chem.275:6453-6461.)

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