Properties of BK(Ca) channels formed by bicistronic expression of hSloalpha and beta1-4 subunits in HEK293 cells.
Large-conductance Ca(2+)-activated K(+) (BK(Ca)) channels are sensitive to both voltage and internal [Ca(2+)] 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 Ca(2+)- and voltage-dependence. Much of this variation results from the association of the pore-forming alpha subunit (hSloalpha) with different beta subunits leading to altered channel properties. Since hSloalpha alone produces functional BK(Ca) channels, we have used a bicistronic expression method to ensure that both alpha and beta subunits are expressed, with the beta subunit being in excess. Using this method we have investigated the effect of four beta subunits (beta1 to beta4) on cloned BK(Ca) channels. The four beta subunits were individually cloned into a vector that had hSloalpha 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 BK(Ca) 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 beta subunits, hKCNMB3 and hKCNMB4. J. Biol. Chem.275:6453-6461.)
Lippiat
J D
JD
Ion Channel Group, Dept. Cell Physiology & Pharmacology, University of Leicester, Leicester LE1 9HN, UK.
Standen
N B
NB
Harrow
I D
ID
Phillips
S C
SC
Davies
N W
NW
D002460:Cell Line; D003001:Cloning, Molecular; D004305:Dose-Response Relationship, Drug; D005786:Gene Expression Regulation / Q000502:physiology; D006801:Humans; D015640:Ion Channel Gating / Q000187:drug effects / Q000502:physiology*; D007668:Kidney / Q000187:drug effects / Q000196:embryology / Q000502:physiology*; D051037:Large-Conductance Calcium-Activated Potassium Channel alpha Subunits; D051038:Large-Conductance Calcium-Activated Potassium Channel beta Subunits; D051036:Large-Conductance Calcium-Activated Potassium Channels; D008564:Membrane Potentials / Q000502:physiology*; D010455:Peptides / Q000494:pharmacology; D024681:Potassium Channels, Calcium-Activated / Q000737:chemistry / Q000235:genetics / Q000502:physiology*; D021122:Protein Subunits; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism
D051037:Large-Conductance Calcium-Activated Potassium Channel alpha Subunits; D051038:Large-Conductance Calcium-Activated Potassium Channel beta Subunits; D051036:Large-Conductance Calcium-Activated Potassium Channels; D010455:Peptides; D024681:Potassium Channels, Calcium-Activated; D021122:Protein Subunits; D011994:Recombinant Proteins; C064719:iberiotoxin