Molecular biology and electrophysiology of calcium-activated potassium channels from lens epithelium.
OBJECTIVE
We structurally and functionally characterized the alpha and beta subunits of the human lens epithelium Ca(++)-activated potassium channel (BK).


METHODS
The two subunits were sequenced following RT-PCR with multiple primer pairs. The subunits were cloned using a PCR approach and were expressed in tsA-201 cells for patch clamp recording. Green fluorescence protein-channel subunit fusion proteins were characterized by patch clamping and were imaged by fluorescence microscopy.


RESULTS
Alpha subunits alone make a large single-channel conductance, potassium-selective channel with modest Ca++ sensitivity. Beta subunits alone make no channel but, when coexpressed with the alpha subunit, make a channel with increased Ca++ sensitivity, although still less than for natural channels of this type. GFP-BK subunit fusion proteins continue to function and result in a fluorescing channel, which can be localized by fluorescence microscopy. The alpha subunit codes for a "minimal" BK channel in that none of its potential alternative splicing sites contains an "extra" exon.


CONCLUSIONS
The Ca(++)-activated potassium channel known as BK has the nucleotide sequences of its alpha and beta subunits represented in messenger RNA of cultured human lens epithelium. It is the first identified channel, to date, which imparts internal Ca++ dependence to lens epithelial potassium conductance.
Rae
J L
JL
Department of Physiology, Mayo Foundation, Rochester, MN 55905, USA. rae.james@mayo.edu
Shepard
A R
AR
D000595:Amino Acid Sequence; D000818:Animals; D002460:Cell Line; D003001:Cloning, Molecular; D017931:DNA Primers / Q000737:chemistry; D004594:Electrophysiology; D004848:Epithelium / Q000378:metabolism; D015870:Gene Expression; D049452:Green Fluorescent Proteins; D006801:Humans; D051037:Large-Conductance Calcium-Activated Potassium Channel alpha Subunits; D051036:Large-Conductance Calcium-Activated Potassium Channels; D007908:Lens, Crystalline / Q000378:metabolism*; D008164:Luminescent Proteins / Q000378:metabolism; D008253:Macaca mulatta; D008969:Molecular Sequence Data; D018408:Patch-Clamp Techniques; D015221:Potassium Channels / Q000235:genetics* / Q000502:physiology*; D024681:Potassium Channels, Calcium-Activated; D012333:RNA, Messenger / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D013552:Swine; D014162:Transfection
D017931:DNA Primers; D051037:Large-Conductance Calcium-Activated Potassium Channel alpha Subunits; D051036:Large-Conductance Calcium-Activated Potassium Channels; D008164:Luminescent Proteins; D015221:Potassium Channels; D024681:Potassium Channels, Calcium-Activated; D012333:RNA, Messenger; D011993:Recombinant Fusion Proteins; D049452:Green Fluorescent Proteins