Selection of stable RNA molecules that can regulate the channel-opening equilibrium of the membrane-bound gamma-aminobutyric acid receptor.
The gamma-aminobutyric acid (GABA(A)) receptor belongs to a superfamily of membrane-bound proteins that regulate signal transmission between cells in the nervous system. It is the target of convulsants such as picrotoxin and is mutated in some forms of epilepsy, a disease affecting approximately 50 million people worldwide. In picrotoxin inhibition and in one form of epilepsy, a decrease in the channel-opening equilibrium of a GABA(A) receptor is responsible for receptor dysfunction. Here we identify compounds that can regulate the channel-opening equilibrium of the GABA(A) receptor. Fluorinated RNA polymers containing a 40-nucleotide region with a randomized sequence were used to select those that can displace picrotoxin from the membrane-bound GABA(A) receptor in the rat forebrain. After 11 selection rounds, two classes of RNA molecules that bind to the GABA(A) receptor with nanomolar affinity were isolated and sequenced. Class I and class II molecules have different consensus sequences and different binding affinities for the receptor. A transient kinetic technique, the cell-flow method, was employed in combination with the whole-cell current-recording technique to determine the affinity of the selected RNA aptamers for the GABA(A) receptor. Class I molecules have a higher affinity for the closed-channel form than for the open-channel receptor form and inhibit the receptor; class II aptamers bind with equal or higher affinity to the open-channel form and alleviate picrotoxin inhibition.
Cui
Yang
Y
Department of Molecular Biology and Genetics, 216 Biotechnology Building, Cornell University, Ithaca, New York 14853-2703, USA.
Rajasethupathy
Priyamvada
P
Hess
George P
GP
D000818:Animals; D004594:Electrophysiology; D005260:Female; D015640:Ion Channel Gating / Q000502:physiology; D007700:Kinetics; D008297:Male; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction; D012313:RNA / Q000378:metabolism*; D051381:Rats; D011956:Receptors, Cell Surface / Q000378:metabolism*; D018079:Receptors, GABA / Q000378:metabolism*
D011956:Receptors, Cell Surface; D018079:Receptors, GABA; D012313:RNA