Genes and channels: patch/voltage-clamp analysis and single-cell RT-PCR.
Technological advances in electrophysiology and molecular biology in the last two decades have led to great progress in ion channel research. The invention of the patch-clamp recording technique has enabled the characterization of the biophysical and pharmacological properties of single channels. Rapid progress in the development of molecular biology techniques and their application to ion channel research led to the cloning, in the 1980s, of genes encoding all major classes of voltage- and ligand-gated ionic channels. It has become clear that operationally defined channel types represent extended families of ionic channels. Several experimental approaches have been developed to test whether there is a correlation between the detection of particular ion channel subunit mRNAs and the electrophysiological response to a pharmacological or electrical stimulus in a cell. In one method, whole-cell patch-clamp recording is performed on a cell in culture or tissue-slice preparation. The biophysical and pharmacological properties of the ionic channels of interest are characterized and the cytoplasmic contents of the recorded cell are then harvested into the patch pipette. In a variant of this method, the physiological properties of a cell are characterized with a two-electrode voltage clamp and, following the recording, the entire cell is harvested for its RNA. In both methods, the RNA from a single cell is reverse-transcribed into cDNA by a reverse transcriptase and subsequently amplified by the polymerase chain reaction, i.e. by the so-called single-cell/reverse transcription/polymerase chain reaction method (SC-RT-PCR). This review presents an analysis of the results of work obtained by using a combination of whole-cell patch-clamp recording or two-electrode voltage clamp and SC-RT-PCR with emphasis on its potential and limitations for quantitative analysis.
Sucher
N J
NJ
Department of Biology, Hong Kong University of Science & Technology, Hong Kong SAR, China.
Deitcher
D L
DL
Baro
D J
DJ
Warrick
R M
RM
Guenther
E
E
D000818:Animals; D017931:DNA Primers; D007473:Ion Channels / Q000235:genetics* / Q000378:metabolism; D009474:Neurons / Q000378:metabolism; D018408:Patch-Clamp Techniques / Q000379:methods*; D016133:Polymerase Chain Reaction / Q000379:methods*; D012313:RNA / Q000096:biosynthesis / Q000302:isolation & purification; D012323:RNA Processing, Post-Transcriptional; D020133:Reverse Transcriptase Polymerase Chain Reaction; D015533:Transcriptional Activation
D017931:DNA Primers; D007473:Ion Channels; D012313:RNA