Genes and channels: patch/voltage-clamp analysis and single-cell RT-PCR

Technological advances in electrophysiology and molecular biology in the la st two decades have led to great progress in ion channel research. The inve ntion of the patch-clamp recording technique has enabled the characterizati on of the biophysical and pharmacological properties of single channels. Ra pid progress in the development of molecular biology techniques and their a pplication to ion channel research led to the cloning, in the 1980s, of gen es encoding all major classes of voltage- and ligand-gated ionic channels. It has become clear that operationally defined channel types represent exte nded families of ionic channels. Several experimental approaches have been developed to test whether there is a correlation between the detection of p articular ion channel subunit mRNAs and the electrophysiological response t o 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-slic e 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 th is method, the physiological properties of a cell are characterized with a two-electrode voltage clamp and, following the recording, the entire cell i s harvested for its RNA. In both methods, the RNA from a single cell is rev erse-transcribed into cDNA by a reverse transcriptase and subsequently ampl ified by the polymerase chain reaction, i.e. by the so-called single-cell/r everse transcription/polymerase chain reaction method (SC-RT-PCR). This rev iew presents an analysis of the results of work obtained by using a combina tion 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.