Quantitative single-cell-reverse transcription-PCR demonstrates that A- current magnitude varies as a linear function of shal gene expression in identified stomatogastric neurons

Abstract

Different Shaker family α-subunit genes generate distinct voltage- dependent K+ currents when expressed in heterologous expression systems. Thus it generally is believed that diverse neuronal K+ current phenotypes arise, in part, from differences in Shaker family gene expression among neurons. It is difficult to evaluate the extent to which differential Shaker family gene expression contributes to endogenous K+ current diversity, because the specific Shaker family gene or genes responsible for a given K+ current are still unknown for nearly all adult neurons. In this paper we explore the role of differential Shaker family gene expression in creating transient K+ current (I(A)) diversity in the 14-neuron pyloric network of the spiny lobster, Panulirus interruptus. We used two-electrode voltage clamp to characterize the somatic I(A) in each of the six different cell types of the pyloric network. The size, voltage-dependent properties, and kinetic properties of the somatic I(A) vary significantly among pyloric neurons such that the somatic I(A) is unique in each pyloric cell type. Comparing these currents with the I(A)s obtained from oocytes injected with Panulirus shaker and shal cRNA (lobster I(shaker) and lobster I(shal), respectively) reveals that the pyloric cell I(A)S more closely resemble lobster I(shal) than lobster I(shaker). Using a novel, quantitative single-cell-reverse transcription-PCR method to count the number of shal transcripts in individual identified pyloric neurons, we found that the size of the somatic I(A) varies linearly with the number of endogenous shal transcripts. These data suggest that the shal gene contributes substantially to the peak somatic I(A) in all neurons of the pyloric network.