Quantitative Single-Cell-Reverse Transcription-PCR Demonstrates That A-Current Magnitude Varies as a Linear Function ofshalGene 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 somaticI_A vary significantly among pyloric neurons such that the somatic I_A is unique in each pyloric cell type. Comparing these currents with theI_As obtained from oocytes injected withPanulirus shaker and shal cRNA (lobsterI_shaker and lobsterI_shal, respectively) reveals that the pyloric cell I_As more closely resemble lobster I_shal than lobsterI_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 somaticI_A varies linearly with the number of endogenous shal transcripts. These data suggest that theshal gene contributes substantially to the peak somaticI_A in all neurons of the pyloric network.