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Volume 17, Number 17, Issue of September 1, 1997 pp. 6597-6610
Copyright ©1997 Society for Neuroscience

Quantitative Single-Cell-Reverse Transcription-PCR Demonstrates That A-Current Magnitude Varies as a Linear Function of shal Gene Expression in Identified Stomatogastric Neurons

Received March 4, 1997; revised May 28, 1997; accepted June 17, 1997.

Deborah J. Baro¹, Robert M. Levini¹, Marshall T. Kim¹, Allan R. Willms², Cathy Cole Lanning¹, Hilda E. Rodriguez¹, and Ronald M. Harris-Warrick¹

¹ Section of Neurobiology and Behavior and ² Center for Applied Mathematics, Cornell University, Ithaca, New York 14850

Different Shaker family -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_As obtained from oocytes injected with Panulirus shaker and shal cRNA (lobster I_shaker and lobster I_shal, respectively) reveals that the pyloric cell I_As 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.

Key words: quantitative; single-cell-RT-PCR; stomatogastric; transient potassium current; Shaker family; potassium channel; gene regulation; Kv; transcriptional control; pyloric network; shal; identified neuron; noncompetitive PCR; invertebrate

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