Expression of α_1D subunit mRNA is correlated with L-type Ca²⁺ channel activity in single neurons of hippocampal “zipper” slices

Abstract

L-type voltage-sensitive Ca²⁺ channels (L-VSCCs) play an important role in developmental and aging processes, as well as during normal function of brain neurons. Here, we tested a prediction of the hypothesis that membrane density of functional L-VSCCs is regulated by the level of gene expression for its α_1D pore-forming subunit. If so, α_1D mRNA and L-VSCC activity should be positively correlated within individual neurons. Conventional methods of aspiration and/or acute cell dissociation used in prior single-cell studies have generally yielded variable and incomplete recovery of intracellular mRNA. Thus, quantitative relationships between channel function and expression have been difficult to define. In this study, we used the partially dissociated (“zipper”) hippocampal slice preparation as a method for collecting a single neuron's mRNA complement. This preparation, developed to expose neuronal somata for recording, also enables the extraction of a neuron with major processes largely intact. Thus, single-cell measures of gene/mRNA expression can be based on approximately the cell's full set of mRNA transcripts. In adult and aged rat hippocampal zipper slices, L-VSCC activity was first recorded in CA1 neurons in cell-attached patch mode. The same neurons were then extracted and collected for semiquantitative reverse transcriptase–PCR analysis of α_1D and calmodulin A (CaM) mRNA content. Across multiple single neurons, a significant, positive correlation was found between the rank orders of L-VSCC activity and of α_1D, but not CaM, mRNA expression. Thus, these studies support the possibility that the level of α_1D gene expression regulates the density of functional L-VSCCs.