Expression of alpha 1D subunit mRNA is correlated with L-type Ca2+ channel activity in single neurons of hippocampal "zipper" slices.
L-type voltage-sensitive Ca(2+) 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 alpha(1D) pore-forming subunit. If so, alpha(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 alpha(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 alpha(1D), but not CaM, mRNA expression. Thus, these studies support the possibility that the level of alpha(1D) gene expression regulates the density of functional L-VSCCs.
Chen
K C
KC
Department of Pharmacology, University of Kentucky Medical Center, 800 Rose Street, Lexington, KY 40536, USA. kueyc@pop.uky.edu
Blalock
E M
EM
Thibault
O
O
Kaminker
P
P
Landfield
P W
PW
D000818:Animals; D001483:Base Sequence; D020746:Calcium Channels, L-Type / Q000235:genetics / Q000378:metabolism* / Q000502:physiology; D017931:DNA Primers; D006624:Hippocampus / Q000166:cytology / Q000378:metabolism*; D008297:Male; D008564:Membrane Potentials; D009474:Neurons / Q000378:metabolism*; D016133:Polymerase Chain Reaction; D012333:RNA, Messenger / Q000235:genetics* / Q000378:metabolism; D051381:Rats; D011916:Rats, Inbred F344
D020746:Calcium Channels, L-Type; D017931:DNA Primers; D012333:RNA, Messenger