Abstract

Depolarization-induced signaling to the nucleus by the L-type voltage-gated calcium channel Cav1.2 is widely assumed to proceed by elevating intracellular calcium. The apparent lack of quantitative correlation between Ca2+ influx and gene activation suggests an alternative activation pathway. Here, we demonstrate that membrane depolarization of HEK293 cells transfected with alpha(1)1.2/beta 2b/alpha 2 delta subunits (Cav1.2) triggers c-Fos and MeCP2 activation via the Ras/ERK/CREB pathway. Nuclear signaling is lost either by absence of the intracellular beta 2 subunit or by transfecting the cells with the channel mutant alpha(1)1.2(W440A)/beta 2b/alpha 2 delta, a mutation that disrupts the interaction between alpha(1)1.2 and beta 2 subunits. Pulldown assays in neuronal SH-SY5Y cells and in vitro binding of recombinant H-Ras and beta 2 confirmed the importance of the intracellular beta 2 subunit for depolarization-induced gene activation. Using a Ca2+-impermeable mutant channel alpha(1)1.2(L745P)/beta 2b/alpha 2 delta or disrupting Ca2+/calmodulin binding to the channel using the channel mutant al alpha(1)1.2(11624A)/beta 2b/alpha 2 delta, we demonstrate that depolarization-induced c-Fos and MeCP2 activation does not depend on Ca2+ transport by the channel. Thus, in contrast to the paradigm that elevated intracellular Ca2+ drives nuclear signaling, we show that Cav1.2-triggered c-Fos or MeCP2 is dependent on extracellular Ca2+ and Ca2+ occupancy of the open channel pore, but is Ca(2+)influx independent. An indispensable beta-subunit interaction with H-Ras, which is triggered by conformational changes at alpha(1)1.2 independently of Ca2+ flux, brings to light a master regulatory role of beta 2 in transcriptional activation via the ERK/CREB pathway. This mode of H-Ras activation could have broad implications for understanding the coupling of membrane depolarization to the rapid induction of gene transcription.