Autism associated mutations in ß2 subunit of voltage-gated calcium channels constitutively activate gene expression.

Membrane depolarization triggers gene expression through voltage-gated calcium channels (VGCC) in a process called Excitation-transcription (ET) coupling. Mutations in the channel subunits α11.2, or ß2d, are associated with neurodevelopmental disorders such as ASD. Here, we found that two mutations S143F and G113S within the rat Cavß2a corresponding to autistic related mutations Cavß2dS197F and Cavß2dG167S in the human Cavß2d, activate ET-coupling via the RAS/ERK/CREB pathway. Membrane depolarization of HEK293 cells co-expressing α11.2 and α2δ with Cavß2aS143F or Cavß2aG113S triggers constitutive transcriptional activation, which is correlated with facilitated channel activity. Similar to the Timothy-associated autistic mutation α11.2G406R, constitutive gene activation is attributed to a hyperpolarizing shift in the activation kinetics of Cav1.2. Pulldown of RasGRF2 and RhoGEF by wt and the Cavß2a autistic mutants is consistent with Cavß2/Ras activation in ET coupling and implicates Rho signaling as yet another molecular pathway activated by Cavα11.2/Cavß2 . Facilitated spontaneous channel activity preceding enhanced gene activation via the Ras/ERK/CREB pathway, appears a general molecular mechanism for Ca2+ channel mediated ASD and other neurodevelopmental disorders.

pH modulates interaction of 14-3-3 proteins with pollen plasma membrane H+ ATPases independently from phosphorylation.

Pollen grains transport the sperm cells through the style tissue via a fast-growing pollen tube to the ovaries where fertilization takes place. Pollen tube growth requires a precisely regulated network of cellular as well as molecular events including the activity of the plasma membrane H+ ATPase, which is known to be regulated by reversible protein phosphorylation and subsequent binding of 14-3-3 isoforms. Immunodetection of the phosphorylated penultimate threonine residue of the pollen plasma membrane H+ ATPase (LilHA1) of Lilium longiflorum pollen revealed a sudden increase in phosphorylation with the start of pollen tube growth. In addition to phosphorylation, pH modulated the binding of 14-3-3 isoforms to the regulatory domain of the H+ ATPase, whereas metabolic components had only small effects on 14-3-3 binding, as tested with in vitro assays using recombinant 14-3-3 isoforms and phosphomimicking substitutions of the threonine residue. Consequently, local H+ influxes and effluxes as well as pH gradients in the pollen tube tip are generated by localized regulation of the H+ ATPase activity rather than by heterogeneous localized distribution in the plasma membrane.

Elevated basal transcription can underlie timothy channel association with autism related disorders.

Timothy syndrome (TS) is a neurodevelopmental disorder caused by mutations in the pore-forming subunit α11.2 of the L-type voltage-gated Ca2+-channel Cav1.2, at positions G406R or G402S. Although both mutations cause cardiac arrhythmias, only Cav1.2G406R is associated with the autism-spectrum-disorder (ASD). We show that transcriptional activation by Cav1.2G406R and Cav1.2G402S is driven by membrane depolarization through the Ras/ERK/CREB pathway in a process called excitation-transcription (ET) coupling, as previously shown for wt Cav1.2. This process requires the presence of the intracellular ß-subunit of the channel. We found that only the autism-associated mutant Cav1.2G406R, as opposed to the non-autistic mutated channel Cav1.2G402S, exhibits a depolarization-independent CREB phosphorylation, and spontaneous transcription of cFos and MeCP2. A leftward voltage-shift typical of Cav1.2G406R activation, increases channel opening at subthreshold potentials, resulting in an enhanced channel activity, as opposed to a rightward shift in Cav1.2G402S. We suggest that the enhanced spontaneous Cav1.2G406R activity accounts for the increase in basal transcriptional activation. This uncontroled transcriptional activation may result in the manifestation of long-term dysregulations such as autism. Thus, gating changes provide a mechanistic framework for understanding the molecular events underlying the autistic phenomena caused by the G406R Timothy mutation. They might clarify whether a constitutive transcriptional activation accompanies other VGCC that exhibit a leftward voltage-shift of activation and are also associated with long-term cognitive disorders.

Ion occupancy of the channel pore is critical for triggering excitation-transcription (ET) coupling.

During membrane depolarization the voltage-gated calcium channel (VGCC) activates gene expression in excitable cells by means of a signal-transduction pathway termed excitation transcription (ET) coupling. The L-type calcium channel Cav1.2 can drive nuclear activity by either the ERK-CREB pathway, the Ca2+/calmodulin-dependent protein kinase II (CaMKII) cascade, or via the Ca2+-dependent protein phosphatase calcineurin. The ERK-CREB pathway mediates nuclear activity via a direct interaction of the intracellular ß subunit of VGCC with the Ras/GRF1 complex. Here we show that ET coupling in HEK293 cells transfected with wt Cav1.2 or the Timothy mutant Cav1.2G406R is mediated by substituting Ca2+ with the impermeable lanthanum (La3+). In the absence of extracellular Ca2+ or La3+, ET coupling was not triggered. This implies that cation occupancy of the selectivity filter, as opposed to calcium influx, plays an essential role in depolarization triggered signaling to the nucleus. ET coupling triggered by membrane depolarization in Cav1.2 transfected HEK293 cells and neuroendocrine PC12 cells was also supported by substituting Ba2+ for Ca2+ as the charge carrier. Since Ba2+ ions do not bind to calmodulin this implies activation of ET coupling via a Ca2+/calmodulin-independent pathway. Together, these results suggest a model whereby nuclear signaling through the ERK-CREB pathway is driven by voltage-dependent conformational change that requires channel pore occupancy and is Ca2+ influx-independent. This model is also consistent with the previous observation that ET coupling can be driven by the Ca2+-impermeable Cav1.2L745P mutant. Thus, the conversion of synaptic stimuli to transcriptional activation is mediated by the metabotropic function (Ca2+-inflow independent) of Cav1.2, similar to the ion-influx independent depolarization-triggered transmitter release and transcription activation mediated by the NMDA receptors.

ß-Subunit of the voltage-gated Ca2+ channel Cav1.2 drives signaling to the nucleus via H-Ras.

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 α11.2/ß2b/α2δ 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 ß2 subunit or by transfecting the cells with the channel mutant α11.2W440A/ß2b/α2δ, a mutation that disrupts the interaction between α11.2 and ß2 subunits. Pulldown assays in neuronal SH-SY5Y cells and in vitro binding of recombinant H-Ras and ß2 confirmed the importance of the intracellular ß2 subunit for depolarization-induced gene activation. Using a Ca2+-impermeable mutant channel α11.2L745P/ß2b/α2δ or disrupting Ca2+/calmodulin binding to the channel using the channel mutant α11.2I1624A/ß2b/α2δ, 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 Ca2+-influx independent. An indispensable ß-subunit interaction with H-Ras, which is triggered by conformational changes at α11.2 independently of Ca2+ flux, brings to light a master regulatory role of ß2 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.