Summary The vascular system is essential for embryonic development and adult life. Aberrant vascularization is associated with numerous diseases, including cancer, atherosclerosis, retinopathy, and stroke. Vascular development begins when mesodermal cells differentiate into endothelial cells, which then form primitive vessels. It has been hypothesized that endothelial-specific gene expression may be regulated combinatorially, but the transcriptional mechanisms governing vascular gene expression remain incompletely understood. Here, we identify a transcriptional code, consisting of Forkhead and Ets factors, which is required and sufficient for vascular development and endothelial gene expression through combinatorial activation of a composite cis-acting element. We show that the presence of this FOX:ETS motif is an effective predictor of endothelial-specific enhancers. These studies establish a paradigm in which two broadly expressed classes of transcription factors regulate tissue specific expression combinatorially through a single composite cis-acting element. This mechanism has broad implications for understanding differentiation and gene expression in many tissues.

G protein-activated inwardly rectifying K ؉ (GIRK) channels regulate neuronal excitability by mediating inhibitory effects of G protein-coupled receptors for neurotransmitters and neuromodulators. Notwithstanding many studies reporting modulation of GIRK channel function, whether neuronal activity regulates GIRK channel trafficking remains an open question. Here we report that NMDA receptor activation in cultured dissociated hippocampal neurons elevates surface expression of the GIRK channel subunits GIRK1 and GIRK2 in the soma, dendrites, and dendritic spines within 15 min. This activity-induced increase in GIRK surface expression requires protein phosphatase-1-mediated dephosphorylation of a serine residue (Ser-9) preceding the GIRK2 Val-13/ Leu-14 (VL) internalization motif, thereby promoting channel recycling. Because activation of GIRK channels hyperpolarizes neuronal membranes, the NMDA receptor-induced regulation of GIRK channel trafficking may represent a dynamic adjustment of neuronal excitability in response to inhibitory neurotransmitters and/or neuromodulators.GIRK ͉ NMDA receptor ͉ trafficking ͉ protein phosphatase-1 ͉ dendrites G protein-activated inwardly rectifying K ϩ (GIRK) channels belong to the Kir3.x subfamily of inwardly rectifying potassium channels, with each subunit containing 2 transmembrane segments and cytoplasmic N-and C-terminal domains (1). They regulate neuronal excitability in response to neurotransmitters and neuromodulators that activate G protein-coupled receptors (GPCRs) coupled to pertussis toxin-sensitive Gi/o proteins, inducing the exchange of GDP for GTP on the G␣ and dissociation of G␣-GTP from G␤␥ (1). GIRK channel activation by direct binding of G␤␥ causes hyperpolarization, thus reducing neuronal excitability (1). GIRK channels are also modulated by intracellular Na ϩ , Mg 2ϩ , phosphatidylinositol 4,5-bisphosphate, G␣i, and regulators of G protein signaling (2).Neuronal GIRK channels in the central nervous system are mostly heterotetramers of GIRK1 and GIRK2 subunits, whereas midbrain dopaminergic neurons express homomeric GIRK2 channels (1). GIRK channels reside predominantly in the soma and dendrites of pyramidal neurons (1), where their current (3, 4) dampens the effects of excitatory synaptic input (5, 6). They also mediate slow inhibitory postsynaptic current upon GABA B receptor activation and account for the hyperpolarization induced by adenosine and serotonin receptors (7). Underscoring the physiologic importance of GIRK channels, mice lacking GIRK2 display sporadic seizures, increased susceptibility to convulsant agents, and hyperactivity, as well as abnormality in cocaine self-administration, pain threshold, response to analgesics including opioids, and sensitivity to ethanol's motivational effects (8).Dynamic regulation of GIRK channel number affords a powerful way to modulate neuronal activity. Because GIRK1 (9) and GIRK2 (10) reside in the dendrites and dendritic spines that harbor the majority of the excitatory synapses, we wondered whether activation o...

SUMMARY Skeletal muscle comprises a heterogeneous population of fibers with important physiological differences. Fast fibers are glycolytic and fatigue rapidly. Slow fibers utilize oxidative metabolism and are fatigue-resistant. Muscle diseases such as sarcopenia and atrophy selectively affect fast fibers, but the molecular mechanisms regulating fiber type-specific gene expression remain incompletely understood. Here, we show that the transcription factor NFATc1 controls fiber type composition and is required for fast-to-slow fiber type switching in response to exercise in vivo. Moreover, MyoD is a crucial transcriptional effector of the fast fiber phenotype, and we show that NFATc1 inhibits MyoD-dependent fast fiber gene promoters by physically interacting with the N-terminal activation domain of MyoD and blocking recruitment of the essential transcriptional coactivator p300. These studies establish a molecular mechanism for fiber type switching through direct inhibition of MyoD to control the opposing roles of MyoD and NFATc1 in fast versus slow fiber phenotypes.

G protein-activated inwardly rectifying potassium (GIRK) channels mediate slow synaptic inhibition and control neuronal excitability. It is unknown whether GIRK channels are subject to regulation by guanine dissociation inhibitor (GDI) proteins like LGN, a mammalian homolog of Drosophila Partner of Inscuteable (mPINS). Here we report that LGN increases basal GIRK current but reduces GIRK activation by metabotropic transmitter receptors coupled to Gi or Go, but not Gs. Moreover, expression of its N-terminal, TPR-containing protein interaction domains mimics the effects of LGN in mammalian cells, probably by releasing sequestered endogenous LGN. In hippocampal neurons, expression of LGN, or LGN fragments that mimic or enhance LGN activity, hyperpolarizes the resting potential due to increased basal GIRK activity and reduces excitability. Using Lenti virus for LGN RNAi to reduce endogenous LGN levels in hippocampal neurons, we further show an essential role of LGN for maintaining basal GIRK channel activity and for harnessing neuronal excitability.