Ion channels lower the energetic barrier for ion passage across cell membranes and enable the generation of bioelectricity. Electrostatic interactions between permeant ions and channel pore helix dipoles have been proposed as a general mechanism for facilitating ion passage. Here, using genetic selections to probe interactions of an exemplar potassium channel blocker, barium, with the inward rectifier Kir2.1, we identify mutants bearing positively charged residues in the potassium channel signature sequence at the pore helix C terminus. We show that these channels are functional, selective, resistant to barium block, and have minimally altered conductance properties. Both the experimental data and model calculations indicate that barium resistance originates from electrostatics. We demonstrate that potassium channel function is remarkably unperturbed when positive charges occur near the permeant ions at a location that should counteract pore helix electrostatic effects. Thus, contrary to accepted models, the pore helix dipole seems to be a minor factor in potassium channel permeation.

The mechanism by which the phytochrome (phy) photoreceptor family transduces informational light signals to photoresponsive genes is unknown. Using a yeast two-hybrid screen, we have identified a phytochrome-interacting factor, PIF3, a basic helix-loop-helix protein containing a PAS domain. PIF3 binds to wild-type C-terminal domains of both phyA and phyB, but less strongly to signaling-defective, missense mutant-containing domains. Expression of sense or antisense PIF3 sequences in transgenic Arabidopsis perturbs photoresponsiveness in a manner indicating that PIF3 functions in both phyA and phyB signaling pathways in vivo. PIF3 localized to the nucleus in transient transfection experiments, indicating a potential role in controlling gene expression. Together, the data suggest that phytochrome signaling to photoregulated genes includes a direct pathway involving physical interaction between the photoreceptor and a transcriptional regulator.

Skeletal muscle differentiation entails the coupling of muscle-specific gene expression to terminal withdrawal from the cell cycle. Several models have recently been proposed which attempt to explain how regulated expression and function of myogenic transcription factors ensures that proliferation and differentiation of skeletal muscle cells are mutually exclusive processes.

The generation of neurons from stem cells involves the activity of proneural basic helix-loop-helix (bHLH) proteins, but the mechanism by which these proteins irreversibly commit stem cells to neuronal differentiation is not known. Here we report that expression of the transcription factors Sox1, Sox2 and Sox3 (Sox1-3) is a critical determinant of neurogenesis. Using chick in ovo electroporation, we found that Sox1-3 transcription factors keep neural cells undifferentiated by counteracting the activity of proneural proteins. Conversely, the capacity of proneural bHLH proteins to direct neuronal differentiation critically depends on their ability to suppress Sox1-3 expression in CNS progenitors. These data suggest that the generation of neurons from stem cells depends on the inhibition of Sox1-3 expression by proneural proteins.

PAS domains are newly recognized signaling domains that are widely distributed in proteins from members of the Archaea and Bacteria and from fungi, plants, insects, and vertebrates. They function as input modules in proteins that sense oxygen, redox potential, light, and some other stimuli. Specificity in sensing arises, in part, from different cofactors that may be associated with the PAS fold. Transduction of redox signals may be a common mechanistic theme in many different PAS domains. PAS proteins are always located intracellularly but may monitor the external as well as the internal environment. One way in which prokaryotic PAS proteins sense the environment is by detecting changes in the electron transport system. This serves as an early warning system for any reduction in cellular energy levels. Human PAS proteins include hypoxia-inducible factors and voltage-sensitive ion channels; other PAS proteins are integral components of circadian clocks. Although PAS domains were only recently identified, the signaling functions with which they are associated have long been recognized as fundamental properties of living cells.

Hairy-related proteins are a distinct subfamily of basic helix-loop-helix (bHLH) proteins that generally function as DNA-binding transcriptional repressors. These proteins act in opposition to bHLH transcriptional activator proteins such as the proneural and myogenic proteins; together, the activator and repressor genes that encode these proteins have co-evolved as a regulatory gene "cassette" or "module" for controlling cell fate decisions. In the development of the Drosophila peripheral nervous system, Hairy-related genes function at multiple steps during neurogenesis, for example, as positional information genes that establish the "prepattern" that controls where "proneural cluster" equivalence groups will form, and later as nuclear effectors of the Notch signaling pathway to "single out" individual precursor cells within the equivalence group. Hairy-related genes also function in the establishment and restriction of other types of equivalence groups, such as those for muscle and Malphigian tubule precursors. This general function in cell fate specification has been conserved from Drosophila to vertebrates and has implications for human disease pathogenesis.

During the past year, targeted mutagenesis in mice has begun to clarify the roles of individual members of the MyoD family of myogenic regulators in vertebrate development. In this review, we discuss these studies both in the context of tissue interactions necessary to induce skeletal muscle precursor cells during embryogenesis and the molecular circuitry that regulates the terminal differentiation of these cells.

Transcription factors, such as those of the basic-helix-loop-helix (bHLH) and homeodomain classes, are primary regulators of cell fate decisions and differentiation. It is considered axiomatic that they control their respective developmental programs via direct binding to cognate DNA sequences in critical targets genes. Here we test this widely held paradigm by in vivo functional assay of the leukemia oncoprotein SCL, a bHLH factor that resembles myogenic and neurogenic proteins and is essential for both hematopoietic and vascular development in vertebrates. Contrary to all expectation, we find that SCL variants unable to bind DNA rescue hematopoiesis from gene-targeted SCL(-)(/)(-) embryonic stem cells and complement hematopoietic and vascular deficits in the zebrafish mutant cloche. Our findings establish DNA-binding-independent functions of SCL critical for transcriptional specification, and should encourage reassessment of presumed requirements for direct DNA binding by other transcription factors during initiation of developmental programs.

We have used the interaction trap, a yeast two-hybrid system, to identify proteins interacting with hairy, a basic-helix-loop-helix (bHLH) protein that represses transcription during Drosophila embryonic segmentation. We find that the groucho (gro) protein binds specifically to hairy and also to hairy-related bHLH proteins encoded by deadpan and the Enhancer of split complex. The C-terminal WRPW motif present in all these bHLH proteins is essential for this interaction. We demonstrate that these associations reflect in vivo maternal requirements for gro during neurogenesis, segmentation, and sex determination, three processes regulated by the above bHLH proteins, and we propose that gro is a transcriptional corepressor recruited to specific target promoters by hairy-related bHLH proteins.

The ubiquitous Ca(2+)-binding protein calmodulin (CaM) is a key protein in Ca2+ homeostasis and activation of eukaryotic cells. CaM is the molecular link between free Ca2+ in the cell and the inhibition, or activation, of numerous enzymes. Many nuclear functions are under Ca2+/CaM control, and some transcriptional activators are known to be Ca2+ modulated indirectly through Ca2+/CaM-dependent protein kinases. But Ca2+/CaM has not yet been found to directly modulate any transcription factor or other DNA-binding protein. Transcription factors of the basic-helix-loop-helix (bHLH) group are important regulators in numerous systems. Here we report that binding of Ca(2+)-loaded CaM to the bHLH domains of several bHLH proteins directly inhibits their DNA binding. Other bHLH proteins are either less sensitive or resistant. Ca2+ ionophore selectively inhibits transcriptional activation by Ca2+/CaM-sensitive bHLH proteins in vivo, implying that Ca2+ can directly influence transcription through differential CaM inhibition of bHLH domains.