Despite recent advances in delineating the mechanisms involved in cardiogenesis, cellular lineage specification remains incompletely understood. To explore the relationship between developmental fate and potential, we isolated a cardiac-specific Nkx2.5(+) cell population from the developing mouse embryo. The majority of these cells differentiated into cardiomyocytes and conduction system cells. Some, surprisingly, adopted a smooth muscle fate. To address the clonal origin of these lineages, we isolated Nkx2.5(+) cells from in vitro differentiated murine embryonic stem cells and found approximately 28% of these cells expressed c-kit. These c-kit(+) cells possessed the capacity for long-term in vitro expansion and differentiation into both cardiomyocytes and smooth muscle cells from a single cell. We confirmed these findings by isolating c-kit(+)Nkx2.5(+) cells from mouse embryos and demonstrated their capacity for bipotential differentiation in vivo. Taken together, these results support the existence of a common precursor for cardiovascular lineages in the mammalian heart.

A longstanding hypothesis is that ion channels are present in the membranes of synaptic vesicles and might affect neurotransmitter release. Here we demonstrate that TRPM7, a member of the transient receptor potential (TRP) ion channel family, resides in the membrane of synaptic vesicles of sympathetic neurons, forms molecular complexes with the synaptic vesicle proteins synapsin I and synaptotagmin I, and directly interacts with synaptic vesicular snapin. In sympathetic neurons, changes in TRPM7 levels and channel activity alter acetylcholine release, as measured by EPSP amplitudes and decay times in postsynaptic neurons. TRPM7 affects EPSP quantal size, an intrinsic property of synaptic vesicle release. Targeted peptide interference of TRPM7's interaction with snapin affects the amplitudes and kinetics of postsynaptic EPSPs. Thus, vesicular TRPM7 channel activity is critical to neurotransmitter release in sympathetic neurons.

Selective permeability in voltage-gated Ca2+ channels is dependent upon a quartet of pore-localized glutamate residues (EEEE locus). The EEEE locus is widely believed to comprise the sole high-affinity Ca2+ binding site in the pore, which represents an overturning of earlier models that had postulated two high-affinity Ca2+ binding sites. The current view is based on site-directed mutagenesis work in which Ca2+ binding affinity was attenuated by single and double substitutions in the EEEE locus, and eliminated by quadruple alanine (AAAA), glutamine (QQQQ), or aspartate (DDDD) substitutions. However, interpretation of the mutagenesis work can be criticized on the grounds that EEEE locus mutations may have additionally disrupted the integrity of a second, non-EEEE locus high-affinity site, and that such a second site may have remained undetected because the mutated pore was probed only from the extracellular pore entrance. Here, we describe the results of experiments designed to test the strength of these criticisms of the single high-affinity locus model of selective permeability in Ca2+ channels. First, substituted-cysteine accessibility experiments indicate that pore structure in the vicinity of the EEEE locus is not extensively disrupted as a consequence of the quadruple AAAA mutations, suggesting in turn that the quadruple mutations do not distort pore structure to such an extent that a second high affinity site would likely be destroyed. Second, the postulated second high-affinity site was not detected by probing from the intracellularly oriented pore entrance of AAAA and QQQQ mutants. Using inside-out patches, we found that, whereas micromolar Ca2+ produced substantial block of outward Li+ current in wild-type channels, internal Ca2+ concentrations up to 1 mM did not produce detectable block of outward Li+ current in the AAAA or QQQQ mutants. These results indicate that the EEEE locus is indeed the sole high-affinity Ca2+ binding locus in the pore of voltage-gated Ca2+ channels.

We have cloned and characterized mouse and human variants of MONaKA, a novel protein that interacts with and modulates the plasma membrane Na,K-ATPase. MONaKA was cloned based on its sequence homology to the Drosophila Slowpoke channel-binding protein dSlob, but mouse and human MONaKA do not bind to mammalian Slowpoke channels. At least two splice variants of MONaKA exist; the splicing is conserved perfectly between mouse and human, suggesting that it serves some important function. Both splice variants of MONaKA are expressed widely throughout the CNS and peripheral nervous system, with different splice variant expression ratios in neurons and glia. A yeast two-hybrid screen with MONaKA as bait revealed that it binds tightly to the ␤1 and ␤3 subunits of the Na,K-ATPase. The association between MONaKA and Na,K-ATPase ␤ subunits was confirmed further by coimmunoprecipitation from transfected cells, mouse brain, and cultured mouse astrocytes. A glutathione S-transferase-MONaKA fusion protein inhibits Na,KATPase activity from whole brain or cultured astrocytes. Furthermore, transfection of MONaKA inhibits 86 Rb ϩ uptake via the Na,KATPase in intact cells. These results are consistent with the hypothesis that MONaKA modulates brain Na,K-ATPase and may thereby participate in the regulation of electrical excitability and synaptic transmission.

The selectivity filter of voltage-gated Ca2+ channels is in part composed of four Glu residues, termed the EEEE locus. Ion selectivity in Ca2+ channels is based on interactions between permeant ions and the EEEE locus: in a mixture of ions, all of which can pass through the pore when present alone, those ions that bind weakly are impermeant, those that bind more strongly are permeant, and those that bind more strongly yet act as pore blockers as a consequence of their low rate of unbinding from the EEEE locus. Thus, competition among ion species is a determining feature of selectivity filter function in Ca2+ channels. Previous work has shown that Asp and Ala substitutions in the EEEE locus reduce ion selectivity by weakening ion binding affinity. Here we describe for wild-type and EEEE locus mutants an analysis at the single channel level of competition between Cd2+, which binds very tightly within the EEEE locus, and Ba2+ or Li+, which bind less tightly and hence exhibit high flux rates: Cd2+ binds to the EEEE locus ∼104× more tightly than does Ba2+, and ∼108× more tightly than does Li+. For wild-type channels, Cd2+ entry into the EEEE locus was 400× faster when Li+ rather than Ba2+ was the current carrier, reflecting the large difference between Ba2+ and Li+ in affinity for the EEEE locus. For the substitution mutants, analysis of Cd2+ block kinetics shows that their weakened ion binding affinity can result from either a reduction in blocker on rate or an enhancement of blocker off rate. Which of these rate effects underlay weakened binding was not specified by the nature of the mutation (Asp vs. Ala), but was instead determined by the valence and affinity of the current-carrying ion (Ba2+ vs. Li+). The dependence of Cd2+ block kinetics upon properties of the current-carrying ion can be understood by considering the number of EEEE locus oxygen atoms available to interact with the different ion pairs.