In the olfactory pathway, as in the limbs, branchial arches, and heart, mesenchymal/epithelial induction, mediated by retinoic acid (RA), FGF8, sonic hedgehog (shh), and the BMPs, defines patterning, morphogenesis, and differentiation. Neuronal differentiation in the olfactory epithelium and directed growth of axons in the nascent olfactory nerve depend critically upon this inductive interaction. When RA, FGF8, shh, or BMP signaling is disrupted, distinct aspects of olfactory pathway patterning and differentiation are compromised. Thus, a cellular and molecular mechanism that facilitates musculoskeletal and vascular development elsewhere in the embryo has been adapted to guide the differentiation of the olfactory pathway in the developing forebrain.

The identification of nearly a dozen ion channel genes involved in the genesis of human atrial and ventricular arrhythmias has been critical for the diagnosis and treatment of fatal cardiovascular diseases. In contrast, very little is known about the genetic and molecular mechanisms underlying human sinus node dysfunction (SND). Here, we report a genetic and molecular mechanism for human SND. We mapped two families with highly penetrant and severe SND to the human ANK2 (ankyrin-B/AnkB) locus. Mice heterozygous for AnkB phenocopy human SND displayed severe bradycardia and rate variability. AnkB is essential for normal membrane organization of sinoatrial node cell channels and transporters, and AnkB is required for physiological cardiac pacing. Finally, dysfunction in AnkB-based trafficking pathways causes abnormal sinoatrial node (SAN) electrical activity and SND. Together, our findings associate abnormal channel targeting with human SND and highlight the critical role of local membrane organization for sinoatrial node excitability.calcium ͉ trafficking ͉ arrhythmia ͉ cytoskeleton

Voltage-gated Nav channels are required for normal electrical activity in neurons, skeletal muscle, and cardiomyocytes. In the heart, Nav1.5 is the predominant Nav channel, and Nav1.5-dependent activity regulates rapid upstroke of the cardiac action potential. Nav1.5 activity requires precise localization at specialized cardiomyocyte membrane domains. However, the molecular mechanisms underlying Nav channel trafficking in the heart are unknown. In this paper, we demonstrate that ankyrin-G is required for Nav1.5 targeting in the heart. Cardiomyocytes with reduced ankyrin-G display reduced Nav1.5 expression, abnormal Nav1.5 membrane targeting, and reduced Na+ channel current density. We define the structural requirements on ankyrin-G for Nav1.5 interactions and demonstrate that loss of Nav1.5 targeting is caused by the loss of direct Nav1.5–ankyrin-G interaction. These data are the first report of a cellular pathway required for Nav channel trafficking in the heart and suggest that ankyrin-G is critical for cardiac depolarization and Nav channel organization in multiple excitable tissues.

Bhasin N, Cunha SR, Mudannayake M, Gigena MS, Rogers TB, Mohler PJ. Molecular basis for PP2A regulatory subunit B56␣ targeting in cardiomyocytes. Am J Physiol Heart Circ Physiol 293: H109-H119, 2007. First published April 6, 2007; doi:10.1152/ajpheart.00059.2007.-Protein phosphatase 2A (PP2A) is a multifunctional protein phosphatase with critical roles in excitable cell signaling. In the heart, PP2A function is linked with modulation of ␤-adrenergic signaling and has been suggested to regulate key ion channels and transporters including Na/Ca exchanger, ryanodine receptor, inositol 1,4,5-trisphosphate receptor, and Na/K ATPase. Although many of the functional roles and molecular targets for PP2A in heart are known, little is established regarding the cellular pathways that localize specific PP2A isoform activities to subcellular sites. We report that the PP2A regulatory subunit B56␣ is an in vivo binding partner for ankyrin-B, an adapter protein required for normal subcellular localization of the Na/Ca exchanger, Na/K ATPase, and inositol 1,4,5-trisphosphate receptor. Ankyrin-B and B56␣ are colocalized and coimmunoprecipitate in primary cardiomyocytes. Using multiple strategies, we identified the structural requirements on B56␣ for ankyrin-B association as a 13 residue motif in the B56␣ COOH terminus not present in other B56 family polypeptides. Finally, we report that reduced ankyrin-B expression in primary ankyrin-B ϩ/Ϫ cardiomyocytes results in disorganized distribution of B56␣ that can be rescued by exogenous expression of ankyrin-B. These new data implicate ankyrin-B as a critical targeting component for PP2A in heart and identify a new class of signaling proteins targeted by ankyrin polypeptides. cytoskeleton; trafficking; phosphatase EFFICIENT EXCITABLE CELL FUNCTION requires precisely orchestrated signaling pathways to modulate the function of diverse membrane, cytosolic, and nuclear proteins. Specifically, the collaboration between finely tuned protein phosphorylation/ dephosphorylation pathways is critical for neuronal communication and cardiac excitation-contraction coupling. Findings over the past decade clearly demonstrate that a local organization of kinase and phosphatase proteins with specific effector protein pathways facilitates efficient signaling. This local organization may occur by direct interaction of kinases and phosphatases with anchoring or scaffolding proteins, cytoskeleton, or even by direct interaction with target ion channels, transporters, or receptors (21,36,42,44,67). However, the cellular pathways underlying the biogenesis/targeting of kinases or phosphatases to specialized membrane protein complexes are not clearly resolved.Protein phosphatase 2A (PP2A) is a multifunctional serine/ threonine phosphatase with critical roles in ion channel/transporter regulation (3, 34, 41, 88), Wnt signaling (39, 63, 72, 84), transformation (2, 6, 27, 75), cell polarization (23), circadian rhythm (5, 68, 85), and cell survival and apoptosis (37,40,64,73,76,81). In heart, PP2A function is neces...

We asked whether mesenchymal/epithelial (M/E) interactions regulate retinoic acid (RA) signaling in the olfactory placode and whether this regulation is similar to that at other sites of induction, including the limbs, branchial arches, and heart. RA is produced by the mesenchyme at all sites, and subsets of mesenchymal cells express the RA synthetic enzyme RALDH2, independent of M/E interactions. In the placode, RA-producing mesenchyme is further distinguished by its coincidence with a molecularly distinct population of neural crest-associated cells. At all sites, expression of additional RA signaling molecules (RARalpha, RARbeta, RXR, CRABP1) depends on M/E interactions. Of these molecules, RA regulates only RARbeta, and this regulation depends on M/E interaction. Expression of Fgf8, shh, and Bmp4, all of which are thought to influence RA signaling, is also regulated by M/E interactions independent of RA at all sites. Despite these common features, RALDH3 expression is distinct in the placode, as is regulation of RARbeta and RALDH2 by Fgf8. Thus, M/E interactions regulate expression of RA receptors and cofactors in the olfactory placode and other inductive sites. Some aspects of regulation in the placode are distinct, perhaps reflecting unique roles for additional local signals in neuronal differentiation in the developing olfactory pathway.

Na/Ca exchanger activity is important for calcium extrusion from the cardiomyocyte cytosol during repolarization. Animal models exhibiting altered Na/Ca exchanger expression display abnormal cardiac phenotypes. In humans, elevated Na/Ca exchanger expression/activity is linked with pathophysiological conditions including arrhythmia and heart failure. Whereas the molecular mechanisms underlying Na/Ca exchanger biophysical properties are widely studied and generally well characterized, the cellular pathways and molecular partners underlying the specialized membrane localization of Na/Ca exchanger in cardiac tissue are essentially unknown. In this report, we present the first direct evidence for a protein pathway required for Na/Ca exchanger localization and stability in primary cardiomyocytes. We define the minimal structural requirements on ankyrin-B for direct Na/Ca exchanger interactions. Moreover, using ankyrin-B mutants that lack Na/Ca exchanger binding activity, and primary cardiomyocytes with reduced ankyrin-B expression, we demonstrate that direct interaction with the membrane adaptor ankyrin-B is required for the localization and post-translational stability of Na/Ca exchanger 1 in neonatal mouse cardiomyocytes. These results raise exciting new questions regarding potentially dynamic roles for ankyrin proteins in the biogenesis and maintenance of specialized membrane domains in excitable cells.

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Development of the frontonasal mass (FNM), branchial arches, heart, and limbs depends on neural crest-mediated epithelial-mesenchymal (E-M) interactions. Teratogenesis by retinoic acid (RA) or blockade of serotonergic (5-HT) signaling by the pan-5-HT(2) receptor antagonist, ritanserin, perturbs development of these embryonic structures. In both cases, resulting phenotypes include forebrain and olfactory placode anomalies, malformations of the face, eye and lens, as well as posterior neural tube and cardiac defects. Similar sites of malformations, together with the presence of RA response elements in the 5-HT(2B) receptor promoter, have led to the suggestion that a negative regulatory relationship may exist between RA and 5-HT(2)-mediated 5-HT signaling at sites of E-M interaction (Choi et al. 1997); however, another possibility is that RA and 5-HT act independently as opposing signals to regulate development of common embryonic targets. Together with recent evidence for opposite effects on chondrogenic differentiation in hindlimb micromass cultures (Bhasin et al. 2003a), results of the present study raise the possibility that these pathways may act as opposing signals for common targets in the mouse embryo. The RA receptors, co-factors and metabolic enzymes, and 5-HT(2B) receptors were found to be are coordinately expressed at sites of E-M interaction, including the FNM, in the embryonic day (E)10.5 mouse. Cell proliferation experiments using [(3)H]thymidine incorporation demonstrated that RA or activation of 5-HT(2B) receptors caused opposite effects in FNM explants, namely stimulation or inhibition of cell proliferation, respectively, 5-HT(2B) receptor activation did not appreciably alter patterning in FNM explants. While RA has been shown to regulate lateral patterning in the FNM (LaMantia et al. 2000), 5-HT(2B) receptor activation did not alter patterning in FNM explants. Quantification of 5-HT(2B) receptor transcripts by real-time PCR provided no evidence of negative regulation of 5-HT(2B) receptor expression by RA in FNM explants, although preliminary studies using in situ hybridization had suggested that this was a possibility in both explants and RA teratogenized embryos. Future studies using quantitative PCR may still show this to be the case in teratogenized embryos. Together with the finding of coordinate expression of 5-HT(2B )receptors and RA signaling molecules, results of the present study suggest that RA, and 5-HT mediated by 5-HT(2B )receptors, may act as opposing signals to regulate cell proliferation during craniofacial development in the mouse embryo.