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Summary: Vann Bennett is interested in how our cells localize ion transporters and other membrane proteins to sites that optimize their physiological efficiency. He currently is focusing on newly discovered ankyrin-dependent pathways responsible for establishing specialized membrane domains in diverse cells, ranging from neurons to epithelial cells.

To perform their physiological roles, membrane-spanning proteins must segregate to specific locations in our cells. For example, to fire action potentials, neurons and heart cells require clustering of voltage-gated sodium channels at high density within excitable membranes. Similarly, transcellular transport by epithelial tissues requires segregation of channels and transporters within distinct apical and lateral membrane domains. Our laboratory seeks to understand how membrane proteins in vertebrate cells are localized at the precise sites required for their physiological functions. This research began with the discovery of the ankyrin family of adapter proteins. We have found that ankyrins are required for organization of a surprisingly diverse set of proteins and membrane domains and are likely to play pervasive roles in vertebrate physiology.

Mutations of Ankyrin-B Cause a New Cardiac Arrhythmia Syndrome
We and our collaborators discovered that a missense mutation in human ankyrin-B causes a dominantly inherited cardiac arrhythmia and is a risk factor for sudden cardiac death. We also demonstrated that chronically elevated calcium transients combined with sympathetic stress provided an electrical mechanism for arrhythmia. A cellular rationale for elevated calcium was the observed coordinated reduction of three ankyrin-binding proteins classically involved in calcium homeostasis: Na⁺/K⁺-ATPase (NKA), Na⁺/Ca²⁺ exchanger (NCX), and IP3 receptor (IP3R). These findings established the principle of a "channelopathy" due to abnormal localization of ion channels/transporters, and provided a novel mechanism for cardiac arrhythmia.

	Ankyrin-B and the Na/Ca exchanger in neonatal cardiomyocytes... FIGQY phosphorylation in adult sciatic nerve...

We next analyzed four additional ankyrin-B mutations that had been identified (following publication of our initial paper) by Mark Keating (Novartis Institute for Biomedical Research) and Sylvia Priori (University of Pavia) in patients with cardiac arrhythmias. We demonstrated that these mutations resulted in loss-of-function of ankyrin-B in cardiomyocytes. Moreover, one of the mutations (R1788W) blocked association of ankyrin-B with Hsp40. The additional clinical data allowed a more precise definition of the ankyrin-B mutant phenotype as "sick sinus syndrome with bradycardia" (OMIM).

We further explored the cellular basis for altered calcium homeostasis due to ankyrin-B mutation. We discovered that ankyrin-B forms a multiprotein complex with NKA, NCX, and IP3R. Moreover, this complex is localized in a microdomain on T tubules distinct from classic dyads and is deficient in ankyrin-B–heterozygous cardiomyocytes. These findings suggest a model where ankyrin-B coordinates NKA, NCX, and IP3R in a configuration that allows for direct cellular export of Ca²⁺ from the sarcoplasmic reticulum without mixing with cytosolic calcium.

The Cardiac Voltage-Gated Sodium Channel Nav1.5 Requires Ankyrin-G for Cellular Targeting
We discovered that ankyrin-G colocalizes with cardiac voltage-gated sodium channels (Nav1.5) at two strategic cellular domains in cardiomyocytes: intercalated discs, where the action potential is transmitted to adjacent cells, and microdomains on the T tubule, close to voltage-gated calcium channels activated by the sodium-based action potential. We then noticed that the E1053K mutation of Nav1.5, cited in a review on Brugada syndrome mutations, was within the ankyrin-binding motif. We next discovered with our collaborators that this E1053K mutation of Nav1.5, in addition to causing arrhythmia, also abolishes both ankyrin binding and targeting of Nav1.5 to the cell surface of cardiomyocytes. E1053K Nav1.5 is electrically active when expressed in 293 cells, demonstrating that the mutation does not interfere with normal folding or processing.

The finding that Nav1.5 requires ankyrin-binding activity for cellular targeting is important for several reasons. First, it further establishes the principle that missorting of ion channels is functionally equivalent to loss of channel activity. It also suggests that the role of ankyrin-G in targeting voltage-gated sodium channels is conserved between cardiomyocytes and neurons, where we have previously shown that ankyrin-G is required to localize the closely related protein Nav1.6 to axon initial segments. More generally, the cellular mechanism for targeting sodium channels may be closely related to the formation of lateral membranes of epithelial cells, which also requires ankyrin-G.

Ankyrin-G Collaborates with β₂-Spectrin in Biogenesis of the Lateral Membrane of Epithelial Cells
We have discovered that epithelial cells depleted of ankyrin-G by small interfering RNA maintain polarity but are converted from columnar to squamous morphology with minimal lateral membrane and expanded apical and basal membranes. Cells depleted of ankyrin-G do not form new lateral membrane during mitosis and as a result cannot complete cytokinesis. These surprising findings suggest that ankyrin-G is required for formation of the entire lateral membrane in epithelial cells.

We have recently found that ankyrin-G collaborates with β₂-spectrin in biogenesis of the lateral membrane in epithelial cells. Ankyrin-G mutants defective in binding β-spectrin target normally to the lateral membrane but do not restore lateral membrane in ankyrin-G–deficient cells. In addition, knockdown of β₂-spectrin phenocopies depletion of ankyrin-G in interphase and mitotic cells.

Ankyrin-G Is a Molecular Partner of E-Cadherin
E-cadherin is a cell adhesion molecule that is required to form the first lateral membrane domains in development and is a ubiquitous component of lateral membranes in epithelial tissues. We have found that E-cadherin targeting to sites of cell-cell contact in epithelial cells and early mouse embryos requires ankyrin-G and β₂-spectrin. Moreover, in vitro, ankyrin-G binds directly to the cytoplasmic domain of E-cadherin and can recruit spectrin. These results are consistent with an earlier report from James Nelson's group (Stanford University) that ankyrin, spectrin, and E-cadherin associate in a complex.

Several observations suggest ankyrin-G and β₂-spectrin work together with microtubules to deliver E-cadherin to the lateral membrane. Depletion of ankyrin-G, β₂-spectrin, or microtubules results in the accumulation of E-cadherin in the trans-Golgi network (TGN). Moreover, ankyrin-G and β₂-spectrin both transiently colocalize with E-cadherin in a post-TGN compartment during rapid membrane biogenesis following recovery of microtubules. These results suggest a model where E-cadherin binds ankyrin-G, which is then coupled to microtubule motor proteins by β₂-spectrin for post-TGN transport along microtubules to sites of cell-cell contact.

Transcellular interactions between E-cadherin molecules in adjacent cells are likely to provide the spatial cues that direct the ankyrin-spectrin machinery. Coupling of E-cadherin to a versatile adapter protein such as ankyrin-G could promote corecruitment of diverse proteins to sites of cell-cell contact. Ankyrin-G also binds to N-cadherin and likely other type 1 classical cadherins. Ankyrin-G and cadherin partners thus could direct formation of a variety of specialized membrane domains at sites of cell-cell contact, ranging from synapses in the nervous system to intercalated discs in cardiomyocytes.

Ankyrin-B and -G Localize the Dystrophin-Glycoprotein Complex to Costameres
The dystrophin-glycoprotein complex (DGC) is an essential component of costameres, which are sites on the sarcolemma of striated muscle where contractile force is transmitted to the extracellular matrix. Loss of the DGC in Duchenne muscular dystrophy causes increased fragility of the sarcolemma. We initially observed that ankyrin-B–null mice exhibit loss of targeting of the DGC, both in muscle and the nervous system. We have subsequently confirmed and extended these findings with two technical innovations: (1) in vivo knockdown of protein expression in skeletal muscle using electroporation to deliver siRNA plasmids, and (2) high-resolution visualization of costameres in isolated muscle fibers. These experiments have revealed that ankyrins-B and -G cooperate in a stepwise pathway for targeting and retention of the DGC at costameres in skeletal muscle. Ankyrin-B is required for initial targeting of the DGC to the sarcolemma. Ankyrin-G, in contrast, is necessary to retain the DGC at costameres but not for its delivery to the sarcolemma. We have found that a class of microtubules associated with costameres is lost following depletion of ankyrin-B. Ankyrin-B was initially identified as a microtubule-binding protein, but this is the first direct functional connection between ankyrin-B and microtubules.

This work has been supported in part by the Muscular Dystrophy Association.

Last updated: September 14, 2007