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Being there: cellular targeting of voltage-gated sodium channels in the heart
Correspondence to Vann Bennett: v.bennett{at}cellbio.duke.edu
Voltage-gated sodium (Nav) channels in cardiomyocytes are localized in specialized membrane domains that optimize their functions in propagating action potentials across cell junctions and in stimulating voltage-gated calcium channels located in T tubules. Mutation of the ankyrin-binding site of Nav1.5, the principal Nav channel in the heart, was previously known to cause cardiac arrhythmia and the retention of Nav1.5 in an intracellular compartment in cardiomyocytes. Conclusive evidence is now provided that direct interaction between Nav1.5 and ankyrin-G is necessary for the expression of Nav1.5 at the cardiomyocyte cell surface.
Being at the right place at the right time is a well-established principle of success for people that also applies to ion channels. Proper localization is especially important for vertebrate Nav channels, which must be clustered at high density to generate and/or propagate action potentials. In cardiomyocytes, Nav channels (principally Nav1.5) are clustered at intercalated discs, where, together with gap junctions, they transmit action potentials between cells, and are also clustered at T tubules, where they activate voltage-sensitive calcium channels (Fig. 1; Cohen, 1996; Scriven et al., 2000). Several findings have implicated the ankyrin family of membrane adaptors in Nav channel clustering and localization in excitable membranes of both neurons and the heart. Vertebrate Nav channels share a conserved ankyrin-binding motif (Garrido et al., 2003; Lemaillet et al., 2003). Moreover, Nav β subunits also exhibit ankyrin-binding activity (Malhotra et al., 2000). Knockout of ankyrin-G in the postnatal mouse cerebellum results in the loss of Nav1.6 from Purkinje neuron axon initial segments (Zhou et al., 1998; Jenkins and Bennett, 2001). Nav1.5 in the heart colocalizes and coimmunoprecipitates with ankyrin-G (Mohler et al., 2004). Furthermore, E1053K mutation in the ankyrin-binding motif of the cardiac Nav1.5 channel abolishes ankyrin binding and causes Brugada Syndrome, a cardiac arrhythmia caused by the loss of function of Nav1.5 (Mohler et al., 2004). The same E1053K mutation also prevents delivery of Nav1.5 to the cardiomyocyte plasma membrane (Mohler et al., 2004).
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Lowe et al. (see p. 173 of this issue) address these issues in the heart with the demonstration that siRNA-mediated knockdown of ankyrin-G but not ankyrin-B abolishes the surface expression of Nav1.5 in neonatal as well as adult cardiomyocytes. The study further demonstrates that loss of cell surface Nav1.5 in ankyrin-G–depleted neonatal cardiomyocytes can be reversed by rescue with a version of ankyrin-G that is resistant to siRNA. Moreover, mutation of ankyrin-G that abolishes the binding activity for Nav1.5 also abolishes the ability to restore cell surface Nav1.5. Lowe et al. (2008) also take the localization of ankyrin-G and Nav1.5 to the ultrastructural level with the demonstration by immunogold labeling of coclusters of Nav1.5 and ankyrin-G in adult cardiomyocyte membranes. These data, together with previous observations (Mohler et al., 2004), satisfy the equivalent of Koch's postulates for physiological interactions between proteins: (1) Nav1.5 and ankyrin-G colocalize at high resolution in cardiomyocytes and coimmunoprecipitate from heart tissue; (2) Nav1.5 localization in cardiomyocytes is lost with (a) a point mutation of Nav1.5 that abolishes binding to ankyrin-G, (b) depletion of ankyrin-G, and (c) mutation of ankyrin-G that abolishes binding to Nav1.5; and (3) mutation of Nav1.5 in an organism (in this case humans) causing the loss of ankyrin binding results in a phenotype that is consistent with the loss of Nav1.5 function (i.e., Brugada Syndrome).
These findings raise the question of whether the ankyrin-G pathway is used by other components of intercalated discs and T tubules. In axon initial segments, ankyrin-G is required for the localization of KCNQ2/3 channels and neurofascin in addition to Nav1.6 (Jenkins and Bennett, 2001; Chung et al., 2006; Pan et al., 2006; Rasmussen et al., 2007). Interestingly, each of these proteins has independently evolved an ankyrin-binding motif (Pan et al., 2006). It is possible that multiple, unrelated proteins in the heart also could engage the ankyrin-G machinery for coordinated localization in the same specialized domain. Given that T tubules and intercalated discs both contain ankyrin-G yet have distinct proteins, additional mechanisms must exist for fine-tuning the composition of these domains.
What is the role of ankyrin-G in the delivery and/or retention of Nav1.5 to the cell surface? One possibility is that ankyrin-G and spectrin act as a scaffold that retains Nav1.5 after delivery and prevents endocytosis. However, studies of ankyrin-G in epithelial cells suggest a more complex mechanism (Kizhatil et al., 2007a,b). Ankyrin-G in these cells collaborates with β2 spectrin in formation of the lateral membrane and in exit of epithelial cadherin from the trans-Golgi network (Kizhatil et al., 2007a,b). It will be important to evaluate the role of ankyrin-G in the assembly of intercalated discs and Nav1.5-enriched domains of T tubules in cardiomyocytes. A current technical challenge is that these cell surface domains are not fully differentiated in neonatal cardiomyocytes, whereas adult cardiomyocytes frequently lose their morphology and viability after several days in culture. Ultimately, it will be of great interest to resolve the cell biology underlying the targeting of Nav1.5 as well as other membrane-spanning proteins, such as gap junction subunits and calcium channels, whose localization in differentiated cardiomyocytes is key to their physiological function.
Acknowledgments
J. Healy is supported by a predoctoral grant from the American Heart Association.
Submitted: 18 December 2007
Accepted: 19 December 2007
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