Structural analysis of the extracellular part of the sodium channel α-β subunit interface
Detlef Bentrop, Physiologisches Inst. II, Hermann-Herder-Str. 7, 79104 Freiburg Scientific background Voltage-dependent sodium channels (Nav) are responsible for the rising phase of the action potential in excitable cells. They are composed of a pore-forming α subunit and at least one smaller β subunit modifying kinetics and voltage-dependence of gating. Four different β subunits of ~35 kDa have yet been identified (Navβ1-β4). Although not closely related in primary structure, all β subunits exhibit a large extracellular domain at the N-terminus, a single transmembrane region and a small intracellular C-terminal domain. The extracellular domains of all Navβs are structurally homologous to proteins of the V-set of the Ig superfamily (such as myelin protein P0) and the Ig-like fold, a sandwich of two β sheets (see Fig. 1), is thought to serve two functions: first, modulation of channel gating via interaction with extracellular loops of the α subunit (Makita et al, 1996; Qu et al, 1999) and second, cell adhesion via interaction with extracellular matrix proteins. McCormick et al (1998) have shown that three acidic residues in the first β strand of the β1 subunit affect the rate of Nav1.2 inactivation and may therefore participate in the α-β1 interaction. The structural details of the underlying protein-protein interactions, however, are presently unknown. Fig. 1: Cartoon of the subunit structure of voltage-gated sodium channels. The predicted structural homology of the extracellular domain of β1 to Ig-like folds and important points of interaction between the α and β subunits are illustrated (adapted from Yu & Catterall, 2003).
Topic-related research In the past we have investigated the structure-function correlation of various functional domains of K+ channels by closely combining NMR spectroscopy of proteins in solution and patch-clamp recordings. Thus, we identified a short helical region in the otherwise unstructured calmodulin-binding domain (CaMBD) of SK channels that controls the proteinprotein interaction with the tightly bound Ca2+-sensor calmodulin (CaM) (Wissmann et al., 2002). Furthermore, we addressed the β-subunit mediated inactivation of BK channels as well as the inactivation of neuronal Kv1.4 channels by determining the solution structures of the respective inactivation domains (Bentrop et al., 2001; Wissmann et al., 2003). Analysis of the solution structure of K+ channel interacting protein 4a and its interaction with the intracellular N-terminus of Kv4.3 is currently in progress.
Outline of objectives and research plan The aim of the proposed project is to identify specific sites of extracellular protein-protein interactions between the Nav1.2 α and the β1 subunit and to obtain direct structural information on the subunit interface by solution NMR spectroscopy. These goals will be achieved by the following steps: - first, localization of interaction domains by yeast-two-hybrid analysis and/or protein binding assays using peptides representing extracellular loops IS5-S6 and IVS5-S6 of Nav1.2. The functional relevance of interactions will be tested by electrophysiological recordings and structure-based mutagenesis. - second, reconstitution of complexes of the Ig-like domain of the β1 subunit with peptides from the extracellular loops of Nav1.2, spectroscopical characterization of the interactions, structure determination of such a complex in solution. A prerequisite for all these studies is the efficient heterologous expression of the β1 Ig-like domain. - third, structural analysis of the C121W mutant of human β1. This mutation is known to cause inherited febrile seizures (Wallace et al, 1998) and may destabilize the Ig-like fold in the extracellular domain of the β1 subunit. Planned subject(s) of thesis - Structure-function analysis of the extracellular interactions between the sodium channel α subunit and its β1 subunit. - Solution structure of the extracellular domain of the Nav β1 subunit bound to its α subunit target peptide. Literature Bentrop D, Beyermann M, Wissmann R, Fakler B (2001) NMR structure of the "ball-andchain" domain of KCNMB2, the β2-subunit of large conductance Ca2+- and voltageactivated potassium channels. J Biol Chem 276:42116-42121. Makita N, Bennett PB, George AL (1996) Molecular determinants of β1 subunit-induced gating modulation in voltage-dependent Na+ channels. J Neurosci 16:7117-7127. McCormick KA, Isom LI, Ragsdale D, Smith D, Scheuer T, Catterall WA (1998) Molecular determinants of Na+ channel function in the extracellular domain of the β1 subunit. J Biol Chem 273:3954-3962. Qu Y, Rogers JC, Chen SF, McCormick KA, Scheuer T, Catterall WA (1999) Functional roles of the extracellular segments of the sodium channel α subunit in voltage-dependent gating and modulation by β1 subunits. J Biol Chem 274:32647-32654. Wallace RH et al (1998) Febrile seizures and generalized epilepsy associated with a mutation in the Na+-channel β1 subunit gene SCN1B. Nat Genet 19:366-370 Wissmann R, Bildl W, Neumann H, Rivard A, Klöcker N, Weitz D, Schulte U, Adelman JP, Bentrop D, Fakler B (2002) A helical region in the C-terminus of small-conductance Ca2+-activated K+ channels controls assembly with apo-calmodulin. J Biol Chem 277:4558-4564. Wissmann R, Bildl W, Oliver D, Beyermann M, Kalbitzer HR, Bentrop D, Fakler B (2003) Solution structure and function of the „tandem inactivation domain“ of the neuronal Atype potassium channel Kv1.4. J Biol Chem 278:16142-50 Yu FY, Catterall WA (2003) Overview of the voltage-gated sodium channel family. Genome Biol 4:207.