A β(IV)-spectrin/CaMKII signaling complex is essential for membrane excitability in mice

Abstract

Ion channel function is fundamental to the existence of life. In metazoans, the coordinate activities of voltage-gated Na(+) channels underlie cellular excitability and control neuronal communication, cardiac excitation-contraction coupling, and skeletal muscle function. However, despite decades of research and linkage of Na(+) channel dysfunction with arrhythmia, epilepsy, and myotonia, little progress has been made toward understanding the fundamental processes that regulate this family of proteins. Here, we have identified β(IV)-spectrin as a multifunctional regulatory platform for Na(+) channels in mice. We found that β(IV)-spectrin targeted critical structural and regulatory proteins to excitable membranes in the heart and brain. Animal models harboring mutant β(IV)-spectrin alleles displayed aberrant cellular excitability and whole animal physiology. Moreover, we identified a regulatory mechanism for Na(+) channels, via direct phosphorylation by β(IV)-spectrin-targeted calcium/calmodulin-dependent kinase II (CaMKII). Collectively, our data define an unexpected but indispensable molecular platform that determines membrane excitability in the mouse heart and brain.

Figure 1. Identification of putative new CaMKII-binding proteins.

(A) Putative CaMKII-targeting proteins identified through a screen of the human genome using a sequence from the CaMKII autoregulatory domain as bait. Candidates with the highest homology to CaMKII autoregulatory domain are listed first. (B) Candidate targeting molecules included cytoskeletal, nuclear, cytoplasmic, and mitochondrial proteins with identified roles in cell metabolism, cytoskeletal dynamics, and signaling. All CaMKII gene products (α, β, γ, and δ) were recognized by the screen; notably, only 1 known CaMKII-binding partner was identified (β2a). Candidates were cloned from human cDNA, and CaMKII-binding activity was assessed by in vitro binding assays using radiolabeled target proteins and activated CaMKII (CaMKII T287D). Notably, only clones underlined in red in A showed robust binding for CaMKII.

Figure 2. β_IV-spectrin is a CaMKII-binding protein in heart.

(A) β_IV-spectrin contains an N-terminal actin-binding domain (NTD), 17 spectrin repeats, and specific and C-terminal domains (SD/CTD). The putative CaMKII-binding site is denoted by an asterisk. (B) The putative CaMKII-binding domain in β_IV-spectrin was homologous to a CaMKIIδ autoregulatory domain motif and conserved across orthologs. (C) CTP-P bound radiolabeled CaMKIIδ; CTP-C and GST beads alone lacked binding. (D) β_IV-spectrin RNA levels in adult rat brain and heart. (E) β_IV-spectrin (Σ1 and Σ6) in ventricular lysates from multiple species. β_IV-spectrin was expressed approximately 8–10 fold higher in cerebellum than in heart. Cardiac β_IV-spectrin migrated approximately 4 kDa larger than did cerebellar β_IV-spectrin. (F) CTP-P, but not CTP-C, associated with CaMKIIδ from rat heart. (G) Endogenous CaMKIIδ and β_IV-spectrin coimmunoprecipitated from adult heart lysate. (H and I) β_IV-spectrin and N-cadherin in rat cardiomyocytes. Nuclei are shown by Topro-3 dye (blue). (J) CaMKIIδ localization in adult rat myocytes. CaMKIIδ localized to the intercalated disc (white arrows) here and to a second population at transverse-tubules (yellow arrows). Ventricular sections stained for (K) β_IV-spectrin, (L) N-cadherin, and (M) CaMKIIδ showed coexpression of these proteins at the intercalated disc (white arrows). (N and P) Ankyrin-G and (O and Q) Na_v1.5 were also found at the intercalated disc (white arrows) in rat myocytes and tissue sections. (R–U) Coimmunoprecipitation studies demonstrate cardiac complex of β_IV-spectrin, CaMKIIδ, ankyrin-G (AnkG), and Na_v1.5. Scale bars: 10 μm (H–Q).

Figure 3. β_IV-spectrin is required for CaMKIIδ targeting.

(A) β_IV-spectrin organization in WT and qv^3J animals. qv^3J animals lacked a CaMKII-binding domain, but retained actin-, ankyrin-, and α-spectrin–binding domains. (B and C) Schematic of control GST–β_IV-spectrin fusion protein encompassing spectrin repeats 13–17 and the CaMKII-binding domain (βIV-WT) and truncated mutant lacking CaMKII-binding domain (βIV-qv^3J). (D) β_IV-spectrin WT and qv^3J GST fusion proteins were incubated with detergent-soluble rat heart lysate and analyzed by immunoblot (CaMKIIδ). L-type Ca²⁺ channel β2a subunit was also expressed as a GST fusion protein and used as positive control. CaMKIIδ bound to the β2a and WT GST fusion proteins, but not to the qv^3J GST fusion protein. Lanes were run on the same gel but were noncontiguous (white line). (E) β_IV-spectrin WT and qv^3J GST fusion proteins retained binding activity for ankyrin-G. (F) Coimmunoprecipitation studies showing association of ankyrin-G and CaMKIIδ in WT, but not qv^3J, hearts. (G) Expression of ankyrin-G, Na_v1.5, CaMKIIδ, β-catenin, and N-cadherin in heart lysates from WT and qv^3J animals. Actin is shown as loading control. (H–Q) Permeabilized adult rat cardiomyocytes from (H–L) WT and (M–Q) qv^3J hearts were immunostained for (H and M) N-cadherin, (I and N) total CaMKIIδ, (J and O) CaMKII-phospho-T287, (K and P) β_IV-spectrin, and (L and Q) Na_v1.5. Localization of CaMKIIδ (total and phospho-T287) to the intercalated disc (white arrows) was disrupted in qv^3J cardiomyocytes. Scale bars: 10 μm (H–Q).

Figure 4. β_IV-spectrin/CaMKII complex is required for myocyte Na⁺ channel function.

(A and B) Whole-cell patch clamp I_Na traces from WT and qv^3J cardiomyocytes. Test pulse potential is listed next to each trace. (C) Current-voltage relationship for cardiomyocytes from WT, qv^3J, and AC3-I mice (n = 8 per group). *P < 0.05 versus WT. (D and E) Voltage-gated Na⁺ channel steady-state inactivation measured from WT, qv^3J, and AC3-I cardiomyocytes (n = 8 per group). *P < 0.05 versus WT. Pulse protocol is shown in the inset of D. (F and G) I_Na,p from WT and qv^3J mice with or without 10 μM mexiletine. Persistent current was determined as amplitude 50 ms after time of peak. (H and I) I_Na,p in WT (n = 10) and qv^3J (n = 17) cardiomyocytes at baseline and in the presence of 10 μm mexiletine or isoproterenol. *P < 0.05. (J) Whole-cell patch clamp Ca²⁺ current-voltage relationship for WT (n = 9) and qv^3J (n = 8) cardiomyocytes. (K) Ca²⁺ current facilitation (peak current in response to a train of depolarizing voltage pulses to 0 mV) in WT (n = 9) and qv^3J (n = 7) cardiomyocytes. Peak current is expressed as percent increase from first pulse. (L and M) Transverse-tubule CaMKII labeling was preserved in qv^3J mouse myocytes. Scale bars: 5 μm (L and M).

Figure 5. β_IV-spectrin/CaMKII regulates Na_v1.5 phosphorylation.

(A) CaMKII consensus phosphorylation sites in intracellular domains of Na_v1.5. (B and C) Na⁺ channel steady-state inactivation measured from WT and Na_v1.5 mutant channels expressed in HEK cells. *P < 0.05 versus WT. (D) Summary data showing depolarizing shift in steady-state inactivation V_1/2 following CaMKII activation for WT and all mutants except S571A. Black, before CaMKII activation; blue, after CaMKII activation. (E) CaMKII phosphorylation assay on intracellular domains of Na_v1.5. β2a was used as positive control. Asterisks denote location of purified proteins. (F and G) Na⁺ channel steady-state inactivation measured from WT, S571A, and S571E (phosphomimetic) channels expressed in HEK cells. *P < 0.05 versus WT. (H) Na_v1.5 S571 antibody recognized WT, but not Na_v1.5 S571A mutant, channels. WT and mutant channels were expressed at equivalent levels in HEK293 cells overexpressing active CaMKIIδ. (I) Immunoblots showing reduced levels of phospho–Na_v1.5 S571, but unchanged total Na_v1.5 levels, in qv^3J versus WT heart lysates. Actin is shown as loading control.

Figure 6. β_IV spectrin/CaMKII signaling complex is critical for normal cardiac cell membrane excitability.

(A and B) Representative APs and (C) APD₉₀ in WT (n = 14) and qv^3J (n = 16) cardiomyocytes paced at 4, 2, 1, or 0.5 Hz. *P < 0.05. (D) Differences in APD₉₀ were eliminated by application of 50 μM mexiletine, but exaggerated by 1 μM isoproterenol (pacing frequency, 0.5 Hz). n = 14 (WT); 16 (qv^3J). *P < 0.05. (E) Representative electrocardiograms recorded from Langendorff-perfused WT and qv^3J hearts and summary data for (F) QRS duration (G), QT interval at 90% repolarization (QT₉₀), and (H) RR interval for qv^3J versus WT. n = 10 (WT); 10 (qv^3J). *P < 0.05.

Figure 7. β_IV-spectrin/CaMKII complex regulates afterdepolarization formation in response to isoproterenol treatment.

Representative APs recorded from WT and qv^3J cardiomyocytes at baseline (A and D) and following application of 1 μM isoproterenol (B and E) or isoproterenol applied with 10 μM mexiletine (C and F). (G) Summary data showing percent cells displaying afterdepolarizations in WT and qv^3J at baseline (n = 10 per genotype) and following application of isoproterenol (n = 10 per genotype) or isoproterenol applied with mexiletine (n = 5 per genotype). *P < 0.05. (H) β_IV-spectrin–based complex targets CaMKII to effector proteins in excitable cells.

Figure 8. β_IV-spectrin targets CaMKII in neurons.

(A and B) β_IV-spectrin associated with both ankyrin-G and CaMKII from mouse cerebellar lysates. (C) CaMKII (red) was observed at AISs (white arrows) in WT, but not qv^3J, cerebellum. (D) AIS integrity (shown by positive ankyrin-G staining) was not markedly affected in qv^3J mice. (E) β_IV-spectrin (red) was present at qv^3J mouse AISs, but at reduced levels compared with WT cerebellum. Calbindin (blue) was used to label the Purkinje cell body in C–E. Scale bars: 10 μm (C–E).