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Article |
Voltage-gated Nav channel targeting in the heart requires an ankyrin-G–dependent cellular pathway
Correspondence to Peter J. Mohler: peter-mohler{at}uiowa.edu
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.
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Introduction |
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In addition to normal biophysical properties, Nav1.5 activity requires proper localization at specialized cardiomyocyte membrane domains. In the vertebrate heart, Nav1.5 is primarily concentrated at the cardiomyocyte intercalated disc (Kucera et al., 2002; Maier et al., 2002; Mohler et al., 2004b). However, Nav1.5 localization at the peripheral sarcolemma and T tubules has also been described previously (Cohen, 1996; Scriven et al., 2000, 2002; Kucera et al., 2002). The molecular mechanisms underlying the targeting and retention of Nav1.5 in cardiac tissue are unknown. Over the past decade, several potential Nav1.5 intermolecular interactions have been hypothesized to regulate Nav1.5 membrane trafficking, stability, and removal (Herfst et al., 2004; Abriel and Kass, 2005). However, the roles for these protein interactions in the context of a cardiomyocyte are unclear.
Ankyrin-G is an adaptor protein required for the targeting of diverse membrane proteins in the central nervous system (Mohler and Bennett, 2005b). Specifically, ankyrin-G is required for the targeting of Nav channel isoforms (Nav1.2 and Nav1.6) to specialized excitable membrane domains (axon initial segments and nodes of Ranvier) in Purkinje and granule cell neurons (Zhou et al., 1998; Jenkins and Bennett, 2001; Garrido et al., 2003; Lemaillet et al., 2003). Mice lacking ankyrin-G expression in the cerebellum display a loss of Nav channel targeting, abnormal neuronal action potentials, and ataxia (Zhou et al., 1998; Jenkins and Bennett, 2001). In 2003, two independent groups identified a small binding motif in the Nav1.2 DII–DIII cytoplasmic loop required for ankyrin-G association (Garrido et al., 2003; Lemaillet et al., 2003).
Our group recently identified 190-kD ankyrin-G expression in the heart (Mohler et al., 2004b). Like Nav1.5, ankyrin-G is expressed at the intercalated disc and transverse tubules and associates with Nav1.5 in coimmunoprecipitation and in vitro binding experiments (Mohler et al., 2004b). The nine–amino acid ankyrin-binding sequence identified in Nav1.2 (Garrido et al., 2003; Lemaillet et al., 2003) is present in Nav1.5 and is required for Nav1.5–ankyrin-G interaction (Mohler et al., 2004b). A human Brugada Syndrome SCN5A variant in the Nav1.5 ankyrin-G–binding motif (E1053K) abolishes ankyrin-G–Nav1.5 interaction, and this Nav channel mutant is not efficiently targeted to cardiomyocyte membranes (Mohler et al., 2004b). Although largely circumstantial, these data support a potential role for an ankyrin-G–dependent pathway in Nav1.5 targeting to excitable membrane domains in the heart.
In this study, we report new data that conclusively links ankyrin-G activity and Nav1.5 membrane expression and localization in cardiomyocytes. Using viral-mediated small hairpin RNA (shRNA) transfer into primary cardiomyocytes, we demonstrate that a full complement of ankyrin-G expression is required for Nav1.5 expression and membrane localization. Specifically, reduced ankyrin-G expression decreases (1) total cellular Nav1.5 expression, (2) efficient membrane localization, and (3) total Na+ membrane current. We also demonstrate that although ankyrin-G is required for normal Nav1.5 membrane expression, reduced ankyrin-G expression does not affect Nav1.5 channel kinetics. Finally, we report the structural requirements for direct ankyrin-G–Nav1.5 interactions and show that direct intermolecular interaction between these two molecules is required for efficient channel membrane localization. Together, these data identify the first clear cellular pathway for Nav channel trafficking in the heart.
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Ankyrin-G is required for normal Nav1.5 expression and localization
Rat cardiomyocytes lacking a full complement of ankyrin-G expression were used to test the role of ankyrin-G for Nav1.5 expression. As shown in Fig. 1, viral transduction of ankyrin-G shRNA significantly reduces ankyrin-G expression (transduction efficiency of 80–95% based on YFP fluorescence; Fig. 2).
Moreover, a striking reduction in Nav1.5 protein levels was observed in the identical cell lysates from rat-specific ankyrin-G shRNA virally transduced myocytes (Fig. 2). This reduction was specific to Nav1.5, as expression differences in cardiomyocyte membrane–associated proteins, including NHERF1, connexin43, ankyrin-B, Cav1.2 (Fig. 2), SERCA2, Na/K ATPase, or Na/Ca exchanger (not depicted), were not observed.
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and S1) establish that the cardiac ankyrin-G–targeting pathway is specific for Nav1.5 channels and that loss of Nav1.5 membrane expression in ankyrin-G–deficient cells is not simply the result of the general impairment of default myocyte membrane biogenesis/trafficking pathways.
Nav1.5 channel inactivation is unaffected in cardiomyocytes with reduced ankyrin-G expression
Human Nav1.5 Brugada Syndrome variant E1053K, which lacks ankyrin-G–binding activity, is not efficiently targeted to the cardiomyocyte intercalated disc (Mohler et al., 2004b). However, when introduced into heterologous HEK293 cells, this mutant channel is present at the plasma membrane but displays abnormalities in Nav1.5 inactivation (Mohler et al., 2004b). Furthermore, similar data examining Nav1.2 biophysical properties in TsA201 cells support the concept that ankyrin-G could potentially regulate the biophysical properties of Nav channels in heterologous cells (Shirahata et al., 2006). These studies did not determine whether the inactivation of wild-type Nav1.5 channels requires correct localization through ankyrin-G. Therefore, we tested whether primary cardiomyocytes with reduced ankyrin-G expression displayed abnormalities in Nav channel inactivation. No significant difference in Nav1.5 channel inactivation was observed in wild-type cardiomyocytes versus cardiomyocytes with reduced ankyrin-G expression (Fig. 6).
These data demonstrate that in the physiological context of a cardiomyocyte, normal Nav channel inactivation does not require ankyrin-G. Surprisingly, inactivation of wild-type cardiomyocyte Nav1.5 is independent of physiological localization. Thus, localization controls current density but not Nav1.5 inactivation. These data strongly demonstrate the critical nature of studying ankyrin biology in the appropriate physiological context.
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helices connected by a β-hairpin loop. The large number of consecutive ANK repeats come together to form a super helix that surrounds a large central cavity (Michaely et al., 2002). Thus, these variable β-hairpin loops are highly accessible to the solvent for potential protein interactions (Fig. 7 C). This strategy has been successfully used to map the binding sites on ankyrin-B for Na/Ca exchanger and InsP3 receptor (Mohler et al., 2004a; Cunha et al., 2007).
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Although the majority of ankyrin-G mutants associated with Nav1.5 His-tagged DII–DIII at levels similar to wild-type GFP–ankyrin-G, there was a significant reduction in DII–DIII binding to ankyrin-G mutants R13, R14, and R15 (Fig. 7, D and E). In fact, GFP–ankyrin-G mutants R14 and R15 displayed a near complete loss of Nav1.5 DII–DIII binding activity in these assays. Together, our findings demonstrate that ankyrin-G–Nav1.5 binding is exclusively dependent on three critical elements (two β-hairpin loops on ankyrin-G and a nine–amino acid motif on Nav1.5; Mohler et al., 2004b).
Human ankyrin-G expression rescues abnormal Nav1.5 localization
We performed rescue assays to determine whether the loss of Nav1.5 localization in cardiomyocytes with reduced ankyrin-G expression was specifically caused by the monogenic loss of ankyrin-G. Rat neonatal cardiomyocytes with reduced levels of ankyrin-G and Nav1.5 (as a result of the presence of rat-specific ankyrin-G shRNA virus) were transfected with cDNA encoding human GFP–ankyrin-G. This GFP–ankyrin-G cDNA is resistant to the rat ankyrin-G shRNA (Fig. 1). As expected, shRNA-transduced cardiomyocytes displayed decreased expression and abnormal localization of Nav1.5 (Fig. 8 B).
In contrast, shRNA-transduced cardiomyocytes expressing human GFP–ankyrin-G cDNA displayed a Nav1.5 distribution similar to wild-type cardiomyocytes (Fig. 8 C). These data demonstrate that abnormal Nav1.5 localization in ankyrin-G–null cardiomyocytes can be rescued by the exogenous expression of human ankyrin-G and reinforce the critical role for ankyrin-G in the subcellular localization of Nav1.5 in cardiomyocytes.
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Ankyrin-G is required for normal Nav1.5 expression in adult cardiomyocytes
To test whether our findings in neonatal myocytes can also be applied to adult cardiomyocytes, we performed ankyrin-G shRNA viral transduction of freshly isolated adult rat cardiomyocytes. Nav1.5 expression is most pronounced at the intercalated disc of adult cardiomyocytes (see Nav1.5 localization in the control myocyte; Fig. S2 A, available at http://www.jcb.org/cgi/content/full/jcb.200710107/DC1). Therefore, we tested whether ankyrin-G expression is required for Nav1.5 localization at the mature cardiomyocyte intercalated disc. Reduced ankyrin-G expression in adult myocytes results in the reduced membrane expression of Nav1.5 (note the loss of Nav1.5 intercalated disc staining in Fig. S2 B), which is consistent with results in neonatal cardiomyocytes. In fact, identical with our findings in neonatal cells, we observed that Nav1.5 localized to the perinuclear region (Fig. S2 B) in adult cardiomyocytes with reduced ankyrin-G expression. These data from isolated adult cardiomyocytes further confirm the role of the ankyrin-G–based protein-targeting pathway for Nav1.5 membrane expression in the heart.
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Discussion |
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Ankyrin polypeptides have likely coordinately evolved to regulate electrical activity in the heart by targeting key ion channels/transporters involved in controlling the cardiac action potential. Ankyrin-G directly associates with and targets Nav1.5 to the membrane surface (Fig. 9 A) to regulate inward Na+ current and, thus, action potential initiation and cardiomyocyte depolarization. Therefore, it is not surprising that human SCN5A variants that disrupt ankyrin-G interactions are associated with the Brugada Syndrome (Priori et al., 2002; Mohler et al., 2004b), a cardiac syndrome associated with reduced inward INa (for review see Napolitano and Priori, 2006). Clinical features of the Brugada Syndrome include fast polymorphic ventricular tachycardia typically occurring at rest or during sleep (Wilde and Priori, 2000). In contrast, ankyrin-B targets distinct ion channels and transporters with central roles in cytosolic calcium extrusion during cardiac repolarization (Mohler et al., 2002, 2003, 2004c, 2005, 2007; Cunha et al., 2007). Ankyrin-B (product of the ANK2 gene) is required for targeting Na/Ca exchanger, Na/K ATPase, and InsP3 receptor to the transverse tubule and sarcoplasmic reticulum in cardiomyocytes (Fig. 9 B; Mohler et al., 2003, 2005).
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In addition to targeting ion channels in cardiomyocytes, ankyrin-B was recently shown to be required for targeting of the regulatory subunit of the PP2A complex in primary cardiomyocytes (Fig. 9 B; Bhasin et al., 2007). This broad targeting role of ankyrin-B for both integral membrane and signaling proteins in the heart suggests that the ankyrin-G–targeting pathway may similarly facilitate the localization of additional myocyte proteins. Moreover, our new findings for ankyrin-G in Nav1.5 targeting identify an attractive, unconventional candidate disease gene for cardiac (arrhythmia and myopathy) and other excitable cell diseases.
Based on the role of ankyrin-R in the erythrocyte (Bennett and Stenbuck, 1979), cardiac ankyrin-G may simply act as a membrane scaffold to link integral membrane proteins (such as Nav1.5) with the underlying actin- and spectrin-based cytoskeleton. In support of this role, we observe a significant level of Nav1.5 clustering on the plasma membrane surface with ankyrin-G as assessed by immunoelectron microscopy of cardiomyocyte membrane sheets (Fig. 10). Alternatively, ankyrin-G may play an active role in the cellular trafficking of Nav1.5 to specific membrane domains. In theory, ankyrin-G could have multiple roles in both trafficking and stabilization/retention of Nav1.5 channels in the heart. Although these findings are beyond the scope of this study, elucidating the specific cellular roles of ankyrin polypeptides in excitable cells is an obvious future goal for the field.
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Our electrophysiological measurements illustrate the importance of studying ankyrin–membrane protein interactions in the physiological context of a native cell. Our previous work suggested that Nav1.5 E1053K lacking ankyrin-G–binding activity displayed minor yet significant abnormalities in activation and inactivation when expressed in HEK293 cells (Mohler et al., 2004b). However, when expressed in native cardiomyocytes, these biophysical abnormalities were inconsequential, as the majority of the channel lacked sufficient targeting information to even reach the plasma membrane (Mohler et al., 2004b). Our new findings demonstrate that in myocytes lacking ankyrin-G, the small number of residual Nav1.5 channels that reach the plasma membrane have effectively normal biophysical characteristics. These findings are in contrast to a recent publication that demonstrated abnormal inactivation gating of Nav1.2 in TsA201 cells (modified HEK293 cell) lacking ankyrin-G (Shirahata et al., 2006). Our new data suggest that these measurements should be reevaluated in the context of a neuron.
The critical role of ion channels and transporters for normal cardiac function has been highlighted by the linkage of gene mutations in cardiac ion channels and associated subunits with human arrhythmia (Lehnart et al., 2007). Specifically, human gene variants in KCNQ1 (KvLQT1), KCNH2 (HERG), SCN5A (Nav1.5), KCNE1 (minK), KCNE2 (MiRP), KCNJ2 (Kir2.1), and CACNA1C (Cav1.2) have been associated with potentially fatal long QT arrhythmias (Lehnart et al., 2007). Most channel variants affect channel biophysics (Lehnart et al., 2007). However, recent findings suggest that dysfunction in channel trafficking mechanisms may explain a significant number of long QT variant cellular phenotypes (Zhou et al., 1999; Mohler et al., 2003; Ye et al., 2003; Gouas et al., 2004; Krumerman et al., 2004; Liu et al., 2005; Anderson et al., 2006; Ballester et al., 2006; Schmitt et al., 2007).
Cardiomyocytes have clearly evolved unique channel trafficking pathways for the precise localization of specific ion channels to unique cardiomyocyte membrane domains. For example, although Nav1.5, Kv4.2, and connexin 43 are concentrated at the intercalated disc (Kanter et al., 1992; Barry et al., 1995; Maier et al., 2002; Mohler et al., 2004b), Kir2.1, Kir2.3, and Cav1.2 are primarily localized to transverse tubule membranes (Carl et al., 1995; Sun et al., 1995;Clark et al., 2001; Melnyk et al., 2002). Moreover, significant populations of KvLQT1, ERG, minK, MiRP, Na/Ca exchanger, and Na/K ATPase are found at transverse tubule and peripheral sarcolemma membranes (Frank et al., 1992; Kieval et al., 1992; McDonough et al., 1996; Pond et al., 2000; Rasmussen et al., 2004; Wu et al., 2006). Finally, high resolution imaging techniques have revealed unique transverse tubule membrane domains that further segregate cardiac ion channel signaling complexes (Scriven et al., 2000). Clearly, the molecular and structural characteristics of cardiac membrane domains represent a central feature for the regulation of local ion channel pathways and represent a relatively unexplored field in cell biology.
Ankyrin-based ion channel trafficking pathways have unique roles in specific cells. Recently, Pan et al. (2006) demonstrated the requirement of ankyrin-G association for the targeting of KCNQ2 and KCNQ3 (encode axonal M currents responsible for stabilizing neuronal resting potential) to axon initial segments. Specifically, ankyrin-G cerebellar-specific null mice display loss of KCNQ2/3 clustering at axonal initial segments (Pan et al., 2006). Moreover, the ankyrin-G–binding motif originally described in Nav1.2 and Nav1.5 (Garrido et al., 2003; Lemaillet et al., 2003; Mohler et al., 2004b) is present in the C-terminal domain of KCNQ2/3 and is required for neuronal KCNQ2/3 targeting (Chung et al., 2006; Pan et al., 2006; Rasmussen et al., 2007). Interestingly, although nearly structurally identical, KCNQ1 (cardiac KvLQT1) lacks the C-terminal ankyrin-G–binding motif (Pan et al., 2006). Consistent with this data, ankyrin-G and KCNQ1 are differentially targeted in ventricular cardiomyocytes (Mohler et al., 2004b; Rasmussen et al., 2004). Therefore, these data strongly suggest that ankyrin-based pathways operate in a cell type–specific manner.
In summary, our new findings show that ankyrin-G is the physiological binding partner for Nav1.5 in cardiomyocytes. Loss of direct interaction between ankyrin-G and Nav1.5 results in abnormal Nav1.5 channel localization in primary cardiomyocytes. Moreover, the loss of ankyrin-G expression affects Nav1.5 expression, membrane localization, and, therefore, whole cell Nav1.5 activity. Based on recent ankyrin findings in the brain (Dubreuil, 2006; Pan et al., 2006; Yang et al., 2007), heart (Lencesova et al., 2004; Mohler and Bennett, 2005a), and skeletal muscle (Bagnato et al., 2003; Kontrogianni-Konstantopoulos et al., 2004, 2006), our new findings likely represent only the first component of a larger ankyrin-G macromolecular signaling complex at cardiomyocyte excitable membrane domains.
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Immunoblots
After viral and/or rescue treatment, cells were collected into PBS, pH 7.4. Cells were lysed using radioimmunoprecipitation assay buffer containing protease inhibitor cocktail. Detergent-soluble fractions were collected after high speed centrifugation, and protein concentrations were determined using a bicinchoninic acid protein assay kit (Thermo Fisher Scientific). Equal quantities of protein (20 µg) were analyzed by SDS-PAGE and immunoblotting. Equal loading was assessed by transfer to Ponceau S and blotting with NHERF1.
Neonatal rat cardiomyocytes
Hearts were dissected from postnatal day 1 rats and cultured as described previously (Bhasin et al., 2007).
Adult cardiomyocytes
Myocytes were isolated from 250–300 g of Sprague-Dawley rats (Grueter et al., 2006). Acutely isolated cardiomyocytes were then plated at high density (
2 x 106 per well), infected with rat-specific ankyrin-G shRNA virus or control virus for 9 h, and maintained for 22 h at 37°C before experiments.
Binding experiments
GFP 190-kD ankyrin-G and mutants were expressed in HEK293 cells and purified using affinity-purified GFP Ig coupled to protein A agarose beads. Cells were lysed in homogenization buffer plus 1.0% Triton X-100 and 0.5% deoxycholate (Mohler et al., 2005). The extract was centrifuged at 100,000 g, and the supernatant was incubated with GFP Ig coupled to protein A–Sepharose. Nav1.5 DII–DIII cytoplasmic loop was subcloned into pET15b for expression and purification as a hexahistidine fusion protein. Purified proteins were incubated with GFP or control Ig coupled to protein A–Sepharose. Protein bound to each mutant GFP 190-kD ankyrin-G was eluted, analyzed by immunoblotting, normalized for relative GFP ankyrin-G expression, and compared with wild-type GFP 190-kD ankyrin-G binding.
Statistics
When appropriate, data were analyzed using a two-tailed t test, and P < 0.05 was considered significant. Values are expressed as the mean ± SD.
Virus generation
Ankyrin-G shRNAs were engineered into the pFIV lentiviral vector and packaged into viral pseudoparticles (System Biosciences, Inc.). Constructs were cotransfected with packaging plasmids into HEK293 cells using Effectene. The pseudoparticle-containing supernatant was concentrated using Centriplus YM-30 columns. The concentrate was stored at –80°C.
Immunostaining/confocal microscopy
Cardiomyocytes were isolated, cultured, and processed for immunofluorescence as described previously (Mohler et al., 2003; Cunha et al., 2007). Secondary antibodies included anti–rabbit and anti–mouse Igs conjugated to AlexaFluor488 or 568 (Invitrogen). Phalloidin-conjugated AlexaFluor633 was used for double-labeling experiments in Fig. S1. After secondary antibody treatment, cells were extensively washed, covered with Vectashield imaging medium (Vector Laboratories), and coverslips (#1) were applied. Images were collected on a confocal microscope (510 Meta; Carl Zeiss, Inc.) with a 63x oil 1.40 NA or 40x oil 1.30 NA lens (pinhole equals 1.0 airy disc; Carl Zeiss, Inc.) using imaging software (release version 4.0 SP1; Carl Zeiss, Inc.). Images were collected using similar confocal protocols at room temperature. In experiments in which both AlexaFluor488 and YFP were analyzed, the emission signal from AlexaFluor488 was collected from 500 to 515 nm, and the emission from YFP was collected from 530 to 565 nm. Because YFP was only used to identify virus-positive cells (versus identifying the localization of a YFP fusion protein), we used minimal laser power and detector gain (thus the minimal YFP image resolution) to collect YFP images to prevent potential signal bleed-through to the AlexaFluor488 image, in which protein immunolocalization was crucial. For images in Figs. 3, Figs.8, and S1, the YFP image was pseudocolored. Images were imported into Photoshop CS (Adobe) for cropping and linear contrast adjustment.
Plasma membrane sheet preparation/immunoelectron microscopy
Ventricular cardiac myocytes from adult Sprague-Dawley rats were obtained as described previously (Shibata et al., 2006). Immunolabeling of the membrane sheets was performed by placing the grids on drops of primary antibody solution for 1 h on ice and rinsing six times for 5 min in PBS containing BSA. Grids were then incubated on drops of gold-conjugated secondary antibody solution (5 nm of goat anti–rabbit diluted 1:50 or 10 nm of goat anti–mouse diluted 1:50 in PBS containing BSA; Electron Microscopy Sciences) for 1 h on ice. Grids were then rinsed three times for 5 min in PBS containing BSA followed by a 2-min fixation in 2.5% glutaraldehyde. Finally, the grids were washed for 5 min in deionized water before being allowed to air dry. Negative controls included grids labeled with secondary antibody alone. Grids were visualized on a transmission electron microscope (H-70000; Hitachi).
Antibodies
Antibodies used include anti-Nav1.5 (Alomone Laboratories), anti-NCX1 (Swant), anti–connexin43 (Invitrogen), anti-Cav1.2 (Affinity BioReagents), anti-NHERF1 (Sigma-Aldrich), affinity-purified Igs against ankyrin-B, ankyrin-G, and GFP (monoclonal and polyclonal), and goat anti–rabbit AlexaFluor568 (Invitrogen).
Electrophysiology experiments
Voltage-dependent Na+ and Ca+2 currents were measured using standard patch clamp techniques. Whole-cell currents were recorded with an amplifier (Axopatch 200B; MDS Analytical Technologies), and the analogue signal was filtered using an eight-pole filter (Bessel) with a bandwidth of 5 kHz and was digitized at a sampling rate of 50 kHz. Borosilicate glass capillaries (VWR Scientific) were used to fabricate patch pipettes. Electrode resistances ranged from 1 to 1.5 M
, and seal resistances were 1–5 G. Pipette seal resistances were compensated to >85% of the uncompensated value. The whole-cell bath solution contained 10 mM NaCl, 130 mM choline chloride, 4.5 mM KCl, 1.8 mM CaCl2, 2.0 mM MgCl2, 10.0 mM Hepes, and 5.5 mM glucose, pH 7.35, titrated with KOH. The pipette solution contained 130 mM CsCl, 0.5 mM CaCl2, 2 mM MgCl2, 5 mM Na2ATP, 0.5 mM GTP, 5 mM EGTA, and 10 mM Hepes, pH 7.3, titrated with CsOH. All electrophysiology experiments were performed at room temperature (21–23°C).
Whole-cell voltage clamp Na+ current data were elicited from a holding potential of –120 mV to membrane potentials ranging from –110 to 30 mV in the presence of 2.0 mM CoCl2. Voltage-dependent steady-state inactivation was determined using a paired two-pulse protocol. Each conditioning voltage was paired with a control after 1.5 s. A 500-ms conditioning pulse from –120 to 20 mV in 10-mV increments was followed by a test pulse to –30 mV. The test pulse in each series was separated form the conditioning pulse by a 2-ms interval to –120 mV. The steady-state inactivation curves were constructed by normalizing currents to the maximal Na+ current elicited from a holding potential of –120 to –30 mV for a duration of 20 ms. The resulting curve was fitted using a Boltzmann distribution equation of the form INa = INa, max/(1 + exp([Vm – V1/2]/k)), where Vm is the conditioning pulse voltage, V1/2 is the voltage at half-inactivation, and k is the slope factor. The whole-cell voltage clamp protocol showing INa and ICa in Fig. 5 F was elicited using the two-pulse paradigm shown in Fig. 5 G. From a holding potential of –100 mV, INa was elicited by a 30-ms pulse to –30 mV. The membrane potential was then hyperpolarized to –70 mV for 10 ms. ICa was then elicited by a 50-ms pulse to 0 mV. This protocol allowed the simple differentiation of INa from ICa as indicated by the unique ICa and INa current signatures in Fig. 5. All presented currents were normalized for cell capacitance. Data were collected and analyzed using pCLAMP 9.0 software (MDS Analytical Technologies) and OriginPro 7.5 (OriginLab Corp.). Analysis of variance was used to compare the nominal change in current density among the means. Statistical significance is defined as P < 0.05.
Online supplemental material
Fig. S1 demonstrates that cardiomyocytes with reduced ankyrin-G expression display normal localization and distribution of Cav1.2 and Na/Ca exchanger. Fig. S2 demonstrates that a full complement of ankyrin-G expression is required for normal Nav1.5 expression in adult rat cardiomyocytes. Online supplemental material is available at http://www.jcb.org/cgi/content/full/jcb.200710107/DC1.
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Acknowledgments |
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Submitted: 16 October 2007
Accepted: 11 December 2007
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