Dynamic reciprocity of sodium and potassium channel expression in a macromolecular complex controls cardiac excitability and arrhythmia

Abstract

The cardiac electrical impulse depends on an orchestrated interplay of transmembrane ionic currents in myocardial cells. Two critical ionic current mechanisms are the inwardly rectifying potassium current (I(K1)), which is important for maintenance of the cell resting membrane potential, and the sodium current (I(Na)), which provides a rapid depolarizing current during the upstroke of the action potential. By controlling the resting membrane potential, I(K1) modifies sodium channel availability and therefore, cell excitability, action potential duration, and velocity of impulse propagation. Additionally, I(K1)-I(Na) interactions are key determinants of electrical rotor frequency responsible for abnormal, often lethal, cardiac reentrant activity. Here, we have used a multidisciplinary approach based on molecular and biochemical techniques, acute gene transfer or silencing, and electrophysiology to show that I(K1)-I(Na) interactions involve a reciprocal modulation of expression of their respective channel proteins (Kir2.1 and Na(V)1.5) within a macromolecular complex. Thus, an increase in functional expression of one channel reciprocally modulates the other to enhance cardiac excitability. The modulation is model-independent; it is demonstrable in myocytes isolated from mouse and rat hearts and with transgenic and adenoviral-mediated overexpression/silencing. We also show that the post synaptic density, discs large, and zonula occludens-1 (PDZ) domain protein SAP97 is a component of this macromolecular complex. We show that the interplay between Na(v)1.5 and Kir2.1 has electrophysiological consequences on the myocardium and that SAP97 may affect the integrity of this complex or the nature of Na(v)1.5-Kir2.1 interactions. The reciprocal modulation between Na(v)1.5 and Kir2.1 and the respective ionic currents should be important in the ability of the heart to undergo self-sustaining cardiac rhythm disturbances.

Fig. 1.

Reciprocal regulation of Na_V1.5 and Kir2.1 in ARVMs. (A and C) Na_V1.5 overexpression increases both I_Na and I_K1 densities. (A) Superimposed I_Na density/voltage relationships (5 mmol/L [Na⁺]_o) for Ad-GFP– (black; N = 1, n = 7) and Ad-Na_V1.5–infected (red; N = 2, n = 6) cells. (B) Voltage-dependent activation and inactivation (m_∞ and h_∞) curves for the data presented in A. (C) Superimposed I_K1 density/voltage relationship for Ad-GFP– (black; N = 3, n = 8) and Ad-Na_V1.5–infected (red; N = 3, n = 8) cells. (Inset) Magnification of the outward component of the I_K1 I–V relationship. (D and E) Kir2.1 overexpression increases both I_K1 and I_Na densities. (D) Superimposed I_K1 density/voltage relationships for Ad-GFP (black; N = 3, n = 8) and Ad-Kir2.1 (red; N = 2, n = 5). (E) Superimposed I_Na density/voltage relationships (20 mmol/L [Na⁺]₀) for Ad-GFP (black; N = 2, n = 6) and Ad-Kir2.1 (red; N = 4, n = 7). (F) Voltage-dependent activation and inactivation (m_∞ and h_∞) curves for the data presented in E. *P < 0.005; ^#P < 0.05; ^δP < 0.01 (unpaired t test with Welch’s correction). N represents the number of animals/litters, and n equals the number of experiments or cells.

Fig. 2.

Role of SAP97 in the reciprocal modulation of I_K1 and I_Na. (A and B) Ad-shSAP97 infection silences SAP97 expression in adult rat ventricular myocytes (ARVMs) by day 3 in culture compared with control. (A) Immunoblot showing immunodetection of SAP97 expression (Top and Middle) and GAPDH (loading control; Bottom) from cells infected with either a control (Ad-515) or shSAP97 (Ad-shSAP97) adenovirus. Middle (SAP97) shows a longer exposure of the same immunoblot shown in Top; the overexposure confirms expression of SAP97 in the Ad-SAP97–silenced cells and shows the contrasting intensities and apparent protein levels between control and SAP97-silenced ARVMs. (B) Densitometric analysis of SAP97 expression normalized to GAPDH levels in control (black) and shSAP97-silenced (red) cells. Values represent data (mean ± SEM) from two different preparations harvested 3 d after infection. SAP97 expression was effectively knocked down by ∼56% on day 3. (C) I_K1 density is reduced after SAP97 silencing in ARVMs. Peak inward current density at −100 mV (control: −7.67 ± 1.52 pA/pF; Ad-shSAP97: −4.55 ± 0.47 pA/pF) and peak outward current density at −60 mV (control: 1.03 ± 0.46 pA/pF; Ad-shSAP97: 0.34 ± 0.03 pA/pF) were significantly different (P = 0.02 and P = 0.04, respectively) between myocytes infected with shSAP97 (N = 4, n = 11) and myocytes infected with Ad-515 (control; N = 2, n = 6). Inset shows the protocol used to measure the current. (D) Effects of SAP97 knockdown on sodium channel current density in ARVMs. Superimposed I_Na density/voltage relationships in control (Ad-515) and SAP97-silenced (Ad-shSAP97) cells. A significant reduction in I_Na density was observed for SAP97-silenced cells at several tested voltages. For both control and silenced conditions, N = 2 and n = 11. (Inset) Voltage clamp step protocol. (E) Voltage-dependent activation and inactivation (m_∞ and h_∞) curves for the data presented in D. *P < 0.005; ^#P < 0.05; ^δP < 0.01 (unpaired t test with Welch’s correction).

Fig. 3.

SAP97 immunolocalizes and immunoprecipitates with Na_V1.5 and Kir2.1 in the rat ventricle. (A) Immunohistochemical analysis and localization for SAP97, Na_V1.5, and Kir2.1 in the adult rat heart. SAP97 localization with Na_V1.5 (a–c) and Kir2.1 (f–h) channels and Na_V1.5 (k and n) localization with Kir2.1 channels (l and o). Double arrows denote a staining pattern reminiscent of a t-tubular distribution. *Intercalated disk. d and e, i and j, and n and o are magnifications of the boxed areas from the merged images shown in c, h, and m, respectively. SAP97 staining is presented in the enlarged d and i images, Na_V1.5 is presented in e and n, and Kir2.1 is presented in j and o; images are shown in black and white for clarity. (Scale bars: 20 μm.) SAP97 (B, Top), Na_V1.5 (C, Top), and Kir2.1 (D, Top) are detected by Western blot after immunoprecipitation with specific antibodies raised to the respective protein, which shows that each immunoprecipitation is specific for the desired protein. Na_V1.5 coimmunoprecipitates with SAP97 (B, Middle), and SAP97 coimmunoprecipitates with Na_V1.5 in the reverse reaction (C, Middle). Kir2.1 coimmunoprecipitates with SAP97 antibodies (B, Bottom), and SAP97 coimmunoprecipitates with Kir2.1 in the reverse reaction (D, Bottom). Na_V1.5 and Kir2.1 each coimmunoprecipitate with the other ion channel protein (C, Bottom and D, Middle, respectively). All (co)immunoprecipitation reactions used membrane-enriched preparations generated from the ventricles of rat heart. IB, antibody used for immunoblotting; IP, antibody used for immunoprecipitation.

Fig. 4.

Biophysical characterization of sodium current in Kir2.1 OE mice. (A) Superimposed I_Na density/voltage relationships in WT (black) and Kir2.1 OE (red) mice. (Inset) Representative examples of I_Na traces in each group. Dotted line denotes 0 pA. WT: N = 4, n = 10; Kir2.1 OE: N = 2, n = 6. *P < 0.005 (unpaired t test with Welch’s correction). (Right) Voltage-dependent activation and inactivation (m∞ and h∞) curves for the data presented in A. (B) The unitary conductance properties of sodium channels were characterized in ventricular myocytes isolated from the Kir2.1 OE mouse. Representative traces from control (littermate WT) and Kir2.1 OE mouse myocytes are illustrated in Inset. Histogram of the unitary events from WT littermates (N = 3, n = 240, seven patches) and Kir2.1 OE mice (N = 3, n = 231, eight patches) are superimposed and plotted.

Fig. 5.

Relative levels of SAP97 and Na_V1.5 are significantly increased in hearts of transgenic mice overexpressing Kir2.1. Crude membrane vesicles were prepared from the ventricles of control (WT) and Kir2.1 OE mice. Samples (16 μg/lane) were analyzed by SDS/PAGE and immunoblotted using specific antibodies for SAP97 or Na_V1.5, as indicated. (A) Representative immunoblots after detection of protein immunoreactivity with HRP-conjugated secondary antibodies and chemiluminescence (Upper). The corresponding Amido Black nitrocellulose (protein stain) is shown in Lower to show analysis of equal total protein. Protein concentrations were also verified by Lowry assay. (B) Densitometric analysis of data shown in A comparing relative protein levels between WT and Kir2.1 OE mice. Results are expressed as mean signal intensity, and they represent data from three animals for each genotype (N = 3 per genotype, ^δP < 0.01, mean ± SEM).

Fig. 6.

Transgenic reduction of Kir2.1 gene expression leads to a significant decrease in relative protein levels of Na_V1.5 and SAP97. Crude membrane vesicles were prepared from the ventricles of control (WT) and Kir2.1^−/+ mice. Fig. 5 has methods describing SDS/PAGE and immunoblotting. (A) Representative immunoblots after detection of protein immunoreactivity with HRP-conjugated secondary antibodies and chemiluminescence (Upper). The corresponding Amido Black nitrocellulose (protein stain) is shown in Lower to show analysis of equal total protein. Protein concentrations were also verified by Lowry assay. (B) Densitometric analysis comparing relative protein levels between WT and Kir2.1^−/+ mice. Results are expressed as mean signal intensity (N = 7 per genotype, ^#P < 0.05, mean ± SEM).

Fig. 7.

Coexpression of Kir2.1-pHluorin with Na_V1.5 promotes cell surface expression of Kir2.1-pHluorin. Quantification of steady-state cell surface Kir2.1-pHluorin in cells cotransfected/coinfected with either empty pcDNA3.1 and DsRed vectors or plasmids/adenoviruses encoding Na_V1.5 and DsRed. Cells were live cell labeled with anti-GFP antibody before incubation for 20 min at 37 °C. Cell surface channels were then labeled as described in SI Materials and Methods. Representative images of surface Kir2.1-pHluorin channels for each condition and cell type are shown. (Scale bars: 10 μm.) *P < 0.005 as determined by unpaired Student t test. (A) Rat neonatal ventricular myocytes (n > 58 myocytes). (B) ARVMs (n > 75 cells). (C) HL-1 cells (n > 56 cells). DNA plasmid transfection was used for rat neonatal ventricular myocytes and HL-1 cells; adenoviral-mediated overexpression was used for ARVMs.

Fig. 8.

Deletion of the PDZ domain of Kir2.1-pHluorin abolishes the Na_V1.5-mediated increase in cell surface channel. (A and B) Coexpression with Na_V1.5 reduces internalization of Kir2.1-pHluorin in HL-1 cells. HL-1 cells expressing Kir2.1-pHluorin were cotransfected with either empty pcDNA3.1 and DsRed vectors or plasmids encoding Na_V1.5 and DsRed. Representative images of surface channels for each of these conditions are shown. Quantification of surface (A) and internalized (B) Kir2.1-pHluorin in HL-1 cells coexpressing either pcDNA3.1 + DsRed or Na_V1.5 + DsRed. (C) Quantification of steady-state cell surface Kir2.1 in HL-1 cells expressing WT Kir2.1, Kir2.1ΔPDZ, or Kir2.1ΔPDZ + Na_V1.5. The absence of an intact PDZ binding consensus sequence significantly decreased cell surface expression of Kir2.1ΔPDZ compared with WT Kir2.1-pHluorin. The reduced surface expression of Kir2.1ΔPDZ was not rescued by concomitant coexpression with Na_V1.5. Cell surface labeling (Materials and Methods and Fig. 7) was conducted 48 h posttransfection (n ≥ 55 cells). Representative images of surface channel for each experimental condition are shown in the indicated panels. (Scale bars: 10 μm.) *P < 0.005; ^#P < 0.05 as determined by unpaired Student t test.

Fig. 9.

Na_V1.5 and Kir2.1 cooverexpression significantly reduces the APD in single NRVMs. (A–D) Action potential recordings from NRVMs infected with adenoviruses encoding (A) GFP (n = 5), (B) Na_V1.5 + GFP (n = 4), (C) Kir2.1 + GFP (n = 4), or (D) Kir2.1 + Na_V1.5 (n = 5) paced at 2 Hz. (E) Summary plots of mean ± SEM. APD₈₀ for each group. Ad-GFP was used as an adenoviral control for single infections to account for the higher viral load expected as a result of Kir2.1 and Na_V1.5 coinfection (red bar). ^#P < 0.05; ^δP < 0.01.

Fig. 10.

Molecular Na_V1.5–Kir2.1 interactions modulate reentry frequency in NRVM monolayers. (A) Phase maps (3, 17, 18) for single rotations from representative optical mapping movies of monolayers infected with Ad-GFP, Ad-Na_V1.5, Ad-Kir2.1, or Ad-Kir2.1 + Ad-Na_V1.5. The color bar indicates the phase in the excitation–recovery cycle. (B) Reentry frequencies in monolayers infected with Ad-GFP (black; n = 11), Ad-Na_V1.5 (blue; n = 13), Ad-Kir2.1 (yellow; n = 11), or Ad-Kir2.1 + Ad-Na_v1.5 (red; n = 13). ^δP < 0.01 (ANOVA).

Fig. P1.

Na_V1.5 and Kir2.1 form a macromolecular complex (a channelosome). The subcellular localization and channel activity of both Na_V1.5 and Kir2.1 are regulated by protein–protein interactions by their respective carboxyl terminal (CT) PDZ binding motifs with such PDZ domain-containing proteins as SAP97 and syntrophin. The CTs of one Na_V1.5 and Kir2.1 molecule each may bind to the same SAP97 molecule but at different PDZ domains. We propose that these interactions result in changes in the expression of Na_V1.5 and/or Kir2.1 and thereby, influence their function in the cell membrane. GK, guanylate kinase-like domain of SAP97; SE/AI, last 3 aa of the Kir2.1 CT, which can be serine and glutamic acid or alanine and isoleucine; SH3, src kinase homology domain of SAP97; SIV, serine, isoleucine, valine (last 3 aa of the NA_V1.5 CT).