Role for myosin-V motor proteins in the selective delivery of Kv channel isoforms to the membrane surface of cardiac myocytes

Abstract

Rationale: Kv1.5 (KCNA5) mediates the ultra-rapid delayed rectifier current that controls atrial action potential duration. Given its atrial-specific expression and alterations in human atrial fibrillation, Kv1.5 has emerged as a promising target for the treatment of atrial fibrillation. A necessary step in the development of novel agents that selectively modulate trafficking pathways is the identification of the cellular machinery controlling Kv1.5 surface density, of which little is yet known.

Objective: To investigate the role of the unconventional myosin-V (MYO5A and MYO5B) motors in determining the cell surface density of Kv1.5.

Methods and results: Western blot analysis showed MYO5A and MYO5B expression in the heart, whereas disruption of endogenous motors selectively reduced IKur current in adult rat cardiomyocytes. Dominant negative constructs and short hairpin RNA silencing demonstrated a role for MYO5A and MYO5B in the surface trafficking of Kv1.5 and connexin-43 but not potassium voltage-gated channel, subfamily H (eag-related), member 2 (KCNH2). Live-cell imaging of Kv1.5-GFP and retrospective labeling of phalloidin demonstrated motility of Kv1.5 vesicles on actin tracts. MYO5A participated in anterograde trafficking, whereas MYO5B regulated postendocytic recycling. Overexpression of mutant motors revealed a selective role for Rab11 in coupling MYO5B to Kv1.5 recycling.

Conclusions: MYO5A and MYO5B control functionally distinct steps in the surface trafficking of Kv1.5. These isoform-specific trafficking pathways determine Kv1.5-encoded IKur in myocytes to regulate repolarizing current and, consequently, cardiac excitability. Therapeutic strategies that manipulate Kv1.5 selective trafficking pathways may prove useful in the treatment of arrhythmias.

Keywords: Kv1.5 potassium channel; arrhythmias, cardiac; connexin 43; heart.

Figure 1. Endogenous MYO5A and MYO5B regulate I_Kur

(A) Representative western blot images of endogenous MYO5A, MYO5B and tubulin from non-transfected HL1 cells and rat heart and brain. (B) Representative images of ventricular myocytes demonstrating fluorescence 24 hr post transduction with mCherry, MYO5aDN or Myo5bDN. (C) Representative traces of voltage protocol, pharmacological steps, and electronic subtraction for isolation of I_Kur current in acutely dissociated myocytes. (D) Quantification of I_Kur and I_ss current, and representative traces of I_Kur current, in acutely dissociated myocytes 24 hr post transduction (n ≥ 7 myocytes). *p < 0.05; **p < 0.01 by one-way ANOVA with Tukey post-test.

Figure 2. Disruption of MYO5A and MYO5B function decreases current density and cell surface levels of Kv1.5

HL-1 cells transfected with Kv1.5-GFP or hERG-GFP, and co-expressing either DsRed vector control, Myo5aDN or Myo5bDN alone or in combination. (A) Representative whole cell voltage clamp current traces and the corresponding IV curve for Kv1.5 current density. (B) Quantification of surface Kv1.5-GFP with representative images for each condition. (C) Quantification of surface hERG-GFP with representative images for each condition. (D) Quantification of surface Kv1.5-GFP with representative images following 48 hr of lentiviral infection with scrambled, Myo5A, or Myo5b shRNA. Scale bars = 10μm. *p < 0.05; ***p < 0.001 by one-way ANOVA with Tukey post-test.

Figure 3. Disruption of MYO5A and MYO5B function decreases current density and cell surface levels of Kv1.5 and increases accumulation at cell-cell borders

Adult rat ventricular myocytes co-transduced with Kv1.5-GFP and either mCherry, Myo5aDN, or Myo5bDN and analyzed 24hrs later. (A) Representative images of live cell imaging. (B) Quantification of images of steady-state surface Kv1.5-GFP levels under each condition. (C) Representative images of fixed and permeabilized cells. (D) Quantification of I_Kur current density. * p < 0.05; *** p < 0.001, by one-way ANOVA with Tukey post-test.

Figure 4

MYO5A is necessary for anterograde movement of connexin-43 (Cx43) from the ER/golgi apparatus to the plasma membrane in neonatal ventricular cardiomyocytes. (A) Representative images of cells co-transduced with Cx43 (green) and either LacZ, MYO5aDN, or Myo5bDN constructs (red), (n=3, 4, and 6 respectively), followed by a 12 hour treatment with Brefeldin A (BFA), to inhibit the anterograde movement of proteins from the ER/Golgi region to the plasma membrane, including 2 and 4 hour post BFA washout. (B and C) Fluorescence intensity profile for Cx43 at the cell-cell border. *p < 0.05, by one-way ANOVA with Tukey post-test.

Figure 5. Processivity of Kv1.5-containing vesicles is lost upon inhibition of MYO5A and MYO5B

(A) Quantification of surface channel and representative images for HL-1 cells 48 hr post transfection with Kv1.5-GFP. Cells were treated with 0.1% DMSO vehicle control or 5 μmol/L cytochalasin D for 0.5, 1, 2, 4, and 6 hr. (B) Representation of the protocol for live cell imaging, and representative image, of Kv1.5-GFP+ vesicle trafficking and retrospective actin filament labeling. (C) Representative images showing the SD map for Kv1.5-GFP+ vesicle motility, rhodamine phalloidin fluorescence, and the merged image. (D) Quantification of the distance traveled and velocity of Kv1.5-GFP+ vesicles trafficking along actin filaments in the periphery of the cell (n = 20 tracks from 16 cells, 17 tracks from 4 cells, and 9 tracks from 2 cells for Kv1.5 alone, Kv1.5 + myosin Va DN, and Kv1.5 + myosin Vb DN, respectively). Scale bars = 10μm. * p < 0.05; ** p < 0.01; *** p < 0.001 by one-way ANOVA with Tukey post-test.

Figure 6. MYO5A acts in the anterograde trafficking, and MYO5B in the post-endocytic recycling of Kv1.5

HL-1 cells transiently expressing Kv1.5-GFP 48 hr post transfection. (A) Quantification and representative images of steady-state surface expression of Kv1.5 upon co-expression with DsRed, MYO5aDN, MYO5aDN + dynS61D, Myo5bDN, or Myo5bDN + dynS61D. (B) Quantification and representative images of newly synthesized Kv1.5-GFP at the surface 6 hr post expression. (C) Quantification and representative images of Kv1.5-GFP recycled to the surface. (D) Co-localization for Kv1.5-GFP, Rab11, and MYO5aDN or Myo5bDN in perinuclear endosomes. Scale bars = 10μm. * p < 0.05; *** p < 0.001 by one-way ANOVA with Tukey post-test.

Figure 7. Recycling of Kv1.5 requires selective interaction of MYO5B with Rab11

Quantification and representative images of HL-1 cells transiently expressing Kv1.5-GFP 48 hr post transfection. (A) Steady-state surface expression of Kv1.5-GFP in the presence of Myo5bYE/QR or Myo5bQL/YC. (B) Quantification and images of Kv1.5-GFP recycled to the surface. (C) Co-localization for Kv1.5-GFP, Rab11, and Myo5bYE/QR in perinuclear endosomes. (D) Steady-state surface Kv1.5 upon co-expression with Rab11-CA, Myo5bYE/QR, or Rab11-CA and Myo5bYE/QR. Scale bars = 10μm. *** p < 0.001 by one-way ANOVA with Tukey post-test.

Figure 8

Proposed model for Kv1.5 trafficking in myocytes.