Role of an inward rectifier K+ current and of hyperpolarization in human myoblast fusion

Abstract

1. The role of K+ channels and membrane potential in myoblast fusion was evaluated by examining resting membrane potential and timing of expression of K+ currents at three stages of differentiation of human myogenic cells: undifferentiated myoblasts, fusion-competent myoblasts (FCMBs), and freshly formed myotubes. 2. Two K+ currents contribute to a hyperpolarization of myoblasts prior to fusion: IK(NI), a non-inactivating delayed rectifier, and IK(IR), an inward rectifier. 3. IK(NI) density is low in undifferentiated myoblasts, increases in FCMBs and declines in myotubes. On the other hand, IK(IR) is expressed in 28% of the FCMBs and in all myotubes. 4. IK(IR) is reversibly blocked by Ba2+ or Cs+. 5. Cells expressing IK(IR) have resting membrane potentials of -65 mV. A block by Ba2+ or Cs+ induces a depolarization to a voltage determined by IK(NI) (-32 mV). 6. Cs+ and Ba2+ ions reduce myoblast fusion. 7. It is hypothesized that the IK(IR)-mediated hyperpolarization allows FCMBs to recruit Na+, K+ and T-type Ca2+ channels which are present in these cells and would otherwise be inactivated. FCMBs, rendered thereby capable of firing action potentials, could amplify depolarizing signals and may accelerate fusion.

Figure 1. Resting potential, non-inactivating (I_K(NI)) and inward rectifier (I_K(IR)) potassium current expression at various stages of differentiation of myogenic cells

A, resting potential in undifferentiated myoblasts (UDMB), fusion-competent myoblasts (FCMB) without I_K(IR) (open column) and fusion-competent myoblasts expressing I_K(IR) (filled column), and myotubes (MT). B, I_K(NI) density in these different types of myogenic cells. I_K(NI) was evaluated at the end of a steady holding voltage at +40 mV lasting 3 min. Leak current at +40 mV was estimated by linear extrapolation from the leak current recorded at −80, −70 and −60 mV, and subtracted. Cell capacitances were: UDMB = 14 ± 2 pF; FCMB = 25 ± 1 pF; MT = 150 ± 22 pF. C, I_K(IR) density in undifferentiated myoblasts (UDMB), fusion-competent myoblasts (FCMB; 37 cells out of 131 expressed I_K(IR) and the density of the current in these cells was not different from that recorded in 3 day-differentiated myotubes), and myotubes (MT; 3 days, 7 days, and 18 days in differentiation medium; all cells expressed I_K(IR)). I_K(IR) was evaluated during a step to −140 mV from a holding potential at −60 mV. Leak current was evaluated either by linear extrapolation from the leak current recorded at −40, −50 and −60 mV or by adding 500 μM Ba²⁺ to the external solution, and subtracted. Cell capacitances were: UDMB = 16 ± 1 pF; FCMB without I_K(IR) = 24 ± 1 pF; FCMB with I_K(IR) = 26 ± 2 pF; 3 day-differentiated MT = 62 ± 7 pF; 7 day-differentiated MT = 192 ± 13 pF; 18 day-differentiated MT = 160 ± 16 pF.

Figure 2. Whole-cell properties of the inward rectifier potassium current (I_K(IR))

A, the voltage of a fusion-competent myoblast expressing I_K(IR) was held steadily at −60 mV and stepped to values between −40 and −140 mV for 400 ms. Leak current was obtained by adding 500 μM Ba²⁺ to the external medium, and subtracted. Cell capacitance was 26 pF. B, fusion-competent myoblast (capacitance 37 pF) which does not express I_K(IR). Same voltage-clamp protocol as in A. C, voltage-clamp protocol applied to the cells illustrated in A and B. D, same cell as in A. The maximum amplitude of I_K(IR) measured during a voltage step is plotted against the potential of the step. □, control currents; ^, currents remaining during the application of 500 μM Ba²⁺ in the external medium. E, voltage-clamp ramps applied to a fusion-competent myoblast bathed in a solution containing either 5 or 30 mM potassium. The potassium concentration in the pipette was 110 mM. Leak current was obtained by adding 500 μM Ba²⁺ to the external medium, and subtracted. The enlargement (upper panel) shows that I_K(IR) can carry a small outward current. I_K(IR) was fitted in both external potassium concentrations using a theoretical equation which is a Boltzmann equation multiplied by the driving force on the ions permeable to the channels:where G_max is the maximum conductance of I_K(IR), Q is the gating charge, V_o is the voltage at half-activation, V is the transmembrane potential, k is the Boltzmann constant, T is the temperature, G_min is the minimum conductance of I_K(IR), and V_rev,K(IR) is the reversal potential of I_K(IR). In 5 mM ${\text{K}}_{\text{o}}^{+}$, G_max = 1.43 nS, Q = 2.8 times the elementary charge, V_o = −96 mV, G_min = 0.05 nS, and V_rev,K(IR) = −72 mV. In 30 mM ${\text{K}}_{\text{o}}^{+}$, G_max = 2.12 nS, Q = 2.8 times the elementary charge, V_o = −39 mV, G_min = 0.047, and V_rev,K(IR) = −33 mV. As expected for an inward rectifier potassium current, the reversal potential of the current follows E_K, and G_max was larger and V_o shifted to a more depolarized value in 30 mM ${\text{K}}_{\text{o}}^{+}$. Cell capacitance was 20 pF. F,I_K(IR) is blocked by Ba²⁺ ions in fusion-competent myoblasts (□; n = 7) and in myotubes (▵; n = 6). Data points were fitted with a Michaelis-Menten equation (fusion-competent myoblasts: K_d = 8 μM; myotubes: K_d = 14 μM). G, I_K(IR) is blocked by Cs⁺ ions in fusion-competent myoblasts and the block depends upon the membrane potential. □, current at −140 mV (K_d = 30 μMn; = 3 or more); ^, current at −100 mV (K_d = 137 μM; n = 3 or more); ▵, current at −60 mV (K_d = 5.5 mM; n = 3).

Figure 3. Contribution of I_K(IR) to the resting potential of fusion-competent myoblasts and myotubes

Membrane resting potential of fusion-competent myoblasts during the progressive block of I_K(IR) with Ba²⁺ (A; n = 3 or more) and Cs⁺ (B; n = 3 or more). Insets: similar experiment done with myotubes (Ba²⁺, n = 5; Cs⁺, n = 4). The resting potentials of fusion-competent myoblasts and myotubes are depolarized by increasing concentrations of Ba²⁺ and Cs⁺. Dotted lines were drawn by eye.

Figure 4. Myoblast fusion is inhibited by Cs⁺, and the effect is reversible

A, fusion was induced with differentiation medium and assessed after 36 h, and 3 and 5 days. ^, fusion in control conditions. In sister cultures, 10 mM Cs⁺ was added to the differentiation medium either during the entire experiment (□) or for a period of 3 days (▵). The upper panel in B shows Haematoxylin-stained culture of myotubes after 3 days in differentiation medium. The lower panel shows a sister culture after the same time in differentiation medium containing 10 mM Cs⁺ (only myoblasts are present). From a morphological point of view, the myoblasts appear perfectly normal despite the presence of 10 mM Cs⁺. Scale bar represents 40 μm.