Mechanisms underlying Ca 2؉ signaling during human myoblast terminal differentiation were studied using cell cultures. We found that T-type Ca 2؉ channels (T-channels) are expressed in myoblasts just before fusion. Their inhibition by amiloride or Ni 2؉ suppresses fusion and prevents an intracellular Ca 2؉ concentration increase normally observed at the onset of fusion. The use of antisense oligonucleotides indicates that the functional T-channels are formed by ␣1H subunits. At hyperpolarized potentials, these channels allow a window current sufficient to increase [Ca 2؉ ]i. As hyperpolarization is a prerequisite to myoblast fusion, we conclude that the Ca 2؉ signal required for fusion is produced when the resting potential enters the T-channel window. A similar mechanism could operate in other cell types of which differentiation implicates membrane hyperpolarization.

An early sign of human myoblast commitment to fusion is the expression of a non-inactivating delayed rectifier K + current, I K(NI) , and an associated membrane potential hyperpolarization. We have isolated the full-length coding region of a human ether-a-go-go K + channel (h-eag) from myoblasts undergoing differentiation. The h-eag gene was localized to chromosome 1q32^41, and is expressed as a V9 kb transcript in myogenic cells and in adult brain tissue. Forced expression of h-eag in undifferentiated myoblasts generates a current with remarkable similarity to I K(NI) indicating that h-eag constitutes the channel responsible for this current in vivo.z 1998 Federation of European Biochemical Societies.

Myoblast fusion is essential to skeletal muscle development and repair. We have demonstrated previously that human myoblasts hyperpolarize, before fusion, through the sequential expression of two K+ channels: an ether-à-go-go and an inward rectifier. This hyperpolarization is a prerequisite for fusion, as it sets the resting membrane potential in a range at which Ca2+ can enter myoblasts and thereby trigger fusion via a window current through α1H T channels.

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. 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. I K(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. I K(IR) is reversibly blocked by Ba2+ or Cs+. 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). Cs+ and Ba2+ ions reduce myoblast fusion. 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.

The skeletal muscle fibre is a postmitotic multinucleated cell formed by the fusion of mononucleated myoblasts. Myoblast fusion is essential to skeletal muscle development and repair, and understanding this process is important for myoblastbased grafts or gene therapies. Our recent work on human fusion-competent myoblasts indicates that an early step in the fusion process is a hyperpolarization of the resting potential. This hyperpolarization is a two-step mechanism involving the sequential expression of two voltage-gated potassium currents. The first is a non-inactivating delayed rectifier current (IK(NI)) that hyperpolarizes the cell to an intermediate resting potential of approximately −32 mV (Bernheim et al. 1996). Then, slightly before fusion, an inward rectifier current is expressed (IK(IR)), which drives the potential further down to approximately −65 mV, i.e. similar to that measured for multinucleated myotubes ).We recently isolated the full-length coding region of a human ether-à-go-go K¤ channel (h_eag) from myoblasts undergoing differentiation . We describe here the properties of the current elicited by h_eag and present data indicating that it constitutes the K(NI) channel responsible for the first hyperpolarization step linked to myoblast differentiation. METHODS Dissociation and cell culturesSamples of human skeletal muscle were obtained during corrective orthopaedic surgery of young patients (9 months to 17 years old) without any known neuromuscular disease, in accordance with the guidelines of the ethical committee of the University Hospital of Geneva (written informed consent was obtained from patients or their legal guardians). Myoblasts (Baroffio et al. 1993) and fusioncompetent myoblasts (Krause et al. 1995) were prepared as Forced expression of h_eag hyperpolarizes undifferentiated myoblasts from −9 to −50 mV, the threshold for the activation of both Ih_eag and IK(NI). Similarly, the higher the density of IK(NI), the more hyperpolarized the resting potential of fusion-competent myoblasts. 6. It is concluded that h_eag constitutes the channel underlying IK(NI) and that it contributes to the hyperpolarization of fusion-competent myoblasts. To our knowledge, this is the first demonstration of a physiological role for a mammalian eag K¤ channel.8405