Insulin‐like growth factor‐1 enhances rat skeletal muscle charge movement and L‐type Ca2+ channel gene expression

We investigated whether insulin-like growth factor-1 (IGF-1), an endogenous potent activator of skeletal muscle proliferation and differentiation, enhances L-type Ca2+ channel gene expression resulting in increased functional voltage sensors in single skeletal muscle cells. Charge movement and inward Ca2+ current were recorded in primary cultured rat myoballs using the whole-cell configuration of the patch-clamp technique. Ca2+ current and maximum charge movement (Qmax) were potentiated in cells treated with IGF-1 without significant changes in their voltage dependence. Peak Ca2+ current in control and IGF-1-treated cells was -7·8 ± 0·44 and -10·5 ± 0·37 pA pF−1, respectively (P < 0·01), whilst Qmax was 12·9 ± 0·4 and 22·0 ± 0·3 nC μF−1, respectively (P < 0·01). The number of L-type Ca2+ channels was found to increase in the same preparation. The maximum binding capacity (Bmax) of the high-affinity radioligand [3H]PN200-110 in control and IGF-1-treated cells was 1·21 ± 0·25 and 3·15 ± 0·5 pmol (mg protein)−1, respectively (P < 0·01). No significant change in the dissociation constant for [3H]PN200-110 was found. Antisense RNA amplification showed a significant increase in the level of mRNA encoding the L-type Ca2+ channel α1-subunit in IGF-1-treated cells. This study demonstrates that IGF-1 regulates charge movement and the level of L-type Ca2+ channel α1-subunits through activation of gene expression in skeletal muscle cells. The dihydropyridine (DHP)-sensitive L-type Ca2+ channel plays a central role in intracellular Ca2+ homeostasis. In particular, this channel is essential in skeletal muscle excitation-contraction coupling (Melzer et al. 1995). When a muscle fibre is activated by a nerve impulse, an action potential spreads throughout the surface and T-tubular sarcolemma. The L-type Ca2+ channel α1-subunit senses changes in membrane voltage. Conformational changes of the channel associated with the movement of positively charged segments of α1-subunits (S4 segments) couple depolarization of the T-tubule and Ca2+ release (Schneider, 1994; Melzer et al. 1995). The L-type Ca2+ channel α1-subunit is the main source of intramembrane current together with the Na+ and T-type Ca2+ channel in skeletal muscle. The L-type Ca2+ channel by itself contributes more than 60 % of the total charge movement recorded in skeletal myotube (Adams et al. 1990). This voltage-dependent non-linear capacitive current is thought to be associated with the voltage sensor function and is termed gating current or charge movement (Schneider & Chandler, 1973). Because the integral of charge movement is an indication of the number of ion channels inserted in the membrane, the level of channel expression is associated with the amplitude of the charge movement recordings. Insulin-like growth factor-1 (IGF-1) is a peptide structurally related to proinsulin and has a primary role in promoting skeletal muscle differentiation and growth in a relatively slow time scale (Florini et al. 1996). IGF-1, as well as some other growth factors, regulates the ion permeation function of various ion channels. Growth/trophic factor-mediated potentiation of Ca2+ permeation through different ion channels in clonal pituitary and neuroblastoma cell lines has been reported (Meza et al. 1994; Selinfreund & Blair, 1994; Delbono & Sonntag, 1996; Blair & Marshall, 1997). In skeletal muscle, we have demonstrated that IGF-1 potentiates Ca2+ current through L-type Ca2+ channels in adult fibres (Delbono et al. 1997), but not in muscles from ageing mice (Renganathan et al. 1997c) probably due to IGF-1 resistance (Dardevet et al. 1994). Using a transgenic mouse model overexpressing IGF-1 exclusively in skeletal muscle (Coleman et al. 1995) we found increased levels of binding sites for the dihydropyridine PN200-110 (Renganathan et al. 1997b). In the present study we examined the influence of IGF-1 on charge movement in cultured muscle cells. The role of IGF-1 in the biochemical expression of the L-type Ca2+ channel α1-subunit was assessed by a high-affinity radioligand binding assay in skeletal muscle cells. The influence of IGF-1 on α1s gene expression was examined in myoballs by combining patch clamp and single cell antisense RNA amplification.