The cardiac hypertrophic factors modulate the function of the cardiac L-type Ca channels

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The leukemia inhibitory factor (LIF) is a member of the IL-6 cytokine family that can induce cardiac hypertrophy. Our group reported that LIF can activate cardiac L-type Ca^<2+> channels blocked by PD98059 (MEK inhibitor). We also showed that LIF induces cardiac hypertrophy in concert with activated ERK, CaMK-IV and calcineurin, which are dependent on increased Ca^<2+> currents through L-type Ca^<2+>. To determine whether LIF phosphorylates the α1 subunits, the main regulator of the channel function of L-type Ca^<2+> channels, primary cultures of neonatal rat cardiomyocytes were stimulated with LIF for 15 min, and the kinase activity measured by the in-gel-kinase assay using recombinant al subunits (GST-fusion proteins containing 800 amino acids of the carboxyl terminal) as the substrate. ERK1/2 phosphorylated the fusion protein containing the certain serine of the ERK1/2 motif. LIF promoted the phosphorylation of the immuno-precipitated α1 subunits, which was metabolically labeled wit … More h P-32, significantly. Phospho-amino acid analysis showed that LIF phosphorylated the al subunits only at the serine position. The point mutant of the target serine of the rabbit α1 subunit was transfected into HEK293 cells. LIF enhanced phosphorylation of the transfected wild-type α1 subunit by significantly, but not that of the mutant. The wild-type α1 subunits which were co-transfected into HEK293 cells with the constitutively active MEK1 were labeled with P-32, and theses were showed enhanced phosphorylation significantly. By the perforated patch-clump method, the L-type Ca^<2+> channels containing the rabbit wild-type α1 subunit showed the prolongation of the Ca^<2+> current after LIP administration, but the channels of the point mutant did not showed the prolongation. These findings indicate that LIF phosphorylates the target serine of the α1 subunit of cardiac L-type Ca^<2+> channels both in vitro and in vivo, and that ERK1/2 may therefore be a potential activator of cardiac L-type Ca^<2+> channels. Less