Emerging Concepts in Ion Channel Biophysics

Emerging Concepts in Ion Channel Biophysics

Poster Abstracts

25-POS Board 25 Effect of Camkii-Mediated Phosphorylation in the Β 1a Subunit of the Voltage-Gated Ca 2+ Channel. Dora Bodnar 1 , Claudio F. Perez 1 , Jose M. Eltit 2 . 2 Virginia Commonwealth University, School of Medicine, Richmond, VA, USA. 1 Brigham and Women's Hospital, Harvard Medical School, Boston, MA, USA, The β 1a subunit is part of the DHPR-RyR1 complex in skeletal muscle. This macro-protein complex transduces electrical signals in the plasma membrane into cytosolic Ca 2+ transients, triggering muscle contraction. We showed that mutations in 489 QVQVLTSLRRNLSFW 503 C- term tail domain of β 1a hinder the conformational communication between the DHPR and the RyR1. The residue Ser 501 is predicted to be a phosphorylation target for various protein kinases (motif NLSFW). In this study, we evaluated the specificity of protein kinases to phosphorylate this site in vitro and we further evaluate its effects on calcium handling in cultured myotubes. The C-term tail of β 1a subunit, encompassing residues V 485 through M 524 , was expressed as a GST-fusion protein and subjected to in vitro phosphorylation by PKC, MAPK and CaMKII. Western blot analysis with an anti-phospho antibody confirmed specific phosphorylation of the C-term domain by CaMKII but not PKC or MAPK. Alanine substitution of residue S 501 (S501A mutation) further prevented CaMKII phosphorylation confirming S 501 as the phosphorylation site. The effect of phosphorylation on Ca 2+ handling was evaluated in β 1 -null myotubes stably transfected with β 1a subunit carrying mutation S501A. Patch-clamp analysis of wt and S501A myotubes showed small to no difference in the L-type Ca 2+ current between both genotypes. However, Ca 2+ imaging studies revealed a significant increase in the amplitude of depolarization-induced Ca 2+ transient (K + stimulation) in myotubes expressing the S501A mutation. In addition, mutant cells displayed significantly higher levels of SR Ca 2+ content and faster rate of decay of the depolarization-induced Ca 2+ transient response. Our findings suggest that residue S 501 of β 1a subunit is a substrate for CaMKII phosphorylation and that its state of phosphorylation modulates Ca 2+ release in skeletal muscle cell, presumably altering the DHPR to RyR1 signaling. Supported by grant NIH-1R01AR06773

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