Programme Contents

In vitro interactions between C-terminal residues of the β_1a subunit of DHPR and RyR1 from skeletal muscle

Excitation-contraction (EC) coupling is the process which links the excitatory action potential to contraction of the muscle. Essential to this process in skeletal muscle is the interaction between two calcium channels: the L-type voltage gated dihydropyridine receptor (DHPR) located in the transverse tubule and the ryanodine receptor (RyR1) located in the sarcoplasmic reticulum (SR). This interaction between the two channels triggers SR Ca²⁺ release through the RyR1, which raises cytosolic [Ca²⁺] from ∼100 nM to 1-10 μM and enables contraction. The physical components of this channel-to-channel interaction are yet to be elucidated.

The last 35 C-terminal residues (V490-M524) and a hydrophobic heptad repeat motif (L478, V485 and V492) of the β_1a subunit of the DHPR have been shown to play an important role in EC coupling (Beurg et al. 1999; Sheridan et al., 2004). However, the importance of the heptad repeat in EC coupling is controversial as Dayal et al. (2010) has recently shown that expression of a heptad repeat mutant (L478A, V485A, V492A) only slightly hinders EC coupling in a β_1a-null zebrafish model. Our aim was to investigate the functional and physical interaction between RyR1 and two 35 residue peptides: β_1a C-tail peptide, corresponding to the last 35 C-terminal residues of the β_1a subunit, (V490-M524; which contains only one of the hydrophobic residues in the heptad repeat) and β_1a A474-A508 peptide, which contains the full heptad repeat.

New Zealand rabbits were euthanized by captive bolt. Fractions of SR membrane, containing native RyR1, were collected from rabbit back and leg muscle by gradient centrifugation. RyR1 was purified by solubilising the SR and further gradient centrifugation. The physical interaction between RyR1 and the biotinylated β_1a peptides was investigated using streptavidin-agarose affinity chromatography. The functional action of β_1a peptides on RyR1 were measured by reconstituting RyR1 into artificial lipid bilayers and assessing channel response to peptide addition, and with [³H]ryanodine binding.

We show that native and purified RyR1 bound to both β_1a peptides. As a functional consequence of the physical interaction between RyR1 and β_1a C-tail peptide, RyR1 activity increased 2 to 3-fold in the presence of the β_1a C-tail peptide (100 pM to 500 nM). Consistently, the β_1a peptide significantly increased [³H]ryanodine binding to native RyR1 at Ca²⁺ concentrations between 1 and 10 μM. Significantly, there was also a 2-fold increase in native RyR1 activity in the presence of 10 and 100 nM β_1a A474-A508 peptide. These results demonstrate that the C-terminus of the β_1a subunit can modulate RyR1 activity by directly binding to the RyR1 and that the heptad repeat is not crucial for the functional interaction between the DHPR and RyR1. Preliminary data however indicate that three hydrophobic residues located along one surface of an α-helix between L496 and W503 are essential for the action of the C-tail peptide on RyR1 channel activity. Overall, the results suggest that through its C-terminus, the β_1a subunit modulates RyR1 activity under Ca²⁺ concentrations that occur in skeletal muscle fibres during EC coupling, but that the heptad repeat is not essential for this regulation.

Beurg M, Ahern CA, Vallejo P, Conklin MW, Powers PA, Gregg RG and Coronado R. (1999) Biophysical Journal 77: 2953-2967.

Dayal A, Schredelseker J, Franzini-Armstrong C and Grabner M. (2010) Cell Calcium 47: 500-506.

Sheridan DC, Cheng W, Carbonneau L, Ahern CA and Coronado R. (2004) Biophysical Journal 87: 929-942.