Coupling of RYR1 and L-type Calcium Channels via Calmodulin Binding Domains

Abstract

In skeletal muscle the L-type Ca 2؉ channel directly controls the opening of the sarcoplasmic reticulum Ca 2؉ release channel (RYR1), and RYR1, in turn, prevents L-type Ca 2؉ channel inactivation. We demonstrate that the two proteins interact using calmodulin binding regions of both proteins. A recombinant protein representing amino acids 1393-1527 (D1393-1527) of the carboxyl-terminal tail of the skeletal muscle L-type voltage-dependent calcium channel binds Ca 2؉ , Ca 2؉ calmodulin, and apocalmodulin. In the absence of calmodulin, D1393-1527 binds to both RYR1 and a peptide representing the calmodulin binding site of RYR1 (amino acids 3609-3643). In addition, biotinylated R3609-3643 peptide can be used with streptavidin beads to pull down [ 3 H]PN200-110-labeled L-type channels from detergent-solubilized transverse tubule membranes. The binding of the L-type channel carboxyl-terminal tail to the calmodulin binding site on RYR1 may stabilize the contact between the two proteins, provide a mechanism for Ca 2؉ and/or calmodulin regulation of their interaction, or participate directly in functional signaling between these two proteins. A unique aspect of this study is the finding that calmodulin binding sequences can serve as specific binding motifs for proteins other than calmodulin.

A Ca2+-Binding Domain in RyR1 that Interacts with the Calmodulin Binding Site and Modulates Channel Activity

“…We previously have shown that a carboxyterminal tail fragment of the skeletal L-type channel (Ca v 1.1) a 1 -subunit, which contains both Ca 21 and CaM binding sites, binds to RyR1. This interaction is blocked by Ca 21 CaM (26). We have also shown that a peptide representing the CaM binding site on RyR1 (amino acids 3614-3643) binds to both the Ca v 1.1 and an expressed fragment of the carboxyterminal tail of its a 1 -subunit (amino acids 1393-1527) (26).…”

“…This interaction is blocked by Ca 21 CaM (26). We have also shown that a peptide representing the CaM binding site on RyR1 (amino acids 3614-3643) binds to both the Ca v 1.1 and an expressed fragment of the carboxyterminal tail of its a 1 -subunit (amino acids 1393-1527) (26).…”

“…Since the CaM binding peptides from these two channels do not themselves interact (unpublished observation), we began to look for sequences within RyR1 that could bind to the carboxy-terminal tail of the Ca v 1.1 a 1subunit. A region of Ca v 1.1 (amino acids 1395-1540) that contains the putative E-F hands was previously predicted by 3D-PSSM (http://www.sbg.bio.ic.ac.uk) to fold like CaM and was shown to interact with CaM binding peptides from RyR1 and Ca v 1.1 (26). The CaM binding peptide from RyR1 has been shown to bind both Ca 21 CaM and apoCaM (22,23).…”

“…We have previously shown that a synthetic peptide matching the CaM binding site on RyR1 bound to Ca v 1.1, and, conversely, that a CaM binding peptide from Ca v 1.1 bound to RyR1 (26). Since the CaM binding peptides from these two channels do not themselves interact (unpublished observation), we began to look for sequences within RyR1 that could bind to the carboxy-terminal tail of the Ca v 1.1 a 1subunit.…”

“…The interaction of R3614-3643 with the Ca v 1.1 is also blocked by Ca 21 CaM (26). Three sequences on the carboxyterminal tail of the cardiac L-type Ca 21 channel (Ca v 1.2), designated A (1609-1628, numbering of the human cardiac channel), C (1627-1652), and IQ (1665-1685) have been implicated in CaM binding (27)(28)(29)(30).…”