A cardiac dihydropyridine receptor II‐III loop peptide inhibits resting Ca2+ sparks in ferret ventricular myocytes

We studied the effect of a peptide (Ac-10C) on cardiac ryanodine receptor (RyR) opening. This decapeptide (KKERKLARTA) is a fragment of the cardiac dihydropyridine receptor (DHPR) from the cytosolic loop between the second and third transmembrane domains (II–III loop). Studies were carried out in ferret ventricular myocytes by simultaneously applying ruptured-patch voltage clamp and line-scan confocal microscopy with fluo-3 to measure intracellular [Ca2+] ([Ca2+]i) and Ca2+ sparks. Inclusion of Ac-10C in the dialysing pipette solution inhibited resting Ca2+ spark frequency (due to diastolic RyR openings) by > 50 %. This occurred without changing sarcoplasmic reticulum (SR) Ca2+ content, which was measured via the caffeine-induced Ca2+ transient amplitude and the caffeine-induced Na+–Ca2+ exchange current (INCX) integral. Ac-10C also reduced slightly the size of Ca2+ sparks. Ac-10C did not alter either resting [Ca2+]i (assessed by indo-1 fluorescence) or DHPR gating (measured as L-type Ca2+ current). The SR Ca2+ fractional release was depressed by Ac-10C at relatively low SR Ca2+ content, but not at higher SR Ca2+ content. A control scrambled peptide (Ac-10CS) did not alter any of the measured parameters (notably Ca2+ spark frequency or SR Ca2+ fractional release). Thus, the Ac-10C effects may be sequence or charge distribution specific. Our results suggest an inhibitory regulation of RyRs at rest via the cardiac DHPR II–III loop N-terminus region. The mechanism of the effect and whether this interaction is important in cardiac excitation-contraction coupling (E–C coupling) per se, requires further investigation. Evidence suggests that the skeletal dihydropyridine receptor (DHPR) directly interacts with the sarcoplamic reticulum (SR) ryanodine receptor (RyR) causing SR Ca2+ release upon sarcolemmal depolarization, independent of Ca2+ entry (Schneider & Chandler, 1973; Flucher & Franzini-Armstrong, 1996). By contrast, Ca2+-induced-Ca2+-release (CICR) is thought to be the central mechanism in cardiac excitation-contraction coupling (E–C coupling), where Ca2+ influx rather than depolarization triggers RyR opening (Wier & Balke, 1999; Bers, 2001). Among the three RyR types, skeletal muscle relies mainly on RyR1 for E–C coupling, while cardiac muscle utilizes RyR2. RyR1 and RyR2 share a highly homologous sequence, the same structural characteristics, and both are located in the junctional SR, which allows an intimate relation with DHPRs of the T-tubular membrane (Block et al. 1988; Franzini-Armstrong et al. 1998). However, evidence of direct DHPR-RyRs interactions is limited. Within the skeletal DHPR a cytosolic region (T671-V790) between the second and third transmembrane domains (II–III loop) may play a crucial role in skeletal DHPR- RyR interaction and E–C coupling, based on expression of chimaeric DHPRs in dysgenic myotubes (Tanabe et al. 1990). The purified skeletal DHPR II–III loop region was found to increase RyR1 open probability (Po) in lipid bilayers and ryanodine binding activity (Lu et al. 1994). Nevertheless, different effects of various regions of the II–III loop have been found by different groups using different technical approaches. For instance, peptide A (T671-L690) located at the N-terminus of the skeletal DHPR II–III loop was found to increase ryanodine binding to RyR1 and Ca2+ release from both SR vesicles and skinned skeletal muscle fibres (El-Hayek et al. 1995, 1998; Lamb et al. 2000). Interestingly, peptide C (F725-P760), located in the middle of the II–III loop, was found to depress the effect of peptide A. However, other studies expressing chimeric DHPRs in dysgenic myotubes suggested that the peptide C region was more important in skeletal muscle E–C coupling than peptide A (Nakai et al. 1998; Proenza et al. 2000). It seems likely that physical linkage between these DHPR subdomains and RyRs is critical in the mechanism of skeletal E–C coupling. The effects of different fragments of the 20-mer peptide A region of both skeletal DHPR (T671-L690) and cardiac DHPR (T793-A812) on RyR gating have been examined (El-Hayek et al. 1998; Marx et al. 1998; Henrikson et al. 1999; Lamb et al. 2000). The carboxyl terminus decamer of peptide A in skeletal DHPR (As-10C) was found to have inhibitory effects on Ca2+ release during depolarization in skinned skeletal muscle fibres, but this skeletal muscle effect was not found with the analogous region in cardiac DHPR, Ac-10C. On the other hand, preliminary data suggested that Ac-10C might inhibit RyR2 gating in single channel recordings (Li et al. 1999; Henrikson et al. 1999). Figure 1 shows the sequence and charge comparison of the peptides mentioned above. Figure 1 Sequences and charge comparisons of several peptides The sequence and co-localization of Ca2+ channels (DHPR and RyR) are homologous, but not identical in cardiac and skeletal muscle. Could there be regulation of RyR gating by DHPR via direct interaction in cardiac muscle? There are fewer studies of this possibility and these are mostly from lipid bilayer and isolated SR vesicle experiments (Slavik et al. 1997; Zhu et al. 1999; Henrickson et al. 1999). The L-type Ca2+ channel agonist Bay K 8644 appears to bind to the cardiac DHPR and alter RyR gating in intact ventricular myocytes, independent of Ca2+ influx (McCall et al. 1996; Satoh et al. 1998; Katoh et al. 2000). This was suggested to reflect a weak physical link between cardiac DHPR and RyR that might function primarily in co-localization rather than E–C coupling per se. Here we investigated the effect of Ac-10C on RyR gating in intact ferret ventricular myocytes using the patch-clamp technique coupled with confocal microscopy and found that Ac-10C peptide inhibited resting RyR opening, and probably E–C coupling as well, without altering SR Ca2+ content, ICa or resting [Ca2+]i.