Abstract
JP2 (junctophilin-2) is believed to hold the transverse tubular and jSR (junctional sarcoplasmic reticulum) membranes in a precise geometry that facilitates excitation–contraction coupling in cardiomyocytes. We have expressed and purified human JP2 and shown using electron microscopy that the protein forms elongated structures ~15 nm long and 2 nm wide. Employing lipid-binding assays and quartz crystal microbalance with dissipation we have determined that JP2 is selective for PS (phosphatidylserine), with a Kd value of ~0.5 μM, with the N-terminal domain mediating this interaction. JP2 also binds PtdIns(3,4,5)P3 at a different site than PS, resulting in the protein adopting a more flexible conformation; this interaction is modulated by both Ca2+ and Mg2+ ions. We show that the S101R mutation identified in patients with hypertrophic cardiomyopathy leads to modification of the protein secondary structure, forming a more flexible molecule with an increased affinity for PS, but does not undergo a structural transition in response to binding PtdIns(3,4,5)P3. In conclusion, the present study provides new insights into the structural and lipid-binding properties of JP2 and how the S101R mutation may have an effect upon the stability of the dyad organization with the potential to alter JP2–protein interactions regulating Ca2+ cycling.
Highlights
IntroductionCardiac contraction is regulated by Ca2+ through a process termed CICR (calcium-induced calcium release)
Cardiac contraction is regulated by Ca2+ through a process termed calcium-induced calcium release (CICR)
In the present study we describe the first overexpression of soluble tagged full-length human JP2 in E. coli which we have purified to homogeneity using column chromatography, with the SDS/PAGE gel in Figure 1(B) showing a single polypeptide band at ∼ 75 kDa
Summary
Cardiac contraction is regulated by Ca2+ through a process termed CICR (calcium-induced calcium release). Disruption of the dyad architecture results in partial uncoupling of the geometric relationship between two of the protein components governing CICR: the sarcolemmal LTCCs (L-type voltage-gated calcium channels) and RyRs (ryanodine receptors) in the jSR, resulting in perturbed Ca2+ dynamics, which is a hallmark of heart failure [5]. Both a gradual drift of the t-t membranes [6] and an expansion of the dyadic cleft [7] have been proposed to lead to the loss of the dyad Ca2+ microdomain and the spatial and temporal restrictions that maintain CICR. The molecular mechanisms that underlie this cellular re-organization remain poorly understood
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