Gain-of-function mutations of dynamin-2, a mechano-GTPase that remodels membrane and actin filaments, cause centronuclear myopathy (CNM), a congenital disease that mainly affects skeletal muscle tissue. Among these mutations, the variants p.A618T and p.S619L lead to a gain of function and cause a severe neonatal phenotype. By using total internal reflection fluorescence microscopy (TIRFM) in immortalized human myoblasts expressing the pH-sensitive fluorescent protein (pHluorin) fused to the insulin-responsive aminopeptidase IRAP as a reporter of the GLUT4 vesicle trafficking, we measured single pHluorin signals to investigate how p.A618T and p.S619L mutations influence exocytosis. We show here that both dynamin-2 mutations significantly reduced the number and durations of pHluorin signals induced by 10 μM ionomycin, indicating that in addition to impairing exocytosis, they also affect the fusion pore dynamics. These mutations also disrupt the formation of actin filaments, a process that reportedly favors exocytosis. This altered exocytosis might importantly disturb the plasmalemma expression of functional proteins such as the glucose transporter GLUT4 in skeletal muscle cells, impacting the physiology of the skeletal muscle tissue and contributing to the CNM disease.

Neurotransmission is critical for brain function, allowing neurons to communicate through neurotransmitters and neuropeptides. RVD-hemopressin (RVD-Hp), a novel peptide identified in noradrenergic neurons, modulates cannabinoid receptors CB1 and CB2. Unlike hemopressin (Hp), which induces anxiogenic behaviors via transient receptor potential vanilloid 1 (TRPV1) activation, RVD-Hp counteracts these effects, suggesting that it may block TRPV1. This study investigates RVD-Hp’s role as a TRPV1 channel blocker using HEK293 cells expressing TRPV1-GFP. Calcium imaging and patch-clamp recordings demonstrated that RVD-Hp reduces TRPV1-mediated calcium influx and TRPV1 ion currents. Molecular docking and dynamics simulations indicated that RVD-Hp interacts with TRPV1’s selectivity filter, forming stable hydrogen bonds and van der Waals contacts, thus preventing ion permeation. These findings highlight RVD-Hp’s potential as a therapeutic agent for conditions involving TRPV1 activation, such as pain and anxiety.

Phospholipase Cζ (PLCζ), a sperm-specific enzyme, plays a critical role in mammalian fertilization. Mutations in PLCζ have been linked to male infertility, as they impair its ability to trigger calcium (Ca2+) oscillations necessary for egg activation and embryo development. During fertilization, PLCζ is introduced into the egg, where it hydrolyzes phosphatidylinositol 4,5-bisphosphate (PIP2) into inositol 1,4,5-trisphosphate and diacylglycerol, leading to Ca2+ release from the endoplasmic reticulum. Human infertility-associated mutations include H233L, H398P, and R553P, which disrupt PLCζ function. To elucidate the molecular consequences of the mutations, we employed full-atom molecular dynamics simulations to analyze structural perturbations and their impact on PIP2 and Ca2+ binding. Our results reveal that H233L and H398P mutations significantly reduce interactions with PIP2, disrupting hydrogen bonding and salt bridge formation, leading to misalignment of the substrate. Additionally, these mutations destabilize Ca2+ binding by altering its positioning within the active site. In contrast, the R553P mutation primarily affects intramolecular stability and enzyme dynamics without impairing substrate or ion binding. Free energy calculations indicate an increased affinity for PIP2 in H233L and H398P mutants, leading to an aberrant substrate positioning and compromised hydrolysis. These structural insights help explain the egg activation failure and infertility of patients carrying these mutations.