SummaryBy vacuolar patch-clamp and Ca2+ imaging experiments, we show that the yeast vacuolar transient receptor potential (TRPY) channel 1 is activated by cytosolic Ca2+ and inhibited by Ca2+ from the vacuolar lumen. The channel is cooperatively affected by vacuolar Ca2+ (Hill coefficient, 1.5), suggesting that it may accommodate a Ca2+ receptor that can bind two calcium ions. Alanine scanning of six negatively charged amino acid residues in the transmembrane S5 and S6 linker, facing the vacuolar lumen, revealed that two aspartate residues, 401 and 405, are essential for current inhibition and direct binding of 45Ca2+. Expressed in HEK-293 cells, a significant fraction of TRPY1, present in the plasma membrane, retained its Ca2+ sensitivity. Based on these data and on homology with TRPV channels, we conclude that D401 and D405 are key residues within the vacuolar vestibule of the TRPY1 pore that decrease cation access or permeation after Ca2+ binding.

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The anterior cruciate ligament (ACL), the principal ligament for stabilization of the knee, is highly predisposed to injury in the human population. As a result of its poor intrinsic healing capacities, surgical intervention is generally necessary to repair ACL lesions, yet the outcomes are never fully satisfactory in terms of long-lasting, complete, and safe repair. Gene therapy, based on the transfer of therapeutic genetic sequences via a gene vector, is a potent tool to durably and adeptly enhance the processes of ACL repair and has been reported for its workability in various experimental models relevant to ACL injuries in vitro, in situ, and in vivo. As critical hurdles to the effective and safe translation of gene therapy for clinical applications still remain, including physiological barriers and host immune responses, biomaterial-guided gene therapy inspired by drug delivery systems has been further developed to protect and improve the classical procedures of gene transfer in the future treatment of ACL injuries in patients, as critically presented here.

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Lesions in the human anterior cruciate ligament (ACL) are frequent, unsolved clinical issues due to the limited self-healing ability of the ACL and lack of treatments supporting full, durable ACL repair. Gene therapy guided through the use of biomaterials may steadily activate the processes of repair in sites of ACL injury. The goal of the present study was to test the hypothesis that functionalized poly(sodium styrene sulfonate)-grafted poly(ε-caprolactone) (pNaSS-grafted PCL) films can effectively deliver recombinant adeno-associated virus (rAAV) vectors as a means of overexpressing two reparative factors (transforming growth factor beta-TGF-β and basic fibroblast growth factor-FGF-2) in primary human ACL fibroblasts. Effective, durable rAAV reporter red fluorescent protein and candidate TGF-β and FGF-2 gene overexpression was achieved in the cells for at least 21 days, especially when pNaSS-grafted PCL films were used versus control conditions, such as ungrafted films and systems lacking vectors or films (between 1.8- and 5.2-fold differences), showing interactive regulation of growth factor production. The expression of TGF-β and FGF-2 from rAAV via PCL films safely enhanced extracellular matrix depositions of type-I/-III collagen, proteoglycans/decorin, and tenascin-C (between 1.4- and 4.5-fold differences) in the cells over time with increased levels of expression of the specific transcription factors Mohawk and scleraxis (between 1.7- and 3.7-fold differences) and without the activation of the inflammatory mediators IL-1β and TNF-α, most particularly with pNaSS-grafted PCL films relative to the controls. This work shows the value of combining rAAV gene therapy with functionalized PCL films to enhance ACL repair.