No abstract

No abstract

The type 2a sarco/endoplasmic reticulum (ER) Ca 2þ -ATPase (SERCA2a) plays a key role in intracellular Ca 2þ regulation in the heart. We have previously shown evidence of stable homodimers of SERCA2a in heterologous cells and cardiomyocytes. However, the functional significance of the pump dimerization remains unclear. Here, we analyzed how SERCA2a dimerization affects ER Ca 2þ transport. Fluorescence resonance energy transfer experiments in HEK293 cells transfected with fluorescently labeled SERCA2a revealed increasing dimerization of Ca 2þ pumps with increasing expression level. This concentration-dependent dimerization provided means of comparison of the functional characteristics of monomeric and dimeric pumps. SERCA-mediated Ca 2þ uptake was measured with the ER-targeted Ca 2þ sensor R-CEPIA1er in cells cotransfected with SERCA2a and ryanodine receptor. For each individual cell, the maximal ER Ca 2þ uptake rate and the maximal Ca 2þ load, together with the pump expression level, were analyzed. This analysis revealed that the ER Ca 2þ uptake rate increased as a function of SERCA2a expression, with a particularly steep, nonlinear increase at high expression levels. Interestingly, the maximal ER Ca 2þ load also increased with an increase in the pump expression level, suggesting improved catalytic efficiency of the dimeric species. Reciprocally, thapsigargin inhibition of a fraction of the population of SERCA2a reduced not only the maximal ER Ca 2þ uptake rate but also the maximal Ca 2þ load. These data suggest that SERCA2a dimerization regulates Ca 2þ transport by improving both the SERCA2a turnover rate and catalytic efficacy. Analysis of ER Ca 2þ uptake in cells cotransfected with human wild-type SERCA2a (SERCA2a WT ) and SERCA2a mutants with different catalytic activity revealed that an intact catalytic cycle in both protomers is required for enhancing the efficacy of Ca 2þ transport by a dimer. The data are consistent with the hypothesis of functional coupling of two SERCA2a protomers in a dimer that reduces the energy barrier of rate-limiting steps of the catalytic cycle of Ca 2þ transport.

Objective Sweet taste receptors (STR) are expressed in the gut and other extra-oral tissues, suggesting that STR-mediated nutrient sensing may contribute to human physiology beyond taste. A common variant (Ile191Val) in the TAS1R2 gene of STR is associated with nutritional and metabolic outcomes independent of changes in taste perception. It is unclear whether this polymorphism directly alters STR function and how it may contribute to metabolic regulation. Methods We implemented a combination of in vitro biochemical approaches to decipher the effects of TAS1R2 polymorphism on STR function. Then, as proof-of-concept, we assessed its effects on glucose homeostasis in apparently healthy lean participants. Results The Ile191Val variant causes a partial loss of function of TAS1R2 through reduced receptor availability in the plasma membrane. Val minor allele carriers have reduced glucose excursions during an OGTT, mirroring effects previously seen in mice with genetic loss of function of TAS1R2. These effects were not due to differences in beta-cell function or insulin sensitivity. Conclusions Our pilot studies on a common TAS1R2 polymorphism suggest that STR sensory function in peripheral tissues, such as the intestine, may contribute to the regulation of metabolic control in humans.

The sodium/potassium-ATPase (NKA) is the enzyme that establishes gradients of sodium and potassium across the plasma membrane. NKA activity is tightly regulated for different physiological contexts through interactions with single-span transmembrane peptides, the FXYD proteins. This diverse family of regulators has in common a domain containing a Phe-X-Tyr-Asp (FXYD) motif, two conserved glycines, and one serine residue. In humans, there are seven tissue-specific FXYD proteins that differentially modulate NKA kinetics as appropriate for each system, providing dynamic responsiveness to changing physiological conditions. Our understanding of how FXYD proteins contribute to homeostasis has benefitted from recent advances described in this review: biochemical and biophysical studies have provided insight into regulatory mechanisms, genetic models have uncovered remarkable complexity of FXYD function in integrated physiological systems, new posttranslational modifications have been identified, high-resolution structural studies have revealed new details of the regulatory interaction with NKA, and new clinical correlations have been uncovered. In this review, we address the structural determinants of diverse FXYD functions and the special roles of FXYDs in various physiological systems. We also discuss the possible roles of FXYDs in protein trafficking and regulation of non-NKA targets.