Diverse residues of intracellular loop 5 of the Na+/H+ exchanger modulate proton sensing, expression, activity and targeting.

The mammalian Na+/H+ exchanger isoform 1 (NHE1) is an integral membrane protein that regulates intracellular pH (pHi) by removing a single intracellular proton in exchange for one extracellular sodium ion. It is involved in cardiac hypertrophy and ischemia reperfusion damage to the heart and elevation of its activity is a trigger for breast cancer metastasis. NHE1 has an extensive 500 amino acid N-terminal membrane domain that mediates transport and consists of 12 transmembrane segments connected by intracellular and extracellular loops. Intracellular loops are hypothesized to modulate the sensitivity to pHi. In this study, we characterized the structure and function of intracellular loop 5 (IL5), specifically amino acids 431-443. Mutation of eleven residues to alanine caused partial or nearly complete inhibition of transport; notably, mutation of residues L432, T433, I436, N437, R440 and K443 demonstrated these residues had critical roles in NHE1 function independent of effects on targeting or expression. The nuclear magnetic resonance (NMR) solution spectra of the IL5 peptide in a membrane mimetic sodium dodecyl sulfate solution revealed that IL5 has a stable three-dimensional structure with substantial alpha helical character. NMR chemical shifts indicated that K438 was in close proximity with W434. Overall, our results show that IL5 is a critical, intracellular loop with a propensity to form an alpha helix, and many residues of this intracellular loop are critical to proton sensing and ion transport.

Sequential and controlled release of small molecules from poly(N-isopropylacrylamide) microgel-based reservoir devices.

Systems composed of a poly(N-isopropylacrylamide)-co-acrylic acid (pNIPAm-co-AAc) microgels (AAc-MG) and poly(N-isopropylacrylamide-3-(acrylamido)phenylboronic acid) (pNIPAm-co-APBA) microgels (APBA-MG) were used to sequentially release small molecules to a system in a controlled and triggered fashion. Specifically, at pH 10.0, methylene blue (MB, positively charged) exhibited strong electrostatic interactions with both the negatively charged AAc and APBA-modified microgels. This resulted in MB uptake into both of the microgels. At pH 7.0, the APBA groups were neutralized, allowing MB to be released from the APBA-MG only. When the solution pH was again lowered to 3.0, the AAc groups are neutralized allowing MB to be released from the AAc-MG. By incorporating the mixed microgels into reservoir devices, and varying their ratio, the small molecule release rate and release amount (dosage) can be easily tuned. Furthermore, two different small molecules can be loaded into the two distinct microgels, which allows for their sequential release at particular pHs. These devices could find use for delivering multiple drugs to a system in a controlled and triggered fashion, which may find a variety of biomedical applications.