26 research outputs found
GAP, an aequorin-based fluorescent indicator for imaging Ca2+ in organelles
ProducciĂłn CientĂficaGenetically encoded calcium indicators allow monitoring subcellular
Ca2+ signals inside organelles. Most genetically encoded calcium
indicators are fusions of endogenous calcium-binding proteins
whose functionality in vivo may be perturbed by competition with
cellular partners.We describe here a novel family of fluorescent Ca2+
sensors based on the fusion of two Aequorea victoria proteins, GFP
and apo-aequorin (GAP). GAP exhibited a unique combination
of features: dual-excitation ratiometric imaging, high dynamic
range, good signal-to-noise ratio, insensitivity to pH and Mg2+,
tunable Ca2+ affinity, uncomplicated calibration, and targetability
to five distinct organelles. Moreover, transgenic mice for endoplasmic
reticulum-targeted GAP exhibited a robust long-term
expression that correlated well with its reproducible performance in
various neural tissues. This biosensor fills a gap in the actual repertoire
of Ca2+ indicators for organelles and becomes a valuable tool
for in vivo Ca2+ imaging applications
Interpretation of comparative LCAs: external normalization and a method of mutual differences
Protective effects of GTM-1 on endoplasmic reticulum stress induced by thapsgargin in rat neurons
GFP-Aequorin Protein Sensor for Ex Vivo and In Vivo Imaging of Ca 2+ Dynamics in High-Ca 2+ Organelles
Stereoselective effects of the enantiomers of a new local anaesthetic, IQB-9302, on a human cardiac potassium channel (Kv1.5)
1. The N-substituent of IQB-9302 has the same number of carbons as bupivacaine, but it exhibits a different spatial localization (n-butyl vs cyclopropylmethyl). Thus, the study of the effects of IQB-9302 enantiomers on hKv1.5 channels will lead to a better knowledge of the determinants of stereoselective block. 2. The effects of the IQB-9302 enantiomers were studied on hKv1.5 channels stably expressed in Ltk(â) cells using the whole-cell configuration of the patch-clamp technique. Drug molecular modelling was performed using Hyperchem software. 3. Block induced by IQB-9302 was stereoselective with the R(+) enantiomer being 3.2-fold more potent than the S(â) one (K(D) of 17.8±0.5âÎŒM vs 58.6±4.0âÎŒM). 4. S(â)- and R(+)IQB-9302 induced-block was time- and voltage-dependent consistent with an electrical distance from the cytoplasmic side of 0.173±0.022 (n=12) and 0.181±0.018 (n=10), respectively. 5. Potency of block of pipecoloxylidide local anaesthetics was linearly related to the length between the cationic tertiary amine and the end of the substituent. 6. Molecular modelling shows that only when S(â) and R(+) enantiomers are superimposed by their aromatic ring, their N-substituents are in opposite directions, which can explain the stereospecific block induced by bupivacaine and IQB-9302 with hKv1.5 channels. 7. These results suggest that: (a) IQB-9302 enantiomers block the open state of hKv1.5 channels, and (b) the length of the N-substituent in these local anaesthetics and not its volume determines the potency and degree of their stereoselective hKv1.5 channel block