Faithful reporting of temporal patterns of intracellular Ca2+ dynamics requires the working range of indicators to match the signals. Current genetically encoded calmodulin-based fluorescent indicators are likely to distort fast Ca2+ signals by apparent saturation and integration due to their limiting fluorescence rise and decay kinetics. A series of probes was engineered with a range of Ca2+ affinities and accelerated kinetics by weakening the Ca2+-calmodulin-peptide interactions. At 37 °C, the GCaMP3-derived probe termed GCaMP3fast is 40-fold faster than GCaMP3 with Ca2+ decay and rise times, t1/2, of 3.3 ms and 0.9 ms, respectively, making it the fastest to-date. GCaMP3fast revealed discreet transients with significantly faster Ca2+ dynamics in neonatal cardiac myocytes than GCaMP6f. With 5-fold increased two-photon fluorescence cross-section for Ca2+ at 940 nm, GCaMP3fast is suitable for deep tissue studies. The green fluorescent protein serves as a reporter providing important novel insights into the kinetic mechanism of target recognition by calmodulin. Our strategy to match the probe to the signal by tuning the affinity and hence the Ca2+ kinetics of the indicator is applicable to the emerging new generations of calmodulin-based probes.
Acute hypoxia is thought to trigger protective responses that, in tissues like heart and carotid body, include rapid (5-10 s) suppression of Ca(2+) and K(+) channels. To gain insight into the mechanism for the suppression of the cardiac l-type Ca(2+) channel, we measured O2-dependent fluorescence in the immediate vicinity of voltage-clamped cardiac cells subjected to rapid exchange of solutions with different O2 tensions. This was accomplished with an experimental chamber with a glass bottom that was used as a light guide for excitation of a thin ruthenium-based O2-sensitive ORMOSIL coating. Fluorescence imaging showed that steady-state Po2 was well controlled within the entire stream from an electromagnetically controlled solution "puffer" but that changes were slower at the periphery of the stream (τ1/2 ∼ 500 ms) than immediately around the voltage-clamped myocyte (τ1/2 ∼ 225 ms) where, in turn, firmly attached cells produced an additional local delay of 50-100 ms. Performing simultaneous voltage clamp and O2 measurements, we found that acute hypoxia gradually and reversibly suppressed the Ca(2+) channel (CaV1.2). Using Ba(2+) as charge carrier, the suppression was significant after 1.5 s, reached ∼10% after 2.5 s, and was nearly completely reversible in 5 s. The described fluorescence measurements provide the means to check and fine tune solution puffers and suggest that changes in Po2 can be accomplished within ∼200 ms. The rapid and reversible suppression of barium current under hypoxia is consistent with the notion that the cardiac Ca(2+) channel is directly modulated by O2.
Familial Alzheimer's disease (FAD) is caused by mutations in presenilin-1 (PS1) gene in approximately 50% of cases. It was found that FAD PS1 mutants disrupt calcium homeostasis in hippocampal neurons disrupting Ca 2þ storage in the lumen of endoplasmic reticulum (ER). Recently calcium sensors of ER STIM1 were found to negatively regulate the activity of L-type voltagegated calcium channels. Therefore it was suggested that FAD PS1 mutants could affect the activity of L-type channels in neurons. To study the activity of voltage-gated calcium channels experiments with a patch-clamp technique in whole-cell mode were performed with human neuroblastoma SK-N-SH cell line transfected with PS1 M146V mutant or PS1 WT. Currents were induced by 10 mV voltage steps per 200 ms from À80 to þ40 mV. PS1 M146V mutant expression enchased the amplitude of integral current at positive potentials comparing to cells with expression of PS1 WT and untransfected control cells. Currents were found to be blocked by application 10 uM of nifedipine. Knock-down of STIM1 with shRNA abolished the difference between cells with mutant and PS1 WT expressions but in the same time mock shRNA left the difference unchanged. Knock-down of STIM1 was controlled by western-blot of cell lysates. Expression PS1 M146V enhanced the amplitude of calcium entry in mouse hippocampal neurons induced by depolarization with 140 KCl in calcium imaging experiments with Fura2-AM comparing to PS1 WT expressing cells. It was concluded that PS1 M146V mutant connected with FAD affect activity of L-type calcium channels through the STIM1 calcium sensors.
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