Significance: Excitatory synapses convert presynaptic action potentials into chemical signals that are sensed by postsynaptic glutamate receptors. To eavesdrop on synaptic transmission, genetically encoded fluorescent sensors for glutamate have been developed. However, even the best available sensors lag behind the very fast glutamate dynamics in the synaptic cleft. Here, we report the development of an ultrafast genetically encoded glutamate sensor, iGlu _u , which allowed us to image glutamate clearance and synaptic depression during 100-Hz spike trains. We found that only boutons showing paired-pulse facilitation were able to rapidly recover from depression. Thus, presynaptic boutons act as frequency-specific filters to transmit select features of the spike train to specific postsynaptic cells.

A fluorescent calmodulin derivative, 2-chloro-[4-(ϵ-amino-Lys⁷⁵)]-[6-(4-diethylaminophenyl)-1,3,5-triazin-4-yl]-calmodulin (TA-calmodulin) [Török and Trentham (1994) Biochemistry 33, 12807–12820], and equilibrium fluorescence methods were used to identify calmodulin-binding domains of connexin subunits of gap junctions. Synthetic peptides corresponding to six extramembrane regions of connexin 32, a major component of rat liver gap junctions, and peptides derived from connexin 43 and 26, were tested. Two cytoplasmically oriented peptides that correspond to an N-terminal 21-amino-acid sequence and a 15-amino-acid sequence at the C-terminal tail of connexin 32 bound TA-calmodulin in a Ca²⁺-dependent manner. The dissociation constants (K_d) of TA-calmodulin binding to GAP 10 (MNWTGLYTLLSGVNRHSTAIG, residues 1–21) and GAP 8M (ACARRAQRRSNPPSR, residues 216–230) were 27 nM and 1.2 μM respectively at 150 mM ionic strength, 2 mM MgCl₂, 100 μM CaCl₂, pH 7.0 and 21 °C. Both halves of each peptide were required for calmodulin binding. Substitution of Trp³ present in all connexins by Tyr increasedK_d for TA-calmodulin by 40-fold. Liver gap junctions (whose connexons contain mainly connexin 32) and recombinant connexons constructed of connexin 26 expressed by baculovirus-infected insect cells exhibited weaker binding of TA-calmodulin with variable Ca²⁺-dependence. These studies identify two calmodulin-binding amino-acid sequences in connexin 32, and provide independent evidence that calmodulin may function as an intracellular ligand, regulating Ca²⁺-dependent intercellular communication across gap junctions.

Calmodulin-based genetically encoded fluorescent calcium indicators (GCaMP-s) are powerful tools of imaging calcium dynamics from cells to freely moving animals. High affinity indicators with slow kinetics however distort the temporal profile of calcium transients. Here we report the development of reduced affinity ultrafast variants of GCaMP6s and GCaMP6f. We hypothesized that GCaMP-s have a common kinetic mechanism with a rate-limiting process in the interaction of the RS20 peptide and calcium-calmodulin. Therefore we targeted specific residues in the binding interface by rational design generating improved indicators with GCaMP6f_u displaying fluorescence rise and decay times (t_1/2) of 1 and 3 ms (37 °C) in vitro, 9 and 22-fold faster than GCaMP6f respectively. In HEK293T cells, GCaMP6f_u revealed a 4-fold faster decay of ATP-evoked intracellular calcium transients than GCaMP6f. Stimulation of hippocampal CA1 pyramidal neurons with five action potentials fired at 100 Hz resulted in a single dendritic calcium transient with a 2-fold faster rise and 7-fold faster decay time (t_1/2 of 40 ms) than GCaMP6f, indicating that tracking high frequency action potentials may be limited by calcium dynamics. We propose that the design strategy used for generating GCaMP6f_u is applicable for the acceleration of the response kinetics of GCaMP-type calcium indicators.