Fast-Response Calmodulin-Based Fluorescent Indicators Reveal Rapid Intracellular Calcium Dynamics.

Nordine Helassa,John Scaringi,Ianina Conte,Elric Esposito,Jonathan Bradley,Martin Morad,Katalin Török,David Ogden,Xiao-Hua Zhang,Thomas Carter

doi:10.1038/srep15978

Abstract

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.

Highlights

GCaMP3fast is suitable for deep tissue studies
We have previously shown that the mutation of the Trp residue to Tyr in a RS20-related peptide increases the Kd of the complex for Ca2+.CaM19,20
Consistent with our predictions, CaM EF-hand and peptide RS-1 mutations of GCaMP3 resulted in increasing the Kd for Ca2+ binding from 330 nM to 1–3 μ M

Summary

Introduction

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. Mutations to the linker regions and to the cpEGFP domain itself resulted in greater fluorescence enhancements on Ca2+ binding by shifting the equilibrium towards the deprotonated state and/or increasing the brightness of the deprotonated form These mutations, left unchanged the high affinity (equilibrium dissociation constant Kd in the range of hundreds of nM) and concomitant slow response kinetics of the probes to Ca2+. In excitable cells monitoring fast Ca2+ fluxes requires millisecond time resolution and fluorescence decay and rise rates of up to 1000 s−1 These issues have begun to be addressed[17,18], to-date there are no GECI probes sufficiently rapid for accurate tracking of fast or high-frequency Ca2+ transients associated with synaptic transmission and activation of skeletal and cardiac muscle

Objectives

Methods

Results

Conclusion