By making targeted changes in Ca2+-sensing components of GCaMP3, we have generated a series of variants termed Fast-GCaMPs. Fast GCaMPs respond to Ca2+ with up to 20-fold improved kinetics and have affinities spanning the range of intracellular neuronal Ca2+ signals while retaining their permolecule brightness.
Recent research leading to improved GCaMP variants involved screening thousands of mutants generated by exhaustive mutagenesis to yield improvements in proteolytic stability and per-molecule fluorescence5,7
. Another effort has led to similar brightness improvements and modest kinetic improvements7
. Although a combinatorial approach can be effective at maximizing one parameter at a time, parameters such as KD
, decay response and rise response present a challenge because they are often linked to one another. Our results demonstrate that functional parameters of a GECI can be engineered without losing existing beneficial features, and can lead to kinetic improvements beyond previous fast-responding GECIs including TN-XL and GCaMP1.6 (decay τ 240–260 ms)30,31
For kinetic optimization, our quantitative evaluation of Fast-GCaMP variants required an evaluation method in which cellular calcium dynamics are not a rate-limiting factor. The time course of cellular fluorescence signals is limited by both calcium dynamics and probe response kinetics. As an illustration of why these factors matter, a recent GCaMP optimization effort12
showed an improvement in physiological off-responses from t1/2
=0.6 s using GCaMP3 to t1/2
=0.4 s using their variants of GCaMP6 and GCaMP8. However, those measurements were done in slice cultures in which calcium removal mechanisms were slower than in acute slices, as evidenced by the slow GCaMP3 responses. Our fastest physiological off-response times were several times faster, t1/2
0.1–0.2 s for RS06 and RS09, and we found that stopped-flow measurements on purified protein were faster still, with t1/2
7–30 ms. In addition, the variant of GCaMP6 produced by Ohkura et al
and GCaMP8 did not have shorter rising t1/2
than GCaMP3, whereas our Fast GCaMPs showed faster-rising responses than GCaMP3, both in Drosophila and in neocortical L2/3 neurons. Although these measurements all point toward our variants having the fastest responses, direct comparison of GCaMP kinetic performance will ultimately require either stopped-flow measurements or an expression system in which calcium signals are extremely rapid (for example, single spikes in unbuffered dendritic spines8
Our finding of a submillisecond response for calcium steps >KD
is consistent with previous observations on GCaMP1.6 (ref. 32
) and calmodulin itself33
. GCaMP may therefore have a low-affinity binding state capable of rapid transition to a high-fluorescence state. A likely rapid-binding candidate is the low-affinity pair of sites at the N-domain33
, an idea that is consistent with our observation that chelation by N-domain loops I and II is necessary to generate a functional probe.
A second target for perturbation was the interaction between CaM and its target. Upon Ca2+
binding, CaM must interact with RS20 to allow a conformational change to a high-fluorescence state. Ca2+
dissociation from the high-fluorescence state is energetically unfavored because RS20 binding increases Ca2+
. Consistent with this concept is the recent observation that alterations in a linker domain led to both strongly increased affinity and considerable slowing of off-responses, indicating that bound Ca2+
is effectively trapped6
. The relatively bright GECI YC-Nano15 has high affinity, making it useful for detecting single action potentials34
; however, high affinity is accompanied by extremely slow off-kinetics, precluding the tracking of successive spikes occurring at high frequency. The same difficulty is apparent for the faster GCaMP5 family of GECIs7
. Our findings demonstrate the converse point: perturbations to CaM—RS20 interactions decreased affinity and led to considerable speeding of off-responses.
We constructed a molecular dynamics model based on our observations. Several conditions had to be satisfied: (1) based on our results, the elimination of Ca2+
binding in any EF-hand loop resulted in reduced Ca2+
affinity, indicating cooperative interactions among the four EF-hand sites (Supplementary Tables S2 and S3
). (2) Deletion of residues from the RS20 peptide can severely disrupt probe activity (Supplementary Tables S2 and S3
), indicating a necessary role for RS20 in reaching both high fluorescence and high Ca2+
affinity. (3) Elimination of either loop I or loop II leads to a significant reduction in Rf
(), indicating a necessity for Ca2+
binding to both sites of the N-lobe to achieve protection of the chromophore and conformational changes that lead to high fluorescence. (4) The elimination of Ca2+
binding to loop III or loop IV led to reduced Ca2+
affinity and left the Rf
intact (), indicating that the C-lobe is required for high-affinity Ca2+
binding but not for chromophore protection. (5) Based on our discovery of the fast, submillisecond rise response and experimental evidence described by Faas et al
, binding of Ca2+
to the N-lobe occurs on a submillisecond timescale, with lower affinity than the slower-binding C-lobe ( and ).
We propose a kinetic model in which GCaMP has two pathways to a high-fluorescence state (). Loops I and II (N-lobe) begin in a low-affinity (1 μM) state, while loops III and IV (C-lobe) begin in a high-affinity (250 nM) state. In ‘C-like’ activation for small Ca2+
would bind to the C-lobe with slow kinetics. The bound C-lobe then acts via interactions with the RS20 domain to increase the calcium affinity of the N-lobe22
. After the N-lobe binds to Ca2+
, the entire CaM-RS20 complex shifts in conformation20
, leading to reduced chromophore-solvent access, leading to a high-fluorescence state.
A functional model for GCaMP molecular dynamics
The submillisecond responses we observe suggest a second possible kinetic pathway, in which high calcium levels can drive rapid binding to the low-affinity state of the N-lobe, which then would be sufficient to drive the CaM–RS20 conformational shift and chromophore protection. In this ‘N-like’ mode, C-lobe binding to Ca2+ is not required, as evidenced by the fact that elimination of loop III or IV Ca2+ binding sites leaves a functional (albeit low-affinity) probe. Finally, after removal of calcium, off-responses are limited in part by dissociation of the CaM–RS20 interface domain followed by Ca2+ unbinding.
A fruitful approach for future improvement would be to take advantage of high-throughput design methods, with which our approach is complementary. The mutations reported here can be integrated with recently reported high-Rf
. The residues altered for high response and high per-molecule fluorescence reside in different probe domains7
, opening the possibility of combinatorial approaches for continued improvement of performance. Another possibility that does not require further design improvements is to co-express multiple variants of differing affinities. Coexpression can expand detection range35
. Such a combination of GECIs would give performance that had lower apparent cooperativity than any single GECI.