Nanometer Resolution of Cav-Generated Ca2+ Gradients

Diffusion theory predicts that each pA of Ca2+ flux through a voltage-gated Ca2+ channel (CaV) should generate ∼100 μM free [Ca2+] in the channel nanodomain, with steep spatial gradient and little sensitivity to intracellular Ca2+ buffers. This is widely quoted, and bears on synaptic transmission, activity-dependent plasticity, and excitation-contraction coupling. Yet, experimental verification has been difficult because diffusible Ca2+ indicators report space-averaged [Ca2+], and CaV-tethered fluorescent indicators cannot distinguish between active and silent channels. Here, we used Ca2+/calmodulin-dependent inactivation (CDI) of CaV1.3 channels themselves as a nearfield indicator of nanodomain [Ca2+], noting that CDI provides an ionic readout based only on active channels. We calibrated the [Ca2+]-sensitivity of CDI by monitoring the decay of Li+ currents following spatially-uniform Ca2+ uncaging steps. We then ran Ca2+ through the same channels to generate local [Ca2+] gradients, and titrated the external Ca2+ concentration until CDI diminished ∼50%. The corresponding unitary flux was resolved through a combination of single-channel and whole-cell recordings, enabling determination of the relationship between unitary Ca2+ flux and nanodomain [Ca2+]. Contrary to theory, nanodomain [Ca2+] signals are larger and more buffer-sensitive than expected: ∼800 μM/pA in 10 mM intracellular BAPTA, and ∼200 μM/pA in 60 mM BAPTA. These findings indicate that diffusion of free Ca2+ ions is ∼10-fold slower in the nanodomain versus other regions of the cell, and that calmodulin (the Ca2+-sensing subunit for CDI) is ∼7 nm from the channel pore. Our results suggest that the nanodomain is a tortuous environment, where a Ca2+ ion would collide with an obstacle in 9 out of every 10 of its Brownian movements. Under physiological buffering, we predict that CaVs, by virtue of their crowded nanodomains, convert small Ca2+ fluxes into enormous Ca2+ concentrations, ∼1 mM/pA over distances of 10-20 nm.