Extrapolating microdomain Ca(2+) dynamics using BK channels as a Ca(2+) sensor.

Ca2+ ions are perhaps the most important second messenger for triggering a variety of biological functions and often signal in a highly compartmentalized manner. For example, fast release of synaptic vesicles from presynaptic terminals is triggered by Ca2+ influx via voltage-gated Ca2+ channels during action potentials, and the local high concentration of Ca2+ transients at the active zones is critical for gating synchronized fusion of synaptic vesicles (SV) and transmitter release. Experimental measurements of global Ca2+ concentrations with Ca2+ indicators and mathematical modeling extrapolate that the peak Ca2+ concentration seen by the Ca2+ sensor on SVs briefly reach tens and even hundreds of micromole levels. Although tremendous progress in recent years has been made with developing novel fluorescent chemical or protein Ca2+ sensors, limited spatiotemporal resolution of these sensors presents a major challenge to directly read out local Ca2+ transients in real-time. Large-conductance Ca-activated potassium channels (BK channels), uniquely sensitive to both membrane potential and intracellular Ca2+, abundantly distributed in the excitable cells, regulate the membrane excitability and electrical signals in response to the Ca2+-influx from the Ca2+-permeable channels1,2. The BK channel encoded by Slo1 gene contains two calcium binding sites in the regulator of conductance for K+ (RCK) domains of the caboxy-terminal region3,4 and may potentially serve as an ideal sensor of local Ca2+ rise. However, the affinity of these binding sites is primarily determined under the circumstance of Ca2+ uniformly sojourning to its binding sites at equilibrium with very little consideration of dynamics of Ca2+ influx or release. Although elaborate Markov models containing multiple parallel open and closed states have been developed to simulate both voltage- and Ca2+ dependent gating kinetics of BK channels well5,6,7, the forward binding rate constant of Ca2+ (kb) remains unknown, making model parameters too unconstrained to meaningfully profile local Ca2+ dynamics. Previous experiments in inside-out patch configuration have attempted to directly measure kb by ultrafast Ca2+ concentration jumps via a piezoelectric stepper of two barrel theta pipette8,9, which enables a solution exchange in less than 1 ms. However, the patch membrane usually invaginates into the pipette tip and forms Ω-shape geometry, slowing the diffusion of Ca2+ (~10 ms) to reach the inner face of the membrane patch where the RCK domain of BK channels situates9. To extrapolate the local Ca2+ dynamics using BK channels as a sensor, it is therefore necessary to develop a superfast approach of Ca2+ delivery mimicking calcium influxes via calcium channels induced by action potentials, and precisely measure kb in order to quantitatively describe the kinetics of BK channels to such fast Ca2+ transients. In this study, we have applied laser flash photolysis technique of the caged-Ca2+ compound (e.g. NP-EGTA) to achieve instantaneous Ca2+ rises, which has been widely used for studying Ca2+-dependent processes such as the secretory responses10. After a UV flash-induced photolysis, the intracellular calcium concentration have two phases of rise, a fast transient Ca2+ rise with peak concentrations up to tens of micromole from the basal [Ca2+]i of ~10–200 nM in sub-milliseconds and a slow uniformly steady-state elevation of global [Ca2+]i 11,12,13. We took advantage of biphasic properties with laser photolysis of the caged-Ca2+ compound to examine both voltage- and calcium-dependent gating behavior, and determined the Ca2+ forward binding rate kb for BK. Our results demonstrate that BK channels have higher calcium-sensitivity capable to follow up to tens of μM transient Ca2+ changes 0.1–0.2 ms, and established a quantitative model for its utility as the fast local Ca2+ sensor to profile the local Ca2+ transients during action potential firing.