A. L. Escobar

Luminal Ca2+ Controls Termination and Refractory Behavior of Ca2+-Induced Ca2+ Release in Cardiac Myocytes

Abstract— Despite extensive research, the mechanisms responsible for the graded nature and early termination of Ca2+-induced Ca2+ release (CICR) from the sarcoplasmic reticulum (SR) in cardiac muscle remain poorly understood. Suggested mechanisms include cytosolic Ca2+-dependent inactivation/adaptation and luminal Ca2+-dependent deactivtion of the SR Ca2+ release channels/ryanodine receptors (RyRs). To explore the importance of cytosolic versus luminal Ca2+ regulatory mechanisms in controlling CICR, we assessed the impact of intra-SR Ca2+ buffering on global and local Ca2+ release properties of patch-clamped or permeabilized rat ventricular myocytes. Exogenous, low-affinity Ca2+ buffers (5 to 20 mmol/L ADA, citrate or maleate) were introduced into the SR by exposing the cells to “internal” solutions containing the buffers. Enhanced Ca2+ buffering in the SR was confirmed by an increase in the total SR Ca2+ content, as revealed by application of caffeine. At the whole-cell level, intra-SR [Ca2+] buffering dramatically increased the magnitude of Ca2+ transients induced by ICa and deranged the smoothly graded ICa-SR Ca2+ release relationship. The amplitude and time-to-peak of local Ca2+ release events, Ca2+ sparks, as well as the duration of local Ca2+ release fluxes underlying sparks were increased up to 2- to 3-fold. The exogenous Ca2+ buffers in the SR also reduced the frequency of repetitive activity observed at individual release sites in the presence of the RyR activator Imperatoxin A. We conclude that regulation of RyR openings by local intra-SR [Ca2+] is responsible for termination of CICR and for the subsequent restitution behavior of Ca2+ release sites in cardiac muscle.

Kinetic properties of DM-nitrophen and calcium indicators: rapid transient response to flash photolysis

Abstract We describe a high temporal resolution confocal spot microfluorimetry setup which makes possible the detection of fluorescence transients elicited by Ca2+ indicators in response to large (50–200 μM), short duration (<100 ns), free [Ca2+] transients generated by laser flash photolysis of DM-nitrophen (DM-n; caged Ca2+). The equilibrium and kinetic properties of the commercially available indicators Fluo-3, Rhod-2, CalciumOrange-5N (COr-5N) and CalciumGreen-2 (CGr-2) were determined experimentally. The data reveal that COr-5N displays simple, fast response kinetics while, in contrast, Fluo-3, Rhod-2 and CGr-2 are characterized by significantly slower kinetic properties. These latter indicators may be unsuitable for tracking Ca2+ signaling events lasting only a few milliseconds. A model which accurately predicts the time course of fluorescence transients in response to rapid free [Ca2+] changes was developed. Experimental data and model predictions concur only when the association rate constant of DM-n is approximately 20 times faster than previously reported. This work establishes a quantitative theoretical framework for the study of fast Ca2+ signaling events and the use of flash photolysis in cells and model systems.

Detection of Ca2+‐transients elicited by flash photolysis of DM‐nitrophen with a fast calcium indicator

A confocal spot detection optical setup was used to record fluorescence signals in response to calcium pulses, elicited by flash photolysis of DM‐nitrophen, with the calcium indicators CaOrange‐5N and Fluo‐3. Our results yield the following conclusions: [Ca2+] changes are almost perfect spikes at pCa 9 and broader transients followed by a step at pCa 7. The [Ca2+] spikes were used to measure the dissociation rate constant of the Ca2+ dyes. Experiments at pCa 7 were used to verify the kinetic rate constants of the dyes and to obtain those of DM‐nitrophen. The association rate constant of this compound was found to be more than one order of magnitude faster than that suggested previously. CaOrange‐5N was able to track changes in [Ca2+] more accurately than Fluo‐3. This latter dye introduced severe distortions which preclude a quantitative deconvolution of the fluorescence transients into changes in the free [Ca2+].

Calcium regulation of single ryanodine receptor channel gating analyzed using HMM/MCMC statistical methods.

Type-II ryanodine receptor channels (RYRs) play a fundamental role in intracellular Ca2+ dynamics in heart. The processes of activation, inactivation, and regulation of these channels have been the subject of intensive research and the focus of recent debates. Typically, approaches to understand these processes involve statistical analysis of single RYRs, involving signal restoration, model estimation, and selection. These tasks are usually performed by following rather phenomenological criteria that turn models into self-fulfilling prophecies. Here, a thorough statistical treatment is applied by modeling single RYRs using aggregated hidden Markov models. Inferences are made using Bayesian statistics and stochastic search methods known as Markov chain Monte Carlo. These methods allow extension of the temporal resolution of the analysis far beyond the limits of previous approaches and provide a direct measure of the uncertainties associated with every estimation step, together with a direct assessment of why and where a particular model fails. Analyses of single RYRs at several Ca2+ concentrations are made by considering 16 models, some of them previously reported in the literature. Results clearly show that single RYRs have Ca2+-dependent gating modes. Moreover, our results demonstrate that single RYRs responding to a sudden change in Ca2+ display adaptation kinetics. Interestingly, best ranked models predict microscopic reversibility when monovalent cations are used as the main permeating species. Finally, the extended bandwidth revealed the existence of novel fast buzz-mode at low Ca2+ concentrations.

Analysis of macroscopic ionic currents mediated by GABAρ1 receptors during lanthanide modulation predicts novel states controlling channel gating

Lanthanide‐induced modulation of GABAC receptors expressed in Xenopus oocytes was studied. We obtained two‐electrode voltage‐clamp recordings of ionic currents mediated by recombinant homomeric GABAρ1 receptors and performed numerical simulations of kinetic models of the macroscopic ionic currents. GABA‐evoked chloride currents were potentiated by La3+, Lu3+ and Gd3+ in the micromolar range. Lanthanide effects were rapid, reversible and voltage independent. The degree of potentiation was reduced by increasing GABA concentration. Lu3+ also induced receptor desensitization and decreased the deactivation rate of GABAρ1 currents. In the presence of 300 μM Lu3+, dose–response curves for GABA‐evoked currents showed a significant enhancement of the maximum amplitude and an increase of the apparent affinity. The rate of onset of TPMPA and picrotoxin antagonism of GABAρ1 receptors was modulated by Lu3+. These results suggest that the potentiation of the anionic current was the result of a direct lanthanide–receptor interaction at a site capable of allosterically modulating channel properties. Based on kinetic schemes, which included a second open state and a nonconducting desensitized state that closely reproduced the experimental results, two nonexclusive probable models of GABAρ1 channels gating are proposed.

Spatial Ca2+ Distribution in Contracting Skeletal and Cardiac Muscle Cells

The spatiotemporal distribution of intracellular Ca(2+) release in contracting skeletal and cardiac muscle cells was defined using a snapshot imaging technique. Calcium imaging was performed on intact skeletal and cardiac muscle cells during contractions induced by an action potential (AP). The sarcomere length of the skeletal and cardiac cells was approximately 2 micrometer. Imaging Rhod-2 fluorescence only during a very brief (7 ns) snapshot of excitation light minimized potential image-blurring artifacts due to movement and/or diffusion. In skeletal muscle cells, the AP triggered a large fast Ca(2+) transient that peaked in less than 3 ms. Distinct subsarcomeric Ca(2+) gradients were evident during the first 4 ms of the skeletal Ca(2+) transient. In cardiac muscle, the AP-triggered Ca(2+) transient was much slower and peaked in approximately 100 ms. In contrast to the skeletal case, there were no detectable subsarcomeric Ca(2+) gradients during the cardiac Ca(2+) transient. Theoretical simulations suggest that the subsarcomeric Ca(2+) gradients seen in skeletal muscle were detectable because of the high speed and synchrony of local Ca(2+) release. Slower asynchronous recruitment of local Ca(2+) release units may account for the absence of detectable subsarcomeric Ca(2+) gradients in cardiac muscle. The speed and synchrony of local Ca(2+) gradients are quite different in AP-activated contracting cardiac and skeletal muscle cells at normal resting sarcomere lengths.