We have used whole-cell and perforated patches to study ionic currents induced by hypotonic extracellular solutions (HTS, 185 mOsm instead of 290 mOsm) in endothelial cells from human umbilical veins. These currents activated within 30-50 s after application of HTS, reached a maximum value after ~50-150 s and recovered completely after re-exposing the cells to normal osmolarity. They slowly inactivated at potentials positive to +50 mV. The same current was also activated by breaking into endothelial cells with a hypertonic pipette solution (377 mOsm instead of 290 mOsm). The reversal potential of these volume-induced currents using different extracellular and intracellular C1-concentrations was always close to the Cl--eqnilibrium potential. These currents are therefore mainly carried by CI-. DIDS only weakly blocked the current (KI = 120 I~M), while another C1--channel blocker, DCDPC (20 IxM) was ineffective.We were unable to record single channel activity in cell-attached patches but we always observed an increased current variance during HTS. From the mean current-variance relation of the whole-cell current records, we determined a single channel conductance of 1.1 pS. The size and kinetics of the current were not correlated with the concomitant changes in intracellular calcium. Furthermore, the currents could still be activated in the presence of 10 mmol/liter intracellular EGTA and are thus Ca 2+ independent.A similar current was also activated with iso-osmotic pipette solutions containing 300 ~mol/liter GTP~/S. Neomycin (1 mmol/liter), a blocker of PLC, did not prevent activation of this current. TPA (4 ixmol/liter) was also ineffective in modulation of this current.The HTS-induced current was completely blocked by 10 Ixmol/liter pBPB, a PLA2 inhibitor. NDGA (4 tzmol/liter) and indomethacin (5 txmol/liter), blockers of lipoxygenase and cyclo-oxygenase respectively, did however not affect the current induced by hypotonic solutions. The effects of arachidonic acid (10 I~mol/liter) were variable. In 12 out of 40 cells it either directly activated a CI-current or potentiated the current activated by HTS. The membrane current was decreased at all potentials

The local control concept of excitation–contraction coupling in the heart postulates that the activity of the sarcoplasmic reticulum ryanodine receptor channels (RyR) is controlled by Ca2+ entry through adjoining sarcolemmal single dihydropyridine receptor channels (DHPRs). One unverified premise of this hypothesis is that the RyR must be fast enough to track the brief (<0.5 ms) Ca2+ elevations accompanying single DHPR channel openings. To define the kinetic limits of effective trigger Ca2+ signals, we recorded activity of single cardiac RyRs in lipid bilayers during rapid and transient increases in Ca2+ generated by flash photolysis of DM-nitrophen. Application of such Ca2+ spikes (amplitude ∼10–30 μM, duration ∼0.1–0.4 ms) resulted in activation of the RyRs with a probability that increased steeply (apparent Hill slope ∼2.5) with spike amplitude. The time constants of RyR activation were 0.07–0.27 ms, decreasing with spike amplitude. To fit the rising portion of the open probability, a single exponential function had to be raised to a power n ∼ 3. We show that these data could be adequately described with a gating scheme incorporating four sequential Ca2+-sensitive closed states between the resting and the first open states. These results provide evidence that brief Ca2+ triggers are adequate to activate the RyR, and support the possibility that RyR channels are governed by single DHPR openings. They also provide evidence for the assumption that RyR activation requires binding of multiple Ca2+ ions in accordance with the tetrameric organization of the channel protein.

Cytosolic calcium concentration in resting cardiac myocytes locally fluctuates as a result of spontaneous microscopic Ca2+ releases or abruptly rises as a result of an external trigger. These processes, observed as calcium sparks, are fundamental for proper function of cardiac muscle. In this study, we analyze how the characteristics of spontaneous and triggered calcium sparks are related to cardiac ryanodine receptor (RYR) gating. We show that the frequency of spontaneous sparks and the probability distribution of calcium release flux quanta of triggered sparks correspond quantitatively to predictions of an allosteric homotetrameric model of RYR gating. This model includes competitive binding of Ca2+ and Mg2+ ions to the RYR activation sites and allosteric interaction between divalent ion binding and channel opening. It turns out that at rest, RYRs are almost fully occupied by Mg2+. Therefore, spontaneous sparks are most frequently evoked by random openings of the highly populated but rarely opening Mg4RYR and CaMg3RYR forms, whereas triggered sparks are most frequently evoked by random openings of the less populated but much more readily opening Ca2Mg2RYR and Ca3MgRYR forms. In both the spontaneous and the triggered sparks, only a small fraction of RYRs in the calcium release unit manages to open during the spark because of the limited rate of Mg2+ unbinding. This mechanism clarifies the unexpectedly low calcium release flux during elementary release events and unifies the theory of calcium signaling in resting and contracting cardiac myocytes.

The local calcium release flux signals (calcium spikes) evoked by membrane depolarization were recorded at high temporal resolution (2000 lines s −1 ) in isolated ventricular myocytes of male rats, using combination of scanning confocal microscopy and the patch-clamp technique. The kinetic properties of calcium spikes were investigated. The time course of calcium spike activation could be described reliably by a model with higher-order (n = 3) kinetics, but not by a first-order exponential process. A model of calcium spike with calcium release termination coupled to its activation was preferential to a model with the release termination independent of its activation. Three fluorescent calcium dyes (OG-5N, fluo-3, and fluo-4) were compared for calcium spike measurements. Experimental measurements as well as simulations showed that the occurrence and latency of calcium spikes could be measured faithfully with all indicators, while the kinetics of calcium spikes was reliably traced only with OG-5N. Calcium spikes evoked by a step depolarization from −50 to 0 mV commenced with a mean latency of 4.1 ± 0.2 ms and peaked 6.7 ± 0.2 ms later. Their full amplitudes were normally distributed. The activation time constant of calcium spikes was 3.1 ± 0.1 ms, and the time constant of termination was 5.5 ± 0.2 ms. A negative correlation was observed between the observed amplitude of calcium spikes and their time constant of activation, but there was no correlation between their observed amplitude and time constant of termination, in agreement with the concept of steep calcium-dependent activation and fateful inactivation of calcium release flux.

Single channel activity of the cardiac ryanodine-sensitive calcium-release channel in planar lipid membranes was studied in order to elucidate the calcium-dependent mechanism of its steady-state behavior. The single channel kinetics, observed with Cs+ as the charge carrier at different activating (cis) Ca2+ concentrations in the absence of ATP and Mg2+, were similar to earlier reports and were extended by analysis of channel modal behavior. The channel displayed three episodic levels of open probability defining three gating modes: H (high activity), L (low activity), and I (no activity). The large difference in open probabilities between the two active modes resulted from different bursting patterns and different proportions of two distinct channel open states. I-mode was without openings and can be regarded as the inactivated mode of the channel; L-mode was composed of short and sparse openings; and H-mode openings were longer and grouped into bursts. Modal gating may explain calcium-release channel adaptation (as transient prevalence of H-mode after Ca2+ binding) and the inhibitory effects of drugs (as stabilization of mode I), and it provides a basis for understanding the regulation of calcium release.