Functional Coupling of Ryanodine Receptors to KCa Channels in Smooth Muscle Cells from Rat Cerebral Arteries

The relationship between Ca2+ release (“Ca2+ sparks”) through ryanodine-sensitive Ca2+ release channels in the sarcoplasmic reticulum and KCa channels was examined in smooth muscle cells from rat cerebral arteries. Whole cell potassium currents at physiological membrane potentials (−40 mV) and intracellular Ca2+ were measured simultaneously, using the perforated patch clamp technique and a laser two-dimensional (x–y) scanning confocal microscope and the fluorescent Ca2+ indicator, fluo-3. Virtually all (96%) detectable Ca2+ sparks were associated with the activation of a spontaneous transient outward current (STOC) through KCa channels. A small number of sparks (5 of 128) were associated with currents smaller than 6 pA (mean amplitude, 4.7 pA, at −40 mV). Approximately 41% of STOCs occurred without a detectable Ca2+ spark. The amplitudes of the Ca2+ sparks correlated with the amplitudes of the STOCs (regression coefficient 0.8; P < 0.05). The half time of decay of Ca2+ sparks (56 ms) was longer than the associated STOCs (9 ms). The mean amplitude of the STOCs, which were associated with Ca2+ sparks, was 33 pA at −40 mV. The mean amplitude of the “sparkless” STOCs was smaller, 16 pA. The very significant increase in KCa channel open probability (>104-fold) during a Ca2+ spark is consistent with local Ca2+ during a spark being in the order of 1–100 μM. Therefore, the increase in fractional fluorescence (F/Fo) measured during a Ca2+ spark (mean 2.04 F/Fo or ∼310 nM Ca2+) appears to significantly underestimate the local Ca2+ that activates KCa channels. These results indicate that the majority of ryanodine receptors that cause Ca2+ sparks are functionally coupled to KCa channels in the surface membrane, providing direct support for the idea that Ca2+ sparks cause STOCs

A Computational Model of the Ionic Currents, Ca2+ Dynamics and Action Potentials Underlying Contraction of Isolated Uterine Smooth Muscle

Uterine contractions during labor are discretely regulated by rhythmic action potentials (AP) of varying duration and form that serve to determine calcium-dependent force production. We have employed a computational biology approach to develop a fuller understanding of the complexity of excitation-contraction (E-C) coupling of uterine smooth muscle cells (USMC). Our overall aim is to establish a mathematical platform of sufficient biophysical detail to quantitatively describe known uterine E-C coupling parameters and thereby inform future empirical investigations of physiological and pathophysiological mechanisms governing normal and dysfunctional labors. From published and unpublished data we construct mathematical models for fourteen ionic currents of USMCs: currents (L- and T-type), current, an hyperpolarization-activated current, three voltage-gated currents, two -activated current, -activated current, non-specific cation current, - exchanger, - pump and background current. The magnitudes and kinetics of each current system in a spindle shaped single cell with a specified surface area∶volume ratio is described by differential equations, in terms of maximal conductances, electrochemical gradient, voltage-dependent activation/inactivation gating variables and temporal changes in intracellular computed from known fluxes. These quantifications are validated by the reconstruction of the individual experimental ionic currents obtained under voltage-clamp. Phasic contraction is modeled in relation to the time constant of changing . This integrated model is validated by its reconstruction of the different USMC AP configurations (spikes, plateau and bursts of spikes), the change from bursting to plateau type AP produced by estradiol and of simultaneous experimental recordings of spontaneous AP, and phasic force. In summary, our advanced mathematical model provides a powerful tool to investigate the physiological ionic mechanisms underlying the genesis of uterine electrical E-C coupling of labor and parturition. This will furnish the evolution of descriptive and predictive quantitative models of myometrial electrogenesis at the whole cell and tissue levels