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

Abstract

The relationship between Ca ²⁺ release (“Ca ²⁺ sparks”) through ryanodine-sensitive Ca ²⁺ release channels in the sarcoplasmic reticulum and K _Ca channels was examined in smooth muscle cells from rat cerebral arteries. Whole cell potassium currents at physiological membrane potentials (−40 mV) and intracellular Ca ²⁺ were measured simultaneously, using the perforated patch clamp technique and a laser two-dimensional (x–y) scanning confocal microscope and the fluorescent Ca ²⁺ indicator, fluo-3. Virtually all (96%) detectable Ca ²⁺ sparks were associated with the activation of a spontaneous transient outward current (STOC) through K _Ca 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 Ca ²⁺ spark. The amplitudes of the Ca ²⁺ sparks correlated with the amplitudes of the STOCs (regression coefficient 0.8; P < 0.05). The half time of decay of Ca ²⁺ sparks (56 ms) was longer than the associated STOCs (9 ms). The mean amplitude of the STOCs, which were associated with Ca ²⁺ sparks, was 33 pA at −40 mV. The mean amplitude of the “sparkless” STOCs was smaller, 16 pA. The very significant increase in K _Ca channel open probability (>10 ⁴-fold) during a Ca ²⁺ spark is consistent with local Ca ²⁺ during a spark being in the order of 1–100 μM. Therefore, the increase in fractional fluorescence (F/F _o) measured during a Ca ²⁺ spark (mean 2.04 F/F _o or ∼310 nM Ca ²⁺) appears to significantly underestimate the local Ca ²⁺ that activates K _Ca channels. These results indicate that the majority of ryanodine receptors that cause Ca ²⁺ sparks are functionally coupled to K _Ca channels in the surface membrane, providing direct support for the idea that Ca ²⁺ sparks cause STOCs.