Functional coupling of ryanodine receptors to K(Ca) channels in smooth muscle cells from rat cerebral arteries

Abstract

The relationship between Ca2+ release ('Ca2+ sparks') through ryanodine-sensitive Ca2+ 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 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 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 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 K(Ca) 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/F(o)) measured during a Ca2+ spark (mean 2.04 F/F(o) or ~310 nM Ca2+) appears to significantly underestimate the local Ca2+ that activates K(Ca) channels. These results indicate that the majority of ryanodine receptors that cause Ca2+ sparks are functionally coupled to K(Ca) channels in the surface membrane, providing direct support for the idea that Ca2+ sparks cause STOCs.