Spontaneous Transient Outward Currents Arise from Microdomains Where BK Channels Are Exposed to a Mean Ca2+ Concentration on the Order of 10 μM during a Ca2+ Spark

Abstract

Ca2+ sparks are small, localized cytosolic Ca2+ transients due to Ca2+ release from sarcoplasmic reticulum through ryanodine receptors. In smooth muscle, Ca2+ sparks activate large conductance Ca2+-activated K+ channels (BK channels) in the spark microdomain, thus generating spontaneous transient outward currents (STOCs). The purpose of the present study is to determine experimentally the level of Ca2+ to which the BK channels are exposed during a spark. Using tight seal, whole-cell recording, we have analyzed the voltage-dependence of the STOC conductance (g(STOC)), and compared it to the voltage-dependence of BK channel activation in excised patches in the presence of different [Ca2+]s. The Ca2+ sparks did not change in amplitude over the range of potentials of interest. In contrast, the magnitude of g(STOC) remained roughly constant from 20 to −40 mV and then declined steeply at more negative potentials. From this and the voltage dependence of BK channel activation, we conclude that the BK channels underlying STOCs are exposed to a mean [Ca2+] on the order of 10 μM during a Ca2+ spark. The membrane area over which a concentration ≥10 μM is reached has an estimated radius of 150–300 nm, corresponding to an area which is a fraction of one square micron. Moreover, given the constraints imposed by the estimated channel density and the Ca2+ current during a spark, the BK channels do not appear to be uniformly distributed over the membrane but instead are found at higher density at the spark site.

RACK1 is a BKCa channel binding protein

“…This is relevant here because in smooth muscle, BK Ca channels open release events from the sarcoplasmic reticulum. These sparks activate nearby BK Ca channels on the plasma membrane, creating outward BK Ca currents known as spontaneous transient outward currents (30,32,60). The frequency and amplitude of spontaneous transient outward currents then influence membrane potential and indirectly smooth muscle tone (16,32,54).…”

“…When the ␣ subunit was expressed with RACK1, the channel's G-V relation with 10 M internal [Ca 2ϩ ] was rightshifted from a half-maximal activation voltage of 59 Ϯ 3.5 (SE) mV to 72 Ϯ 4.4 (SE) mV (P ϭ 0.037) (Fig. 5C) ] was used in this experiment because that is the Ca 2ϩ concentration BK Ca channels are thought to be exposed to in smooth muscle during their natural stimulus, the Ca 2ϩ spark (34,35,60). Surprisingly, however, when the same experiment was performed with the BK Ca ␤1 subunit present, RACK1 no longer shifted the channel's G-V relation (Fig.…”

Inositol 1,4,5-trisphosphate receptors modulate Ca2+ sparks and Ca2+ store content in vas deferens myocytes

“…It is well established that Ca 2ϩ sparks can activate Ca 2ϩ -sensitive conductances in the adjacent plasma membrane of smooth muscle cells. In the case of Ca 2ϩ -activated K ϩ channels, the high [Ca 2ϩ ] generated by localized Ca 2ϩ release into the microdomain adjacent to the membrane increases K ϩ channel open probability, resulting in a STOC (38,39). It follows, therefore, that interventions that increase spark activity are also likely to promote STOCs.…”

The BK channel: a vital link between cellular calcium and electrical signaling

“…Although it was initially unclear whether ER Ca 2+ release was sufficient to activate BK channels, it was discovered that highly localized Ca 2+ release events, known as "Ca 2+ sparks", are responsible. A Ca 2+ spark results from concerted opening of several ryanodine receptor (RYR) channels, which form apparent microdomains where the ER membrane in close proximity to the plasma membrane, localized near BK channels (ZhuGe et al, 1998(ZhuGe et al, , 2002. Within these microdomains, local [Ca 2+ ] can reach 10 μmol/L, which is sufficient for activation of BK channels leading to membrane repolarization (ZhuGe et al, 2002).…”

“…A Ca 2+ spark results from concerted opening of several ryanodine receptor (RYR) channels, which form apparent microdomains where the ER membrane in close proximity to the plasma membrane, localized near BK channels (ZhuGe et al, 1998(ZhuGe et al, , 2002. Within these microdomains, local [Ca 2+ ] can reach 10 μmol/L, which is sufficient for activation of BK channels leading to membrane repolarization (ZhuGe et al, 2002). Through this repolarization effect, BK channel activity deactivates voltage-dependent Ca 2+ channels at the plasma membrane, limiting both Ca 2+ influx and subsequent smooth muscle contraction (Fig.…”