Small conductance Ca2+-activated K+ channels are regulated by Ca2+–calmodulin-dependent protein kinase II in murine colonic myocytes

Ca2+ regulates the activity of small conductance Ca2+-activated K+ (SK) channels via calmodulin-dependent binding. We investigated whether other forms of Ca2+-dependent regulation might control the open probability of SK channels.Under whole-cell patch-clamp conditions, spontaneous openings of SK channels can be resolved as charybdotoxin-insensitive spontaneous transient outward currents (STOCs). The Ca2+–calmodulin-dependent (CaM) protein kinase II inhibitor KN-93 reduced the occurrence of charybdotoxin-insensitive STOCs.The charybdotoxin-insensitive STOCs are related to spontaneous, local release of Ca2+. KN-93 did not affect spontaneous Ca2+-release events.KN-93 and W-7, a calmodulin inhibitor, decreased the open probability of SK channels in on-cell patches but not in excised patches.Application of autothiophosphorlated CaM kinase II to the cytoplasmic surface of excised patches increased the open probalibity of SK channels. Boiled CaM kinase II had no effect.We conclude that CaM kinase II regulates SK channels in murine colonic myocytes. This mechanism provides a secondary means of regulation, increasing the impact of a given Ca2+ transient on SK channel open probability

Intracellular calcium events activated by ATP in murine colonic myocytes

ATP is a candidate enteric inhibitory neurotransmitter in visceral smooth muscles. ATP hyperpolarizes visceral muscles via activation of small-conductance, Ca2+-activated K+(SK) channels. Coupling between ATP stimulation and SK channels may be mediated by localized Ca2+release. Isolated myocytes of the murine colon produced spontaneous, localized Ca2+release events. These events corresponded to spontaneous transient outward currents (STOCs) consisting of charybdotoxin (ChTX)-sensitive and -insensitive events. ChTX-insensitive STOCs were inhibited by apamin. Localized Ca2+transients were not blocked by ryanodine, but these events were reduced in magnitude and frequency by xestospongin C (Xe-C), a blocker of inositol 1,4,5-trisphosphate receptors. Thus we have termed the localized Ca2+events in colonic myocytes “Ca2+puffs.” The P2Yreceptor agonist 2-methylthio-ATP (2-MeS-ATP) increased the intensity and frequency of Ca2+puffs. 2-MeS-ATP also increased STOCs in association with the increase in Ca2+puffs. Pyridoxal-phospate-6-azophenyl-2′,4′-disculfonic acid tetrasodium, a P2receptor inhibitor, blocked responses to 2-MeS-ATP. Spontaneous Ca2+transients and the effects of 2-MeS-ATP on Ca2+puffs and STOCs were blocked by U-73122, an inhibitor of phospholipase C. Xe-C and ryanodine also blocked responses to 2-MeS-ATP, suggesting that, in addition to release from IP3receptor-operated stores, ryanodine receptors may be recruited during agonist stimulation to amplify release of Ca2+. These data suggest that localized Ca2+release modulates Ca2+-dependent ionic conductances in the plasma membrane. Localized Ca2+release may contribute to the electrical responses resulting from purinergic stimulation.</jats:p

Dissociation between electrical and mechanical responses to nitrergic stimulation in the canine gastric fundus

We examined the relationships between membrane potential, intracellular [Ca2+] ([Ca2+]i), and tension in muscles of the canine gastric fundus in response to nitrergic stimulation by NO donors and electrical field stimulation (EFS) of intrinsic enteric inhibitory neurons when adrenergic and cholinergic responses were blocked.NO donors reduced [Ca2+]i and tension in a concentration-dependent manner. A close relationship was noted between these parameters.In terms of the [Ca2+]vs. force relationship, relaxation responses to EFS differed from responses to NO donors. EFS resulted in smaller decreases in [Ca2+]i to produce a given relaxation compared with responses to NO donors. Thus, muscles stimulated with EFS were less sensitive to [Ca2+]i than muscles stimulated with exogenous NO.When membrane potential, [Ca2+]i and tension were monitored simultaneously in the same muscles, a temporal dissociation was noted between the electrical responses and changes in [Ca2+]i and tension. Brief electrical responses were associated with more sustained changes in [Ca2+]i and tension.Further dissociation between electrical and mechanical effects was noted. Changes in [Ca2+]i and tension caused by sodium nitroprusside and EFS were blocked by arginine analogues and by oxyhaemoglobin, but electrical responses were unaffected. 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ), an inhibitor of soluble guanylyl cyclase, also blocked the effects of nitrergic stimulation on [Ca2+]i and tension, without affecting hyperpolarization. Thus, in the presence of continued hyperpolarization, the reductions in [Ca2+]i and tension caused by nitrergic stimulation were blocked.Block of hyperpolarization in response to nitrergic stimulation with tetrapentylammonium chloride (TPEA) had relatively little effect on the [Ca2+]i and tension responses. Thus, hyperpolarization is not required for nitrergic effects on [Ca2+]i and tension.In summary, reduction in [Ca2+]i and tension in response to nitrergic stimulation of the canine gastric fundus does not depend upon electrical hyperpolarization. Non-electrical mechanisms such as enhanced uptake of Ca2+ by the sarcoplasmic reticulum or reduction in the Ca2+ sensitivity of the contractile apparatus may be the primary mechanisms mediating nitrergic responses in these muscles