SUMMARY1. Currents evoked by elevated intracellular free Ca2+ in Xenopus laevis oocytes were studied using the two-electrode voltage clamp technique. The elevation in Ca2+ concentration was achieved in three ways: by the use of the divalent cation ionophore A23187; by application of Ca2+-mobilizing neurotransmitters serotonin and acetylcholine (ACh); by the entry of Ca2+ through voltage-dependent channels.2. In most experiments, the membrane was permeabilized to Ca2+ by a 15 min pretreatment with A23187 in a Ca2+-free solution. Exposure of the ionophore-treated oocytes to external Ca2+ elicited an inward current (at holding potentials of -40 to -60 mV). At external Ca2+ concentrations ([Ca2+]) between 0.1 and 1 mm, the current had a time-to-peak of at least 10 s, and slowly decayed over tens of seconds. At [Ca2+] > 2 mm, the inward current had two distinct kinetic components, a fast and transient one (If.t) and a slow one (Islow).3. The main carrier of the Ca2+-evoked inward current was Cl-. Several data indicate the existence of a tetraethylammonium (TEA)-sensitive K+ conductance.No evidence for a Na+ current was found.4. The two components of the Ca2+-evoked inward current in ionophorepermeabilized oocytes, and the two components of the current evoked by ACh and serotonin (the latter in oocytes injected with rat brain RNA but untreated with A23187), were blocked by intracellular injection of the Ca2+ chelator, ethyleneglycolbis-(,8-aminoethyl ether)-N,N,N'N'-tetraacetic acid (EGTA). The two components of these currents displayed different sensitivity to Ca2+ buffering; higher doses of EGTA were necessary to inhibit the slow component than the fast one.5. One to two minutes of treatment with 2 mM-9-anthracene carboxylic acid (9-AC) fully blocked Ca2+-dependent Cl-current evoked by Ca2+ influx through voltagedependent Ca2+ channels in intact (untreated with A23187) oocytes. In ionophoretreated oocytes, block of If.t was observed at holding potentials at which the current was outward (i.e. due to Cl-influx); Islow was inhibited only partially. The block of Ca2+-evoked Cl-efflux by 9-AC developed much more slowly and was less potent. To explain these results, the existence of two sites of 9-AC action is proposed.6. Exposure of the ionophore-permeabilized oocytes to 0-1-0-2 mm [Ca2+] strongly * To whom correspondence should be sent.

Effects of purtfied subtypes I. II and III of protem kmase C (PKC) on voltage-dependent transient K+ (A) and Na+ channels were studied m Xcnopus oocytes injected with chick bram RNA. The experiments were performed in the constant presence of 10 nM p-phorbol 12-myrtstate-13-acetate (PMA) Intracellular injectton of subtype I (5) reduced the A-current (IA). wtth no effect on Na+ current (INa). PKC subtype II (J, +/3,) and III (n) reduced both currents PKC did not affect the response to kainate Inacttvated (heated) or unacttvated (injected m the absence of PMA) enzyme and vehtcle alone had no effect. Our results strongly suggest that 1,, and IA m vertebrate neurons are modulated by PKC. all PKC subtypes exert a stmilar effect on the A-channel while only subtypes II and III modulate the Na+ channel

In Xenopus oocytes injected with rat brain RNA, serotonin (5HT) and acetylcholine (ACh) evoke membrane responses through a common biochemical cascade that includes activation of phospholipase C, production of inositol 1,4,5-trisphosphate (Ins1,4,5-P3), release of Ca2+ from intracellular stores, and opening of Ca-dependent Cl- channels. The response is a Cl- current composed of a transient component (5HT1 or ACh1) and a slow, long-lasting component (5HT2 or ACh2). Here we show that only the fast, but not the slow, component of the response is subject to desensitization that follows a previous application of the transmitter. The recovery of 5HT1 from desensitization is biphasic, suggesting the existence of two types of desensitization: short-term desensitization (STD), which lasts for less than 0.5 h; and long-term desensitization (LTD) lasting for up to 4 h. The desensitization between 5HT and ACh is heterologous and long-lasting. We searched for (a) the molecular target and (b) the cause of desensitization. (a) Pre-exposure to 5HT does not reduce the response evoked by intracellular injection of Ca2+ and by Ca2+ influx. Cl- current evoked by intracellular injection of Ins1,4,5-P3 was reduced shortly after application of 5HT, but fully recovered 30 min later. Thus, the Cl- channel is not a target for desensitization. Neither Ins1,4,5-P3 receptor nor the Ca2+ store is a target of LTD but they may be the targets of STD. (b) Ca2+ injection did not inhibit the 5HT response, suggesting that Ca2+ is not a sole cause of STD or LTD.(ABSTRACT TRUNCATED AT 250 WORDS)

Inactivation of Ca2(+)-induced Cl- currents was studied in Xenopus oocytes using the two-electrode voltage-clamp technique. In oocytes permeabilized to Ca2+ by treatment with the ionophore A23187, Ca2+ influx caused by the addition of 2.5-5 mM Ca2+ to the extracellular solution elicited Cl- currents consisting of two components: a fast, transient one (Ifast) and a slow one (Islow). In response to a subsequent application of the same dose of Ca2+, Ifast and Islow were reduced (inactivation phenomenon). The inactivation did not depend on the direction of current flow, but did depend on the duration of the first exposure to Ca2+. The extent of inactivation of Ifast was more significant than that to Islow. Both Ifast and Islow fully recovered from inactivation in less than 30 min. Intracellular injections of 100-400 pmol CaCl2 evoked large inward currents but did not reduce the amplitude of currents evoked by Ca2+ influx. The activator of protein kinase C, beta-phorbol dibutyrate, caused full inhibition of Ifast without any change in Islow. H-7 (1,5-isoquinolinesulfonyl-1,2 methylpiperazine), an inhibitor of protein kinases, strongly reduced the extent of inactivation. Our results suggest that elevation of intracellular Ca2+ by Ca2+ influx through the plasma membrane causes inactivation of the Ca2(+)-dependent Cl- conductance via activation of a Ca2(+)-dependent protein kinase, possibly protein kinase C, whereas Ca2+ arriving at the membrane from inside the cell does not initiate the processes leading to inactivation.

Upon two repettttve deep mjections of Ca2+ mto Xenopu.c oocyte (20&300 pm under the membrane).the amphtude of the transient Cl-current mduced by the second InJectIon is several-fold higher than that of the first one. This 'potentlatlon' perststs even at 60-90 mm mtervals between mjecttons. However, m oocytes permeabthzed to CaLi by the ronophore A23187 m a Ca"+ -free solutton, the potenttatton completely dtsappedra after 30 mm. It 1s proposed that the Injected Ca:+ ts largely taken up by the stores, whereas followmg the second mJectton. a htgher proportton of Ca'+ reaches the membrane. since the stores are already loaded In tonophore-treated oocytes. the stores lose the accumulated Ca'+ over several mmutes and are then ready to take up Ca'+ agatn. hmdering Its arrtval at the membrane