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L Toro

Publications and source records attributed to L Toro.

At least 73 records · Page 4Linked to original sources
PubMed1993

Characterization of large-conductance, calcium-activated potassium channels from human myometrium.

OBJECTIVES: The purpose of our study was to detect and characterize potassium channels in the plasma membrane of smooth muscle cells from human myometrium. STUDY DESIGN: Plasma membrane vesicles were incorporated into lipid bilayers to record single potassium channel activity. RESULTS: We predominantly found a "maxi" calcium-activated potassium channel (261 picosiemens). This channel was calcium (micromoles per liter range) and voltage sensitive, highly selective for K+ over Na+ and Cs+, and was sensitive to external tetraethylammonium (dissociation constant approximately 220 mumol/L) and charybdotoxin (dissociation constant approximately 23 nmol/L). External apamin and 4-aminopyridine had no effect on this channel. Another type of potassium channel that was less frequently observed was also identified. It had a smaller conductance (142 picosiemens) and it seemed to be calcium independent (up to 50 nmol/L). CONCLUSION: Human myometrium possesses abundant "maxi" calcium-activated potassium channels. This channel shares common characteristics with other "maxi" calcium-activated potassium channels, including calcium and voltage gating, high conductance and selectivity, and channel pharmacologic profile.

Calcium↗
PubMed1993

Calcium-activated K+ channels as modulators of human myometrial contractile activity.

The role of Ca(2+)-activated potassium (KCa) channels in the regulation of membrane potential, intracellular free calcium ([Ca2+]i) and contraction was investigated in uterine smooth muscle and myometrial cells. In an immortalized human myometrial cell line, oxytocin increased [Ca2+]i and [3H]inositol phosphate formation. Relaxin attenuated the oxytocin-induced increase in [Ca2+]i. In cell-attached patches, membrane depolarization activated a large-conductance KCa channel (179 +/- 4 pS). Iberiotoxin (IbTX), a potent blocker of "maxi" KCa channels (A. Galvez, G. Gimenez-Gallego, J. P. Reuben, L. Roy-Contanciin, P. Feigenbaum, G. J. Kaczorowski, and M. L. Garcia. J. Biol. Chem. 265: 11083-11090, 1990) produced long closed events (approximately 6 min) in these channels. In agreement with this blockage, IbTX depolarized the cells by 9.8 +/- 2.8 mV and caused a dose-dependent increase in [Ca2+]i with a half-maximal effective concentration of 0.79 nM. IbTX also caused phasic contractions in human myometrial strips and increased both the frequency and force of spontaneous contractions in estrogen-primed rat myometrial strips. Moreover, myometrial contractility was also affected by 1 mM tetraethylammonium, a concentration that blocks uterine smooth muscle KCa channels when applied to the extracellular side (G. J. Perez, L. Toro, S. D. Erulkar, and E. Stefani. Am. J. Obstet. Gynecol. 168: 652-660, 1993). These results strongly suggest that the large conductance KCa channels may actively participate in the control of human myometrial cell membrane potential and [Ca2+].

Calcium↗
PubMed1993

Modulation of coronary smooth muscle KCa channels by Gs alpha independent of phosphorylation by protein kinase A.

The occupancy of beta-receptors in the smooth muscle membrane of the coronary arteries produces vasodilation and a concomitant hyperpolarization. Large conductance calcium-activated K (KCa) channels are likely to be involved in such hyperpolarization, since they are densely distributed in coronary myocytes, and they are targets of beta-adrenergic stimulation in other smooth muscles. We sought to explore if coronary smooth muscle KCa channels are modulated by beta-agonists and we studied the mechanisms of their activation. We found that KCa channels reconstituted into lipid bilayers were activated in the presence of GTP by the beta-adrenergic receptor agonist isoproterenol. KCa channels were also stimulated on non-specific activation of an endogenous G protein(s) with guanosine 5'-O-(3-thiotriphosphate) (GTP gamma S), on addition of a purified activated stimulatory G protein (Gs alpha), and when the catalytic subunit of protein kinase A (PKA) was added. Inhibition of PKA activity prevented KCa channel stimulation by PKA, but not by endogenous G protein or by exogenous Gs alpha. These results indicate that beta-adrenoceptor activation of coronary smooth muscle KCa channels results from a dual control: 1) a membrane delimited, possibly direct action of Gs, independent of PKA-mediated phosphorylation; and 2) by PKA-dependent phosphorylation.

Animals↗
PubMed1992

Internal blockade of a Ca(2+)-activated K+ channel by Shaker B inactivating "ball" peptide.

Shaker B inactivating peptide ("ball peptide", BP) interacts with Ca(2+)-activated K+ (KCa) channels from the cytoplasmic side only, producing inhibition of channel activity. This effect was reversible and dose and voltage dependent (stronger at depolarized potentials). The inhibition of KCa channels by BP cannot be mimicked by an inactive point mutation of the BP, L7E. BP binds to KCa channels in a bimolecular reaction (dissociation constant of 95 microM at +40 mV). The binding site is probably located in the internal "mouth" or conduction pathway, since both external K+ and internal tetraethylammonium relieve BP-induced inhibition. These results suggest that KCa channels possess a binding site for the BP with some properties similar to the ball receptor found in Shaker B K+ channels.

Amino Acid Sequence↗
PubMed1992

Novel voltage clamp to record small, fast currents from ion channels expressed in Xenopus oocytes.

The present report describes a novel technique for voltage-clamping amphibian oocytes in which part of the membrane is isolated by a vaseline gap and the cytoplasmic fluid is exchanged by cutting or permeabilizing the remaining membrane. The main features of this open-oocyte, vaseline-gap voltage clamp are: (a) low current noise (1 nA at 3 kHz), (b) control of the ionic composition of both the internal and external media, (c) fast time resolution (20-100 microseconds time constant of decay of the capacity transient) and (d) stable recordings for several hours. These features allow reliable measurements of tail or gating currents and the new method is especially suitable when either of these currents must be measured to test the effects of mutations introduced into the cDNAs of cloned ion channels.

Animals↗
PubMed1992

Progesterone increases Ca2+ currents in myometrial cells from immature and nonpregnant adult rats.

We examined the role of ovarian steroids in regulating Ca2+ channels in rat uterine smooth muscle. Ca2+ currents (ICa) in myometrial cells from nonpregnant adult rats and immature rats injected with either estrogen or progesterone or estrogen plus progesterone were measured with the whole cell patch-clamp method. ICa was more prominent in cells from diestrous rats than in cells from estrous rats. In cells from immature rats the ICa density was significantly greater in cells from progesterone-injected rats than in cells from estrogen-injected or noninjected rats. ICa in cells from rats injected simultaneously with progesterone and the progesterone antagonist RU-486 was not significantly greater than those from noninjected rats. These increases in ICa density are not the result of changes in ICa activation kinetics or voltage dependence, since both are unaffected by steroid injection. The kinetics and voltage dependence of the ICa current in cells from immature and nonpregnant adult rats are similar, suggesting that they represent a single population of Ca2+ channels.

Aging↗
PubMed1992

U46619, a thromboxane A2 agonist, inhibits KCa channel activity from pig coronary artery.

Thromboxane A2 (TxA2) is a potent vasoconstrictor derived from the metabolism of arachidonic acid. Because potassium channels are involved in the contraction of vascular smooth muscle, their blockade could contribute to the TxA2-induced contraction. To test this possibility, we studied the effect of the TxA2 stable analogue U46619 on calcium-activated potassium (KCa) channels from coronary artery reconstituted into lipid bilayers. Addition of U46619 (50-150 nM) to the external but not to the internal side of the channel decreased the channel open probability (Po) between 15 and 80% of the control value. The inhibitory effect of U46619 affected both the open and closed states of the channel and could be reversed by internal calcium. Thromboxane B2, the inactive hydrolysis derivative of TxA2, did not affect channel activity. SQ 29548, a TxA2 receptor antagonist, was able to prevent the inhibition by U46619. Furthermore, SQ 29548 added after U46619 could restore channel activity to near control values. These results suggest that TxA2 could be a regulatory factor of KCa channels from coronary smooth muscle and that this regulation could be related to its action as a vasoconstrictor.

15-Hydroxy-11 alpha,9 alpha-(epoxymethano)prosta-5↗
PubMed1992

Ca(2+)-activated K+ channels in pregnant rat myometrium: modulation by a beta-adrenergic agent.

The properties of Ca(2+)-activated K+ currents and channels were characterized in pregnant rat myometrium in whole cell and cell-attached patches and in lipid bilayers. Membrane depolarization of cultured myometrial cells from a holding potential of -50 to +70 mV in 10-mV steps under voltage-clamp conditions (whole cell mode) activated K+ outward currents (IK). At +70 mV, in the presence of 0.2 mM external Ca2+, the amplitude and activation time constant of IK were 15.0 +/- 2.1 microA/microF and 1.5 +/- 0.2 ms, respectively. Addition of 1 microM A23187 to the external solution increased the current from a control value of 16.0 +/- 2.0 to 67.9 +/- 9.1 microA/microF. Charybdotoxin, a blocker of Ca(2+)-activated K (KCa) channels, and a low concentration of tetraethylammonium chloride (TEA; 1 mM) decreased the amplitude of IK by 47 and 62%, respectively. In cell-attached patches from these cells, 1 microM A23187 increased the open time probability of a 143 +/- 6.0 pS K+ channel. Incorporation of plasma membrane vesicles from pregnant myometrium into lipid bilayers resulted in one predominant type of K+ channel. The unitary conductance of the K+ channel was 326 +/- 9.0 pS in symmetrical 450 mM KCl. The channel activation was both voltage and Ca2+ dependent. TEA inhibited the channel activity with a dissociation constant (Kd) of 378 +/- 10 microM at -60 mV or 1,477 +/- 80 microM at +60 mV. The whole cell currents were found to be stimulated by isoproterenol, a beta-adrenergic agent.(ABSTRACT TRUNCATED AT 250 WORDS)

Adrenergic beta-Agonists↗
PubMed1991

Evidence for direct interaction of Gs alpha with the Ca2+ channel of skeletal muscle.

The alpha subunits of heterotrimeric GTP-binding (G) proteins act upon ion channels through both cytoplasmic and membrane-delimited pathways (Brown, A. M., and Birnbaumer, L. (1990) Annu. Rev. Physiol. 52, 197-213). The membrane pathway may involve either a direct interaction between G protein and ion channel or an indirect interaction involving a membrane-delimited second messenger. To distinguish between the two possibilities, we tested whether a purified G protein could interact with a purified channel protein in a defined system to produce changes in channel currents. We selected the alpha subunit of Gs and the dihydropyridine (DHP)-sensitive Ca2+ channel of skeletal muscle T-tubules, the DHP binding protein (DHPBP), because: 1) a membrane-delimited interaction between the two has been shown (Brown, A. M., and Birnbaumer, L. (1990) Annu. Rev. Physiol. 52, 197-213; Yatani, A., Imoto, Y., Codina, J., Hamilton, S. L., Brown, A. M., and Birnbaumer, L. (1988) J. Biol. Chem. 263, 9887-9895); and 2) at the present time, these Ca2+ channels are the only putative G protein channel effectors which, following purification, still retain channel function. We used a defined system in which purified components were studied by direct reconstitution in planar lipid bilayers. Just as we had found in crude skeletal muscle T-tubule membranes (Yatani, A., Imoto, Y., Codina, J., Hamilton, S. L., Brown, A. M., and Birnbaumer, L. (1988) J. Biol. Chem. 263, 9887-9895), alpha*s but not alpha*i-3 stimulated Ca2+ currents. However, in the reconstituted system, this probably represents a direct interaction between Gs alpha and Ca2+ channels. To establish whether the two proteins were physically associated in the native T-tubule membrane, we examined the ability of either endogenous G proteins or exogenous alpha*s to purify with detergent-solubilized DHPBP through a wheat germ agglutinin affinity column and a sucrose gradient. Small amounts of a labeled G protein were found to co-purify with DHPBP. In addition, partially purified DHPBP increased the sedimentation rate of purified alpha*s but not alpha*i-3. G proteins were immunoprecipitated with an antibody to the alpha 1 subunit of the DHPBP, and, in addition, both alpha s and the beta subunit of Gs were detected in Western blots of the partially purified DHPBP. The results suggest that Gs and Ca2+ channels are closely associated in the T-tubule plasma membrane, and we conclude that skeletal muscle Ca2+ channels are direct effectors for Gs.

Blotting, Western↗
PubMed1991

Calcium-activated K+ channels: metabolic regulation.

Calcium-activated potassium (KCa) channels are highly modulated by a large spectrum of metabolites. Neurotransmitters, hormones, lipids, and nucleotides are capable of activating and/or inhibiting KCa channels. Studies from the last few years have shown that metabolites modulate the activity of KCa channels via: (1) a change in the affinity of the channel for Ca2+ (K 1/2 is modified), (2) a parallel shift in the voltage axis of the activation curves, or (3) a change in the slope (effective valence) of the voltage dependence curve. The shift of the voltage dependence curve can be a direct consequence of the change in the affinity for Ca2+. Recently, the mechanistic steps involved in the modulation of KCa channels are being uncovered. Some interactions may be direct on KCa channels and others may be mediated via G-proteins, second messengers, or phosphorylation. The information given in this review highlights the possibility that KCa channels can be activated or inhibited by metabolites without a change in the intracellular Ca2+ concentration.

Animals↗
PubMed1991

Potassium currents in freshly dispersed myometrial cells.

K+ currents in freshly dispersed cells from rat myometrium at estrus were studied with the patch-clamp technique (whole cell). Three types of K+ currents were identified: 1) a fast-activating current (IKf), 2) a slowly activating current (IKs), and 3) a transient current (IKt). IKf had a half-activation voltage of 12 mV and a time constant of activation (tau on) of approximately 3 ms at +50 mV. IKs had a tau on of approximately 9 ms at +50 mV and a half-activation potential of 33 mV. Both IKf and IKs were sustained and became potentiated by the entrance of Ca2+ from the patch pipette. These two Ca(2+)-activated K+ currents were inhibited by 100 nM external charybdotoxin and were blocked by external tetraethylammonium (TEA, 2-20 mM). The third current (IKt) was transient, had a faster tau on (approximately 1 ms), and a decay phase with a time constant of approximately 8 ms at +50 mV. This current had a half-activation potential of 22 mV. IKt was not potentiated by intracellular Ca2+, was sensitive to 4-aminopyridine (1 mM), was insensitive to external charybdotoxin (100 nM) and TEA (2 mM), and spontaneously decreased with time.

Animals↗
PubMed1991

Calcium-activated potassium channels from coronary smooth muscle reconstituted in lipid bilayers.

This work is the initial characterization of Ca(2+)-activated K+ (KCa) channels from coronary smooth muscle reconstituted into lipid bilayers. The channels were obtained from a surface membrane preparation of porcine coronary smooth muscle. KCa channels were the predominant K+ channels in this preparation. The conductance histogram (n = 137 channels) revealed two main populations of "maxi" KCa channels with conductances of 245 and 295 pS. Each population could be subdivided in two "isoforms" or "isochannels" with different functional properties (voltage and Ca2+ sensitivities and kinetics). The analysis of "burst" probability of opening showed that at pCa 4 the two isochannels of 245 pS (KCa-1 and KCa-1') had half-activation potentials (V1/2) of -80 and 6 mV, respectively. The isochannels of 295 pS (KCa-2 and KCa-2') had V1/2 of -28 and -66 mV, respectively. KCa-1 had the highest Ca2+ sensitivity; at -60 mV, the concentration of half-activation value for Ca2+ was 1.2 +/- 0.3 microM (n = 5). External tetraethylammonium reduced channel amplitude in a voltage-dependent manner; dissociation constant was 180 +/- 6 and 466 +/- 41 microM at -40 and +80 mV, respectively (n = 5). Charybdotoxin (5-50 nM) produced typical long closings. These effects were similar in all the channels. We conclude that coronary smooth muscle possesses isoforms of maxi KCa channels with Ca2+ and voltage sensors with different properties, which may confer to each channel a specific functional role.

Action Potentials↗
PubMed1990

Hormonal regulation of potassium currents in single myometrial cells.

Three potassium currents (IK) were recorded from myometrial cells isolated from the uterus of rats at estrus and diestrus and kept in culture for 1-6 days. IK were differentiated by their modulation with norepinephrine and/or by their onset kinetics. At +50 mV the activation time constants were about 0.7 ms, 6 ms, and 15 ms for the fast, the intermediate, and the slow IK, respectively. Norepinephrine (1 microM) potentiated the fast IK and reduced the intermediate IK. In addition, differences were found with respect to cells from animals at estrus and diestrus. The fast IK was preferentially expressed in cultures from animals at estrus, whereas the intermediate IK was more frequent in cells from rats at diestrus. These results indicate that K+ channels from myometrial cells are multiregulated. Regulation may occur by short-term signals (neurotransmitters) and/or by preferentially expressing distinct types of channels depending on the hormonal status of the animal.

Animals↗
PubMed1990

GTP-dependent regulation of myometrial KCa channels incorporated into lipid bilayers.

The regulation of calcium-activated K (KCa) channels by a G protein-mediated mechanism was studied. KCa channels were reconstituted in planar lipid bilayers by fusion of membrane vesicles from rat or pig myometrium. The regulatory process was studied by exploring the actions of GTP and GTP gamma S on single channel activity. KCa channels had a conductance of 260 +/- 6 pS (n = 25, +/- SE, 250/50 mM KCl gradient) and were voltage dependent. The open probability (Po) vs. voltage relationships were well fit by a Boltzmann distribution. The slope factor (11 mV) was insensitive to internal Ca2+. The half activation potential (V1/2) was shifted -70 mV by raising internal Ca2+ from pCa 6.2 to pCa 4. Addition of GTP or GTP gamma S activated channel activity only in the presence of Mg2+, a characteristic typical of G protein-mediated mechanisms. The Po increased from 0.18 +/- 0.08 to 0.49 +/- 0.07 (n = 7, 0 mV, pCa 6 to 6.8). The channel was also activated (Po increased from 0.03 to 0.37) in the presence of AMP-PNP, a nonphosphorylating ATP analogue, suggesting a direct G protein gating of KCa channels. Upon nucleotide activation, mean open time increased by a factor of 2.7 +/- 0.7 and mean closed time decreased by 0.2 +/- 0.07 of their initial values (n = 6). Norepinephrine (NE) or isoproterenol potentiated the GTP-mediated activation of KCa channels (Po increased from 0.17 +/- 0.06 to 0.35 +/- 0.07, n = 10). These results suggest that myometrium possesses beta-adrenergic receptors coupled to a GTP-dependent protein that can directly gate KCa channels. Furthermore, KCa channels, beta-adrenergic receptors, and G proteins can be reconstituted in lipid bilayers as a stable, functionally coupled, molecular complex.

Adenosine Triphosphate↗
PubMed1990

ANG II inhibits calcium-activated potassium channels from coronary smooth muscle in lipid bilayers.

Angiotensin II (ANG II) is a powerful vasoconstrictor of coronary vessels and other smooth muscles. One of the actions of ANG II is the inhibition of K+ currents, possibly contributing to depolarization and contraction. Therefore, we investigated the role of ANG II on the regulation of K+ channels at the single-channel level. We studied its effect on calcium-activated potassium (KCa) channels (congruent to 250 pS) from coronary smooth muscle incorporated into lipid bilayers. KCa channels were sensitive to externally applied ANG II at voltages from -20 to -70 mV and pCa between 6.5 and 4. The dose-response curve gave a concentration of half-inhibition (Ki1/2) of 58 nM and a Hill coefficient of 2.2, indicating a minimum of two sites in the process. ANG II modified the open and closed states of the channel, affecting their proportions and their values. In addition, a new much slower (congruent to 1 s) closed or "blocked" state appeared. We conclude that one of the mechanisms by which ANG II causes vasoconstriction of the coronary vessels is a direct inhibition of KCa channels contributing to depolarization and contraction.

Angiotensin II↗
PubMed1990

Functional studies of Ca2+ channels from plasmalemma and sarcoplasmic reticulum membranes in muscle cells.

Physiological and biochemical studies (channel characteristics, intracellular Ca2+ determinations and, channel purification, cloning and expression) of the different components involved in the regulation of intercellular Ca2+ have provided new information about their specific role. Recent information favors a major role for plasmalemma Ca2+ channels in E-C coupling of cardiac muscle, while a major role for sarcoplasmic reticulum Ca2+ release channels (ryanodine receptors) is proposed for E-C coupling of skeletal muscle. In smooth muscle, both plasmalemma and sarcoplasmic reticulum (IP3 receptors) Ca2+ channels are involved in E-C coupling. These studies will be comparatively discussed for skeletal, cardiac and smooth muscle cells.

Animals↗