Biomedical subjects
Publications and source records attributed to P Labarca.
We recorded single channel currents activated by agonist binding to purified acetylcholine receptors from the electric organ of Torpedo californica reconstituted in planar lipid bilayers. Analysis of single channel records indicate that the acetylcholine receptor channel displays two kinetically distinct open states which differ in their mean open times but have similar channel conductances.
A voltage-dependent, K+-selective ionic channel from sarcoplasmic reticulum of rabbit skeletal muscle has been studied in a planar phospholipid bilayer membrane. The purpose [corrected] of this work is to study the mechanism by which the channel undergoes transitions between its conducting and nonconducting states. Thermodynamic studies show that the "open" and "closed" states of the channel exist in a voltage-dependent equilibrium, and that the channel displays only a single open state; the channel conductance is 120 pmho in 0.1 M K+. The channel's gating process follows single exponential kinetics at all voltages tested, and the individual opening and closing rate constants are exponentially dependent on voltage. The individual rate constants may also be determined from a stochastic analysis of channel fluctuations among multiple conductance levels. Neither the thermodynamic nor the kinetic parameters of gating depend on the absolute concentration of channels in the bilayer. The results are taken as evidence that the channel gates by an unusually simple two-state conformational mechanism in which the equivalent of 1.1 net charges are moved across the membrane during the formation of the open channel.
The ratio of the rate of transepithelial sodium transport, JNa, across the isolated toad urinary bladder to the simultaneously measured rate of transport-dependent metabolism, JsbCO2, has been measured as a function of the transepithelial electrical voltage, deltapsi. The ratio remains constant with a mean value of 18 to 20 over the range of imposed voltages of 0 to +70 mV. With increasing hyperpolarization of the bladder, JNa decreases and the calculated electromotive force or apparent "ENa" of the sodium pump increases. From thermodynamic and kinetic arguments it is shown that the apparent "ENa" approaches the maximal electrochemical potential gradient, ENa, against which sodium can be transported by this tissue only when JNa approximately 0. At this unique condition F ENa (in which F is the Faraday constant) is the maximal free energy of the chemical reaction driving sodium transport and thus equal to the maximal extramitochondrial phosphorylation potential and the maximal free energy of the mitochondrial respiratory chain within the transporting cells.
The metabolic cost of active sodium transport was determined in toad bladder at different gradients of transepithelial potential. Deltapsi, by continuous and simultaneous measurements of CO2 production and of transepithelial electric current. Amiloride was used to block active sodium transport in order to assess the nontransport-linked, basal, production of CO2 and the passive permeability of the tissue. From these determinations active sodium transport, Jna, and suprabasal CO2 production, Jsb CO2, were calculated. Since large transients in Jna and Jsb CO2 frequently accompanied any abrupt change in deltapsi, steady state conditions were carefully defined. Some 20 to 40 min were required after a change in deltapsi before steady state of transport activity and of CO2 production were achieved. The metabolic cost of sodium transport proved to be the same whether the bladder expended energy moving sodium against a transepithelial electrical potential grandient of +50 mV or whether sodium was being pulled through "the active transport pathway" by an electrical gradient of -50 mV. In both cases the value of the ratio Jna/Jsb CO2 averaged some 20 sodium ions transported per molecule of CO2 produced. When the Na pump was blocked by 10(-2) M ouabain, the perturbations of the transepithelial electrical potential did not elicit changes of Jna nor, consequently of Jsb CO2. The independence of the ratio Jna/Jsb CO2 from deltapsi over the range+/-50 mV indicates a high degree of coupling between active sodium transport and metabolism.
The possibility that sodium from the serosal bathing medium "back diffuses" into the active sodium transport pool within the mucosal epithelial cell of the isolated toad bladder was examined by determining the effect on the metabolism of the tissue of removing sodium from the serosal medium. It was expected that if recycling of serosal sodium did occur through the active transepithelial transport pathway of the isolated toad bladder, removal of sodium from the serosal medium would reduce the rate of CO2 production by the tissue and enhance of stoichiometric ratio of sodium ions transported across the bladder per molecula of sodium transport dependent CO2 produced simultaneously by the bladder (JNa/JCO2). The data revealed no significant change in this ratio (17.19 with serosal sodium and 16.13 after replacing serosal sodium with choline). Further, when transepithelial sodium transport was inhibited (a) by adding amiloride to the mucosal medium, or (b) by removing sodium from the mucosal medium, subsequent removal of sodium from the serosal medium, or (c) addition of ouabain failed to depress the basal rate of CO2 production by the bladder [(a)rate of basal, nontransport related, CO2 production (JbCO2) equals 1.54 +/- 0.52 with serosal sodium and 1.54 +/- 0.37 without serosal sodium; (b) Jb CO2 equals 2.18 +/- 0.21 with serosal sodium and 2.09 +/- 0.21 without serosal sodium; (c) 1.14 +/- 0.26 without ouabain and 1.13 +/- 0.25 with ouabain; unite of JbCO2 are nmoles mg d.w.-1 min-1]. The results support the hypothesis that little, if any, recycling of serosal sodium occurs in the total bladder.