Equilibrium Potential For Chloride Ions Research Articles

Calcium-activated chloride current in rat vascular smooth muscle cells in short-term primary culture.

Isolated cells from rat portal vein smooth muscle in short-term primary culture were studied using patch-clamp technique (whole-cell configuration). In order to study a calcium-activated chloride current, the potassium currents were blocked by intracellular cesium diffusion. Without EGTA in the pipette solution, depolarizing voltage pulses from a holding potential of -70 mV to positive potentials activated an early inward and a late outward current. The latter persisted as a long-lasting inward tail current when the membrane was repolarized to -70 mV. The outward current measured at the end of the pulse and the tail current were blocked by extracellular cobalt, after replacement of external calcium with barium, after removal of external calcium, and when the calcium concentration of the pipette solution was less than 0.5 microM, suggesting that they were calcium-dependent. The tail current decay was voltage sensitive, becoming faster with hyperpolarization. The reversal potential of the calcium-activated current was near the equilibrium potential for chloride ions, and was shifted as predicted by the Nernst equation when the extracellular or intracellular chloride concentration was changed. The calcium-activated current was blocked by adding micromolar concentrations of niflumic acid or millimolar concentrations of DIDS. This effect of compounds known to interfere with chloride channels together with the data on the equilibrium potential for chloride ions indicated above suggested the existence of a calcium-activated chloride current in vascular smooth muscle cells.

Kinetic analysis of acetylcholine-induced chloride current in isolated snail neurons.

1. Kinetics of activation and desensitization phases of the acetylcholine (ACh)-induced chloride current (ICI) were studied using isolated single neurons of Japanese land snail and the "concentration clamp" technique. 2. The dose-response curve for the peak ICI gave a dissociation constant of 7.1 x 10(-6) M and a Hill coefficient of 1.8. 3. The current-voltage relationship was linear in the voltage range examined (-60 to +10 mV) and the reversal potential (EACh) was -7.2 +/- 1.5 mV (N = 10). The value was close to the calculated equilibrium potential for chloride ions (ECI). 4. Both activation and desensitization phases of the ACh-induced ICI consisted of a single exponential at concentrations less than 3 x 10(-6) M and a double exponential at higher concentrations. The time constants of both phases decreased with increasing ACh concentrations but showed no potential dependency. 5. The recovery from desensitization of the ICI induced by 5 x 10(-6) M ACh proceeded double exponentially, with time constants of 11 and 114 sec at a holding potential of -30 mV. 6. Noise analysis was performed on a steady-state current induced by 3 x 10(-7) to 2 x 10(-6) M ACh. The mean open time was about 60 msec at 10(-6) M ACh and the single-channel conductance was 14 PS. 7. These results suggest that the ACh receptor-Cl channel complex in snail neurons has two binding sites with the dissociation constant of 7.1 x 10(-6) M and is rapidly activated and desensitized to a steady level in the presence of the agonist.

Gaba-activated conductance of neurons isolated from rat cerebellum and sensory ganglia

Currents activated by gamma-aminobutyric acid (GABA) were recorded in single Purkinje cells isolated from the rat cerebellum and trigeminal ganglia. All neurons tested were GABA-sensitive. Reversal potential of GABA-activated current matched equilibrium potential for chloride ions as determined by Nernst's equation. The dose-response curve was described by Langmuir's isotherm with a dissociation constant (Kd) of 1.4·10−4 M. Nembutal did not just increase the amplitude of GABA-activated current but also activated matching ionic conductance in the absence of GABA. Ionic currents activated by GABA were reversibly blocked by bicuculline methiodide and isocoryne.

Kinetic analysis of acetylcholine-induced chloride current in isolated Aplysia neurones.

(1) Kinetics of activation and desensitization phases of the ACh-induced chloride current (ICl) were studied in isolated single neurones of Aplysia kurodai, using the 'concentration clamp' technique which combines internal perfusion and rapid exchange of the external solution within a few milliseconds (2) The dose-response curve for the peak ICl gave a dissociation constant of 6.7 X 10(-6) M and a Hill coefficient of 1.7. (3) The current-voltage relationship was linear in the voltage range examined (-70 to +30 mV). The reversal potential (EACh) was -7.1 +/- 1.8 mV (n = 14). The value was close to the calculated equilibrium potential for chloride ions (ECl). (4) The activation phase of the ICl was single exponential and the desensitization proceeded double exponentially to a steady state level. The time constants of both phases decreased with increasing concentrations of ACh but showed no potential dependency. The desensitizing component of the ICl was generated by activation of a single population of the receptor-channel complex. (5) The recovery from desensitization of the ICl induced by 6 X 10(-6) M ACh proceeded double exponentially, with time constants of 6.5 and 43 s at a holding potential of -30 mV. (6) Noise analysis performed on the steady state of ICl induced by low concentrations of ACh (3 X 10(-7) M to 3 X 10(-6) M) showed that the steady ICl was due to activation of a single population of the receptor-channel complex with a single channel conductance of 23.3 +/- 4.3 pS (n = 9).(ABSTRACT TRUNCATED AT 250 WORDS)

Calcium-activated chloride current in cultured sensory and parasympathetic quail neurones.

1. Sensory (trigeminal and dorsal root) and autonomic (ciliary) ganglia from embryonic quail were dissociated and the neurones were grown in tissue culture. 2. Intracellular recordings were made in voltage clamp using patch electrodes and the whole-cell recording technique. In order to investigate a calcium-activated chloride current, the sodium and potassium currents were blocked. 3. Depolarizing voltage steps from a holding potential of -100 mV to a test potential of +20 mV triggered an early inward and a delayed outward current. The latter persisted as a long-lasting inward tail current when the membrane was depolarized to -100 mV. 4. These currents were all blocked by extracellular cobalt suggesting that they were calcium dependent. During a test depolarization to +20 mV, in the presence of intracellular EGTA (20 mM), the inward current persisted but the outward current was suppressed. EGTA (20 mM) also suppressed the long-lasting inward tail current at -100 mV. This suggested the presence of a calcium-activated current. 5. The reversal potential of the calcium-activated current was near the equilibrium potential for chloride ions and was shifted as predicted by the Nernst equation when the extracellular chloride concentration was changed. 6. The calcium-activated current was partially blocked by adding 4-acetamido-4'-isothiocyanatostilbene-disulphonic acid (SITS) at a concentration of 1 mM to the external superfusion medium. This effect of a compound known to interfere with chloride channels together with the results of point (5) suggested the existence of a calcium-activated chloride current (ICl(Ca)). 7. ICl(Ca) could be activated by transient and sustained components of the calcium current present in the cultured neurones. 8. ICl(Ca) was present in 80% of the sensory neurones but only in 10% of the parasympathetic neurones.

Chloride channels activated by γ-aminobutyric acid in normal bovine lactotrophs

The effect of γ-aminobutyric acid (GABA) on normal bovine lactotrophs was investigated using the patch clamp technique. GABA application (1–10 μM) induced fluctuations of membrane current, accompanied by an increase in inward current. The mean conductance of GABA-activated channels was estimated to be 19 pS from statistical analysis of these fluctuations whereas the mean conductance for GABA-activated single ion channels was 16 pS. The reversal potential of the current was near to the equilibrium potential for chloride ions. These results indicate that the one possible mechanism for inhibition of prolactin release induced by GABA is probably mediated by activation of chloride channels.