Full Open State Research Articles

Single chloride-selective channel from cardiac sarcoplasmic reticulum studied in planar lipid bilayers

The behavior of single Cl- channel was studied by fusing isolated canine cardiac sarcoplasmic reticulum (SR) vesicles into planar lipid bilayers. The channel exhibited unitary conductance of 55 pS (in 260 mM Cl-) and steady-state activation. Subconductance states were observed. Open probability was dependent on holding potentials (-60 to +60 mV) and displayed a bell-shaped relationship, with probability values ranging from 0.2 to 0.8 with a maximum at -10 mV. Channel activity was irreversibly inhibited by DIDS, a stilbene derivative. Time analysis revealed the presence of one time constant for the full open state and three time constants for the closed states. The open and the longer closed time constants were found to be voltage dependent. The behavior of the channel was not affected by changing Ca2+ and Mg2+ concentrations in both chambers, nor by adding millimolar adenosine triphosphate, or by changing the pH from 7.4 to 6.8. The presence of sulfate anions decreased the unit current amplitude, but did not affect the open probability. These results reveal that at the unitary level the cardiac SR anion-selective channel has distinctive as well as similar electrical properties characteristic of other types of Cl- channels.

Properties of single chloride selective channel from sarcoplasmic reticulum.

The behavior of single chloride channels in sarcoplasmic reticulum of rabbit and trout skeletal muscle was examined by fusing isolated vesicle fractions into planar lipid bilayers. The channel exhibited a full open state with a unit conductance of 65 pS (in 100 mM Cl-) and several subconductance states with reversal potentials which were dependent on the chloride gradient across the bilayer. Open probability was 0.6-0.95 for membrane potentials ranging from -60 to +60 mV. The kinetic behaviour could be described by assuming one time constant for the fully conducting channel, and at least two time constants for the non-conducting channel. In the presence of methane sulfonate, sulfate and phosphate anions, a decrease in the unit current amplitude but not open time argued in favor of a competition between these anions and Cl- at the transport site of the channel. Chloride channel activity was not affected by variations of Ca2+ concentration in both chambers or by the presence of Mg2+. Similarly, neither millimolar ATP nor the presence of the drugs taurine (up to 10 mM), lidocaine (2-40 microM) or the calmodulin antagonist W7 (5-150 microM), modified channel behavior. Finally, pH variations between 6.8 to 8 were without effect.

Characterization of the channel properties of tetanus toxin in planar lipid bilayers

A detailed characterization of the properties of the channel formed by tetanus toxin in planar lipid bilayers is presented. Channel formation proceeds at neutral pH. However, an acidic pH is required to detect the presence of channels in the membrane rapidly and effectively. Acid pH markedly lowers the single-channel conductance, for phosphatidylserine at 0.5 M KCl gamma = 89 pS at pH 7.0 while at pH 4.8, gamma = 30 pS. The toxin channel is cation selective without significant selectivity between potassium and sodium (gamma [K+]/gamma [Na+] greater than or equal to 1.35). In all the lipids studied gamma is larger at positive than at negative voltages. The toxin channel is voltage dependent both at neutral and acidic pH: for phosphatidylserine membranes, the probability of the channel being open is much greater at positive than at negative voltage. In different phospholipids the channel exhibits different voltage dependence. In phosphatidylserine membranes the channel is inactivated at negative voltages, whereas in diphytanoylphosphatidylcholine membranes channels are more active at negative voltages than at positive. The presence of acidic phospholipids in the bilayers increases both the single-channel conductance as well as the probability of the channel being open at positive voltage. A subconductance state is readily identifiable in the single-channel recordings. Accordingly, single-channel conductance histograms are best fitted with a sum of 3 Gaussian distributions corresponding to the closed state, the open subconductance state and the full open state. Channel activity occurs in bursts of openings separated by long closings. Probability density analysis of the open dwell times of the toxin channel indicate the existence of a single open state with a lifetime greater than or equal to 1 ms in all lipids studied. Analysis of intra-bursts closing lifetimes reveals the existence of two components; the slow component is of the order of 1 ms, the fast one is less than or equal to 0.5 ms. The channel activity induced by tetanus toxin in lipid bilayers suggests a mechanism for its neurotoxicity: a voltage dependent, cation selective channel inserted in the postsynaptic membrane would lead to continuous depolarization and, therefore, persistent activation of the postsynaptic cell.