Multiple Open States Research Articles

Conductance properties of the Na(+)‐activated K+ channel in guinea‐pig ventricular cells.

1. The Na(+)-activated K+ channel current was recorded from inside-out membrane patches excised from single ventricular cells of guinea-pig hearts. 2. The single channel current-voltage relations showed inward-going rectification with an asymptotic conductance of 180-210 pS for the inward current at 150 mM [K+]o, when [K+]i was changed between 5.4 and 150 mM. The reversal potential indicated the PNa/PK of about 0.02. 3. The amplitude of outward current was reduced by increasing [Mg2+]i or [Na+]i, but no obvious blocking noise was recorded. The outward current, which remained shortly after quick removal of both [Na+]i and [Mg2+]i, revealed an ohmic conductance of the K+ channel. 4. The [Mg2+]i and [Na+]i block was increased e-fold by depolarizing the membrane by 49 mV, while the inward current was not blocked. 5. The Na(+)-activated K+ channel showed frequent subconductance levels. The variance-mean analysis resolved at least ten major sublevels. The density distribution of the sublevels were measured by composing the conventional amplitude histogram, excluding clear closed state currents, and then dividing the histogram into five segments. The probability of staying in each segment (Pn) was almost always voltage independent, and the grand averages were P1 = 9.5 +/- 5.9%, P2 = 6.3 +/- 2.1%, P3 = 4.2 +/- 1.8%, P4 = 7.8 +/- 2.5%, and P5 = 39.3 +/- 5.6%, from the lowest segment, respectively. 6. The values of Pn in partially blocked conditions by Na+ and Mg2+ (outward current) were not clearly different from those without any channel block (inward current). The values of Pn, measured before and after applying Ba2+ in the pipette, were also very similar. 7. The above findings indicate that the inward-going rectification of the Na(+)-activated K+ channel is due to the Na+ and Mg2+ block. The subconductance of the channel is not due to any channel block by Na+ or Mg2+, but may be attributable to multiple open states of a single-barrel channel, which has a large conductance. The channel may be blocked from any open conformation with an equal probability and with very fast kinetics.

Mechanism of inactivation of single sodium channels after modification by chloramine-T, sea anemone toxin and scorpion toxin.

Single sodium-channel currents were measured in neuroblastoma cells after inhibition of inactivation by chloramine-T (CHL-T), sea anemone toxin II (ATX-II) and scorpion toxin (SCT). The decaying phase of the averaged single-channel currents recorded with 90-msec pulses in cell-attached patches was clearly slower than that of the ummodified channels, suggesting inhibition of macroscopic inactivation. Each substance caused repetitive openings and a moderate increase in the channel open time. At Vm = RP + 20 mV and T = 12 degrees C, the mean channel open times were 1.4, 1.6 and 1.8 msec for CHL-T, ATX-II and SCT, respectively, as opposed to 1.07 msec for native channels. Open-time histograms could be best fitted by the sum of two exponentials. The time constants of the fits were similar for histograms constructed from single openings and from openings during bursts. This suggests that the population of channels is homogeneous and that in bursts the same open conformations of channels occur as in single openings. Mean burst durations for bursts consisting of more than one opening at Vm = RP + 20 mV were 4.9, 5.8 and 6.1 msec for CHL-T, ATX-II and SCT, respectively. Burst open-time histograms constructed from two or three openings were fitted by the gamma function. The different time constants of the fits obtained for ATX-II and SCT suggested multiple open conformations of channels for openings of bursts. However, significantly different open-time histograms constructed from the first, second and third openings of bursts could not be obtained systematically. A positive correlation was found for the dwell time of the first and the second, as well as for the second and the third opening of bursts with each substance, but a negative one for the dwell time of an opening and the neighboring closing of bursts with ATX-II. The results suggest a model with multiple open and inactivated states. In this model the inactivated states are weakly absorbing.

Evidence for multiple open states of sodium channels in neuroblastoma cells

Open times of voltage-gated sodium channels in neuroblastoma cells were measured during repolarization (following a short depolarizing conditioning pulse) and during moderate depolarization. Conditional and unconditional channel open-time histograms were best fitted by the sum of two exponentials. (The conditional open time was measured from the end of the conditioning pulse until an open channel shuts provided it was open at t = 0). Time constants of both histograms depended on the post-pulse and were shifted to more positive potentials with increasing conditioning pulse potential. This shift could be explained by assuming more than two time constants in the histograms, which could not be separated. Channel open-time histograms from single-pulse experiments showed a maximum at t greater than 0. These histograms could be best fitted by an exponential function with three time constants. One term of this function included the difference of two exponentials resulting in a maximum at t greater than 0. Open-time histograms showed a definite time dependence. At 2 to 6.5 msec after the beginning of the depolarization the best fit could be obtained by the difference of two exponentials. To these components another term had to be added at 0 to 2 msec. Between 6.5 and 14.0 msec the sum of two exponentials, and after 14.0 msec a single exponential resulted in a good fit. The results support the hypothesis that sodium channels in neuroblastoma cells may have multiple open states. Two of these states are irreversibly coupled.

Large depolarization induces long openings of voltage-dependent calcium channels in adrenal chromaffin cells

Single Ca2+-channel currents in bovine adrenal chromaffin cells were studied with the patch-clamp technique using Ba2+ as the charge carrier. Depolarizing pulses to voltages less than +10 mV from holding voltage of -60 mV elicited short openings with a mean life time of less than 1 msec. Depolarization to more positive voltages elicited longer openings with a mean life time of about 3 msec in addition to the short openings similar to those observed at less positive voltages. Following large depolarizing prepulses, 2 types of "tail" openings, one with a mean duration of less than 1 msec and the other with a mean duration of 4 msec, were observed. In the presence of a dihydropyridine BAY K 8644, openings with a mean duration of more than 12 msec were present. Depolarization-induced long openings and BAY K 8644-produced long openings differed in the first latency and open-time properties. The results could be explained in terms of multiple open states of one type of Ca2+ channel. A kinetic model with at least 2 open states is required to explain activation of Ca2+ channels in chromaffin cells.

Antibody activates cationic channels via second messenger Ca 2+

Patch-clamp recordings were used to study single channels permeable to multiple cations in a macrophage cell line. At least three conductance levels were found, consistent with the existence of several types of nonselective cation channels or a single channel with multiple open states. The activity of the channels depended very little on voltage but was affected by internal Ca 2+ concentration. Specific subclasses of immunoglobulins (IgG1 and IgG2b) bound to an Fc receptor on the surface of these macrophages. When an IgG2b was applied to the cell exterior after a patch pipette had been sealed in the cell-attached mode, the nonselective cation channels within the patch were activated. Thus, these channels must be modulated by a second messenger. Since antibodies binding to the Fc receptor have been shown to produce a rise in intracellular Ca 2+, this cation must be considered a candidate as a second messenger that amplifies the effect of antibody in gating these channels.

Kinetics of unliganded acetylcholine receptor channel gating

Open- and closed-state lifetimes of unliganded acetylcholine receptor channel activity were analyzed by the method of likelihood maximazation. For both open times and closed times, the best-fitting density is most often a sum of two exponentials. These multiple open states cannot depend on the number of receptor binding sites occupied since they are observed in the absence of ligand. The rate of spontaneous opening and the faster decay constant of closing increased as the membrane was hyperpolarized. The voltage dependence of the rate of spontaneous opening is stronger than that for curare-liganded channels. Evidence that the acetylcholine receptor channel can open spontaneously in the absence of ligand has been presented previously (Sanchez et al, 1983; Brehm et al, 1984; Jackson, 1984). To add to this evidence, alpha-bungarotoxin was added to the patch electrode, causing the frequency of openings to decay with time. The rate constant determined from this decay is similar to rate constants reported for the binding of iodinated alpha-bungarotoxin to the acetylcholine receptor. The frequency of unliganded channel opening has been estimated as 2 X 10(-3) s-1 per receptor. A comparison of carbamylcholine-liganded and spontaneous gating transition rates suggests that ligand binding increases the rate of opening by a factor of 1.4 X 10(7). Carbamylcholine binding increases the mean open time by a factor of 5. Thus, a cholinergic agonist activates the acetylcholine receptor by destabilizing the closed state. The liganded and unliganded channel gating rates were used to analyze the energetics of ligand activation of the acetylcholine receptor channel, and to relate the open channel dissociation constant to the closed channel dissociation constant.

Alamethicin channels incorporated into frog node of ranvier: calcium-induced inactivation and membrane surface charges.

Alamethicin, a peptide antibiotic, partitions into artificial lipid bilayer membranes and into frog myelinated nerve membranes, inducing a voltage-dependent conductance. Discrete changes in conductance representing single-channel events with multiple open states can be detected in either frog node or lipid bilayer membranes. In 120 mM salt solution, the average conductance of a single channel is approximately 600 pS. The channel lifetimes are roughly two times longer in the node membrane than in a phosphatidylethanolamine bilayer at the same membrane potential. With 2 or 20 mM external Ca and internal CsCl, the alamethicin-induced conductance of frog nodal membrane inactivates. Inactivation is abolished by internal EGTA, suggesting that internal accumulation of calcium ions is responsible for the inactivation, through binding of Ca to negative internal surface charges. As a probe for both external and internal surface charges, alamethicin indicates a surface potential difference of approximately -20 to -30 mV, with the inner surface more negative. This surface charge asymmetry is opposite to the surface potential distribution near sodium channels.

Mechanisms of action of transmitters and other substances on smooth muscle.

Mechanisms of action of transmitters and other substances on smooth muscle.