Kinetics of 9-aminoacridine block of single Na channels.

Abstract

The kinetics of 9-aminoacridine (9-AA) block of single Na channels in neuroblastoma N 1 E-115 cells were studied using the gigohm seal, patch clamp technique, under the condition in which the Na current inactivation had been eliminated by treatment with N-bromoacetamide (NBA) . Following NBA treatment, the current flowing through individual Na channels was manifested by square-wave open events lasting from several to tens of milliseconds . When 9-AA was applied to the cytoplasmic face of Na channels at concentrations ranging from 30 to 100 /M, it caused repetitive rapid transitions (flickering) between open and blocked states within single openings of Na channels, without affecting the amplitude of the single channel current . The histograms for the duration of blocked states and the histograms for the duration of open states could be fitted with a single-exponential function . The mean open time (To) became shorter as the drug concentration was increased, while the mean blocked time (Tb) was concentration independent . The association (blocking) rate constant, k, calculated from the slope of the curve relating the reciprocal mean open time to 9-AA concentration, showed little voltage dependence, the rate constant being on the order of 1 x 10' M's' . The dissociation (unblocking) rate constant, l, calculated from the mean blocked time, was strongly voltage dependent, the mean rate constant being 214 s' at 0 mV and becoming larger as the membrane being hyperpolarized . The voltage dependence suggests that a first-order blocking site is located at least 63% of the way through the membrane field from the cytoplasmic surface . The equilibrium dissociation constant for 9-AA to block the Na channel, defined by the relation of 11k, was calculated to be 21 uM at 0 mV. Both To' and Tb' had a Q10 of 1 .3, which suggests that binding reaction was diffusion controlled . The burst time in the presence of 9-AA, which is the sum ofopen times and blocked times, was longer than the lifetime of open channels in the absence of drug. All of the features of 9-AA block of single Na channels are compatible with the sequential model in which 9-AA molecules block open Na channels, and the blocked channels could not close until 9-AA molecules had left the blocking site in the channels .

On the mechanism of drug-induced blockade of Na+ currents: interaction of antiarrhythmic compounds with DPI-modified single cardiac Na+ channels.

“…In contrast to 9-aminoacridine 14 and the blocking antiarrhythmic drugs tested in the present experiments, STX was shown to be incapable of flicker blocking open modified Na + channels.…”

“…Consequently, normal Na + channels reach the nonconductive configuration more rapidly than blocking molecules with slow association kinetics, such as antiarrhythmic drugs, can interact with them. Flicker block in DPIor NBA-modified Na + channels 14 seems primarily related to their severalfold prolonged channel lifetime, although it cannot be definitely excluded that channel modification per se might finally alter the drug sensitivity of the open state.…”

“…The blocking rate constants found with propafenone and prajmalium are in the same order of magnitude as those obtained with several local anesthetics 26 or octylguanidine 31 in acetylcholine-activated channels or with 9-aminoacridine in NBA-modified neuronal Na + channels. 14 ) were taken as arguments that diffusion controls the binding reaction. This is not inconsistent with a drug specificity of block kinetics that is suggested by the observed differences between the propafenone-induced and the prajmalium-induced open-channel blockade.…”

Responsiveness of cardiac Na+ channels to antiarrhythmic drugs: The role of inactivation

“…Another, second type of block having a quite different biophysical phenomenology and not being involved in the conventional IN~ blockade operates additionally in kinetically modified Na + channels. After pharmacological or chemical removal of Na + inactivation, they become blocked repetitively during their open state [17,37,38]. This microscopic or flicker blockade visualizes the arrival and departure of a blocking molecule and may be considered to reflect the reaction kinetics of the drug-receptor interaction that underlies the channel blockade.…”

Switching Between Two Types of Bursting Activity of Single Ca+2‐Activated K+ Channels in Dissociated Neurons

“…In contrast, we detect the two types of bursts with similar frequency in a given patch. We propose the following 'parallel scheme' to explain our results: (12)(13)(14)(15). In the Ca2+-activated K + channels, some intracellular cations such as Na', Cs+ and TEA' produce rapid conductance fluctuations during openings of the single channel (16,17).…”