Statistical analysis of single sodium channels. Effects of N-bromoacetamide

Abstract

Currents were obtained from single sodium channels in outside-out excised patches of membrane from the cell line GH3. The currents were examined in control patches and in patches treated with N- bromoacetamide ( NBA ) to remove inactivation. The single-channel current-voltage relationship was linear over the range -60 to + 10 mV, and was unaffected by NBA . The slope conductance at 9.3 degrees C was 12 pS, and the Q10 for single channel currents was about 1.35. The currents in both control and NBA -treated patches showed evidence of a slow process similar to desensitization in acetylcholine-receptor channels. This process was especially apparent at rapid rates of stimulation (5 Hz), where openings occurred in clusters of records. The clustering of records with and without openings was analyzed by runs analysis, which showed a statistically significant trend toward nonrandom ordering in the responses of channels to voltage pulses. NBA made this nonrandom pattern more apparent. The probability that an individual channel was "hibernating" during an activating depolarization was estimated by a maximum likelihood method. The lifetime of the open state was also estimated by a maximum likelihood method, and was examined as a function of voltage. In control patches the open time was mildly voltage-dependent, showing a maximum at about -50 mV. In NBA -treated patches the open time was greater than in the control case and increased monotonically with depolarization; it asymptotically approached that of the control patches at hyperpolarized potentials. By comparing channel open times in control and NBA -treated patches, we determined beta A and beta I, the rate constants for closing activation gates and fast inactivation gates. Beta I was an exponential function of voltage, increasing e-fold for 34 mV. beta A had the opposite voltage dependence. The probability of an open channel closing its fast inactivation gate, rather than its activation gate, increased linearly with depolarization from -60 to -10 mV. These results indicate that inactivation is inherently voltage dependent.

Quantification of exponential Na+ current activation in N‐bromoacetamide‐treated cardiac myocytes of guinea‐pig.

“…Removal of the Na+ current inactivation The removal of Na+ inactivation using NBA was accompanied by a decrease in the current amplitude as described previously in different preparations (Oxford et al 1978;Horn et al 1984;Gonoi & Hille, 1987). The decrease was progressive as long as the application of NBA was continued.…”

“…In this study, we removed inactivation of the Na' current by the intracellular perfusion of single myocytes with N-bromoacetamide (NBA) taking advantage of the oil-gap method. The reduction of the amplitude of Na+ current (Oxford, Wu & Narahashi, 1978;Horn, Vandenberg & Lange, 1984;Gonoi & Hille, 1987) was minimized by washing out the NBA quickly after a partial reduction in the inactivation. The adequacy of in' kinetics was confirmed and the rate constants for the rate-limiting step between a closed state and an open state were thus determined based on a new activation model.…”

Statistical properties of ion channel records. Part II: Estimation from the macroscopic current

“…To evaluate how closely the calibrated nonstationary Markov model represents the stochastic process of channel gating and generates the statistics of single-channel records, we compare other independent statistical properties of single channel sweeps, not used for calibration: the pdf of the latency to first opening, F(t), the cdf of closed duration, R(t), and the distribution of the number of openings per record, E(n). (14), (18) and (19) respectively, where the φ function is defined by Equations (16) and (17). These three statistical properties are measured from the data and compared with the model prediction.…”

“…However, Markov models have been shown to predict experimental data better than fractal models [8][9][10][11][12][13] and are the accepted statistical models commonly used in the scientific literature. Statistical analysis of single channel records has been used to construct and calibrate appropriate Markov models [14][15][16][17][18][19][20][21] and also to relate macroscopic current fluctuations to single channel properties [22][23][24][25]. Effects of different environmental conditions (e.g.…”

Mode switching characterizes the activity of large conductance potassium channels recorded from rat cortical fused nerve terminals

“…In order to determine whether the appearance of high levels of channel activity occurred in a random manner or were clustered in defined periods, due to mode switching, the data were examined for statistical correlation in the patterns of gating by using the procedures of 'runs' analysis (Horn et al 1984) and Fisher's exact test (Plummer & Hess, 1991). The runs test was used as follows:…”