Chloramine-T effect on sodium conductance of neuroblastoma cells as studied by whole-cell clamp and single-channel analysis

Abstract

Patch-clamp experiments were done on sodium channels of neuroblastoma cells (N1E-115) in the presence of tetraethylammonium ions to block potassium channels. In Ringer solution whole-cell records revealed a diphasic I _Na inactivation with the fast (τ₀) component being clearly larger than the slow (τ₁ ≈ 3 τ₀) component. In single-channel studies on inside-out patches the mean open time, ¯t₀, turned out to be only a fraction of τ₀ and almost independent of membrane potential. After external application of chloramine-T I _Na inactivation of whole cells was delayed with both τ₀ and τ₁ increased, and incomplete, i.e. a persistent current component emerged. The latter was maximal at a more positive membrane potential than the peak current. Also, after chloramine-T treatment the peak I _Na increased, particularly at weak depolarizations. In inside-out patches the equally effective internal application of chloramine-T led to bursting channel openings with mean burst times (¯t_b) ≈ 6 ms, and gap times (¯t_g) ≈ 20 ms, where gap is defined as a closure of ⩾ 1.5 ms. Within the bursts (¯t_o) was approximately 2 ms, again clearly shorter than τ₀; the mean close time, ¯t_c was approximately 0.5 ms. The single-channel conductance was approximately 13 pS and unaffected by chlopramine-T. Diphasic I _Na inactivation and the fact that ¯t_o < τ₀ led to an extension of the model of Aldrich and Stevens [J Neurosci 7:418–431 (1987)], in which overall kinetics is determined by the openings rather than closures of the sodium channels. The extension comprises two inactivated states in series with the open state. Estimates are given of all rate constants of the transition between states that describe the single-channel results as well as whole-cell I _Na (t), both in the control and in chloramine-T.