Figure 1.
Comparison of the kinetic characteristics of Kv1.3 channels between the experimental data and simulations.
(A) Kinetic model for Kv1.3 channels. The voltage-dependent rates α, β,η and φ are in an exponential form, in which the a, b, c and d are the pre-exponential factor in ms−1 and the m, n, p and q are the exponential factor in mV; the rates A and B are constants in ms−1; the v is voltage in mV. (B) The activation currents of Kv1.3 channels expressed in HEK 293 cells were recorded from a whole-cell patch. Activation was elicited by voltage steps from −70 to +50 mV with increment of 15 mV from holding potential at −90 mV applied every 90 s. The voltage protocol is plotted at the bottom. Red lines are model simulations (N = 7465). (C) Steady-state inactivation was determined by measuring the peak current at +50 mV, following the 60-s pre-conditional pulses between −80 mV and −10 mV in increments of 5 mV. The voltage protocol is plotted at the bottom. Red lines are model simulations (N = 4518). (D) Cells were depolarized to +50 mV for 7 ms from a holding −90 mV and then repolarized to various voltages between −100 and −30 mV in increments of 10 mV. The voltage protocol is placed at the bottom. Red lines are model simulations (N = 4605). (E) Statistics of activation and steady-state inactivation. For activation, V50 = −28.4±1.6 mV, s = 4.9±0.5 mV (n = 9); for inactivation, V50 = −43.5± 3.7 mV, s = 3.0±0.3 mV (n = 5). (F) Time constants of activation (n = 9) and inactivation (n = 5) were plotted as the function of voltages. (G) Time constants of deactivation (n = 10) were plotted as the function of voltages. The red and black lines (or symbols) are for the simulations and experimental data, respectively.
Figure 2.
Comparison of the recovery of Kv1.3 channels between the experimental data and simulations.
(A–B) The representative currents of recovery from inactivation were obtained in whole-cell patch mode at −90 mV and −45 mV. Two-pulse protocol: from a holding potential of −90 mV, the first pulse 1 (P1) was applied for 1 s at +50 mV, and then the second +50-mV pulse (P2) was applied for 150 ms after a variable interpulse interval at −90 mV in (A) and −45 mV in (B). The detailed recovery currents are shown in the box. The voltage protocol is placed at the bottom. Red lines are fits and black ones are data. The channel numbers for simulations are 3788 for −90 mV and 2740 for −45 mV. (C) Comparison of the time courses of recovery between the experimental data (black) and the simulations (red) at −100 mV, −90 mV, −80 mV and −45 mV. (D) Comparison of time constants of recovery between the experimental data (black) and the simulations (red). The time constants of recovery (mean ± SEM) are 8.6±2.7 s (n = 5) at −45 mV, 15.6±1.6 s (n = 3) at −80 mV, 14.6±2.4 s (n = 7) at −90 mV, 14.6±4.7 s (n = 3) at −100 mV, respectively.
Table 1.
Parameters of Kv1.3 model.
Figure 3.
The ADWX-1 toxin blocked the Kv1.3 currents without changing the channel kinetics.
(A–B) Kv1.3 activation currents were recorded before (black) and after (red) applying with 30 pM ADWX-1. Voltage protocol is placed at the top. (C) The activation G-V curves of Kv1.3 (n = 6), before (black) and after (red) applying with 30 pM ADWX-1. (D) The time constants of activation and inactivation of Kv1.3 (n = 6), before (black) and after (red) applying with 30 pM ADWX-1.
Figure 4.
The number of Kv1.3 channels regulates the membrane potential of native T lymphocyte cells.
(A) The Kv1.3 currents in a T lymphocyte cell, bathed in normal saline, were stimulated by a voltage step from −100 to 50 mV in the whole-cell mode, in the presence of 0 (black), 30 (blue) and 300 (green) pM ADWX-1. The inset shows a picture of a T cell. The measured membrane capacitance Cm = 1.52 pF. The voltage protocol is placed at the bottom. (B) A T lymphocyte cell showed the changes in membrane potentials by an injection current of 15 pA, in the presence of 0 (black), 30 (blue) and 300 (green) pM ADWX-1, respectively. A model T cell, composed of Kv1.3, CRAC, IK and TASK channels, conferred the simulations (red) corresponding to the changes of membrane potentials resulted from the Kv1.3 conductance of 0.4, 2.0 and 4.5 nS, respectively. The resting potential of model cells was −55 mV. (C) The Kv1.3 currents from a native T lymphocytes cell (black) and a model T lymphocytes cell (red) were respectively elicited by a voltage command (cyan), conferring a number of Kv1.3 n = 330. Here the background currents were subtracted by 1 nM ADWX-1. (D) Simulation for membrane potentials in a model T cell stimulated by an injection current of 15 pA, while varying the number of Kv1.3 channels. The steady-state values (Vss) of membrane potentials went up to 35.7 mV for 0.4 nS (dark blue), 19.8 mV for 2 nS (green), −0.62 mV for 4.5 nS (dark yellow), −35.9 mV for 9 nS (pink) and −43.1 mV for 22.5 nS (light blue) at the time of 10 s (gray dotted line), respectively. (E) Simulations of intracellular calcium concentrations with the different numbers of Kv1.3 channels. The steady-state calcium concentrations [Ca2+]ss were 3.48 µM for 0.4 nS (dark blue), 4.89 µM for 2 nS (green), 7.01 µM for 4.5 nS (dark yellow), 10.47 µM for 9 nS (pink) and 11.48 µM for 22.5 nS (light blue) at the moment of 10 s (gray dotted line), respectively. (F) The time course of the intracellular Ca2+ concentrations, stimulated by low osmolarity solution labeled in a cyan bar, in a native T lymphocyte cell. The inset shows the Kv1.3 currents recorded from the same T lymphocyte cell.
Figure 5.
A putative activation mechanism of T cells.
Motile polarized T cells naturally use their T-cell receptor (TCR) to come into contact with antigen(Ag)-presenting cell (APC) via peptide–major histocompatibility complex (MHC); T cells tagged activate the smaller amount of Ca2+ influx via CRAC channel, probably evoked by IP3-induced depletion of Ca2+ stores, to slightly increase the expression of Kv1.3; more Ca2+ influx via CRAC channel enters into T cell due to membrane repolarization by augment of Kv1.3; The higher Ca2+ in T cell triggers the IL-2 promoter production program.