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. 2019 Dec 9;8:e51409. doi: 10.7554/eLife.51409

Figure 4. Influence of β4 N-terminus on Slo1 gating.

(A) Voltage-dependent channel activation of the human Slo1 channel alone and co-expressed with the β1 or β4 subunit. Representative current traces recorded using two-electrode voltage clamp (TEVC) are shown. Recording buffer: HEPES 5 mM, KCl 98 mM, CaCl2 0.3 mM, and MgCl2 1 mM. Voltage protocol: holding potential 0 mV, step from −80 to 120 mV in 10 mV incremental steps, step back to 40 mV. A superposition of one single sweep (stepping to 100 mV) from the left three recordings is shown on the right. (B) A schematic drawing of the β4 subunit showing the 10 regions divided based on the atomic structure. (C) Quantification of channel activation and deactivation kinetics by fitting with a single exponential function (see Materials and methods). (D) Comparison of three pairs of mutants demonstrated that the nature of N-tail is correlated with the activation kinetics: the presence of β4 N-tail (left column) results in slower activation kinetics than β1 N-tail (right column). Schematic drawing of each corresponding mutant is shown next to the representative current traces. Voltage protocols are the same as in panel (A).

Figure 4.

Figure 4—figure supplement 1. Identification of the β4 regions with critical functional effects with TEVC.

Figure 4—figure supplement 1.

(A, B) Bar graphs of the activation time constant (τ_on) (A) and deactivation time constant (τ_off) (B) of all the mutants in an ascending order from left to right. The activation and deactivation time constants were measured at voltage of 100 mV and 40 mV, respectively. Data are represented as mean ± SEM (n = 3–5). α alone (i.e. Slo1), β1 wt and β4 wt are colored dark gray, red and blue respectively. The two dashed lines indicate the time constant value of 50% and 2-fold of β4 wt. Mutants with time constant below 50% of β4 wt are colored light gray; more than 2-fold of β4 wt colored magenta; between 50% and β4 wt colored orange; between β4 wt and 2-fold of that colored cyan. (C) Schematic drawings and representative TEVC recordings of those mutants with significantly slower activation kinetics are shown.