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. 2020 Dec 4;9:e59055. doi: 10.7554/eLife.59055

Figure 2. MthK IR is intrinsically heat sensitive.

(A) Representative single-channel recordings of purified MthK IR reconstituted in soybean polar lipids with 0.1 mM calcium at various temperatures. All traces were recorded at −100 mV from the same patch. (B) Current amplitude histograms (left) and open-dwell time (right) of single-channel recordings with the same color scheme as in (A). Inset: The current amplitude histograms corresponding to open channels are enlarged for clarity. (C) Open-dwell time (solid black line) and unitary conductance (dashed blue line) of MthK IR plotted with respect to temperature. (D) Box graph of open probability at various temperatures. The boxes indicate 25–75 percentiles of the data; the whiskers show 5–95 percentiles of the data. Within each box, the line indicates the median value and red symbol marks the mean. Each individual data point is shown as circles. Error bars for (C) are SEM calculated from n = 10 (21°C), 4 (26°C), 4 (32°C), 5 (37°C) independent measurements.

Figure 2—source data 1. Source data for each open probability, open dwell time and condctance data point in the presence of 0.1 mM Ca2+.

Figure 2.

Figure 2—figure supplement 1. Purification and single-channel recordings of MthK IR.

Figure 2—figure supplement 1.

(A) Size exclusion chromatography (SEC) profile of MthK IR in Superdex 200 column. (B) SDS-PAGE analysis of the peak SEC fraction. (C) Extended single-channel recordings of MthK IR at 0.1 mM calcium. The two arrows mark the region shown in Figure 2A.
Figure 2—figure supplement 1—source data 1. Source data for gel filtration profile of MthK IR.
Figure 2—figure supplement 2. Open-dwell time histogram fitted with exponentials.

Figure 2—figure supplement 2.

(A) The open-dwell time of MthK IR recorded at 39°C shown in Figure 2B was fitted using Clampfit. Based on the most likelihood method, the best fitting model is three term exponential with time constants: τ1=0.60 ms, τ1=0.60ms, and τ2=3.87ms.
Figure 2—figure supplement 2—source data 1. Source data for bin plot of open dwell time.
Figure 2—figure supplement 3. Heat activation of MthK channel is reversible.

Figure 2—figure supplement 3.

(A) Representative single-channel recordings of temperature reversibility of purified MthK IR reconstituted in soybean polar lipids with 0.1 mM calcium. All traces were recorded at −100 mV from the same patch. (B) Box graph of open probability of MthK IR at 21°C before and after heat activation. The boxes represent 25–75 percentiles of the data; the whiskers indicate 5–95 percentiles of the data. Within each box, the line indicates the median value and red symbol marks the mean. Each individual data point is shown as circles, black for pre-heating and blue for post-heating. .
Figure 2—figure supplement 3—source data 1. Source data for reversibility of MthK IR.