Figure 7. Mg²⁺removal and Nudix box mutations little affect steady-state single-channel gating kinetics of nvTRPM2 channels.

(A–C) Steady-state channel currents from patches containing (A) twelve WT (B) seven E1443I/F1444L and (C) nine E1446K/E1447K nvTRPM2 channels, activated by exposure to 125 μM Ca²⁺ and 100 μM ADPR (bars) in the presence of 2 mM cytosolic Mg²⁺; channel number was estimated in each patch as the maximum number of simultaneously open channels (see Materials and methods). In (A), WT channels were reopened by a second application of ADPR in the absence of bath Mg²⁺ (orange bar: Mg²⁺ removal + addition of 100 μM CDTA). Narrow yellow boxes highlight segments of record shown to the left/right at expanded time/current scales; note well resolved gating transitions. Bandwidth, 200 Hz. (D–F) Open probabilities (D), mean burst (E) and interburst (F) durations obtained from multi-channel fits (see Figure 7—figure supplement 1), for WT nvTRPM2 with (green) or without (orange) Mg²⁺, and for E1443I/F1444L (red) and E1446K/E1447K (blue) nvTRPM2 with Mg²⁺. Data are shown as mean ± SEM from 6 to 12 patches. See also Figure 7—figure supplement 1.

Figure 7—figure supplement 1. Kinetic analysis of multichannel patches by a simultaneous maximum likelihood fit to the dwell-time histograms of all conductance levels.

(A–D) Logarithmically binned dwell-time histograms (colored bar charts) of steady-state current segments in 125 μM Ca²⁺plus 100 μM ADPR for the recordings shown in Figure 7A–C, plotted individually for each conductance level (cf., dashed lines in Figure 7A–C), obtained by half-amplitude threshold crossing analysis using an imposed fixed dead time of 0.9 ms. The histogram sets in (A) (WT, +Mg²⁺), (B) (WT, −Mg²⁺), (C) (E1443I/F1444L), and (D) (E1446K/E1447K) contain 4560, 7061, 3505, and 1418 events, respectively, in total. Families of solid black curves illustrate the simultaneous maximum likelihood fits of the sets of dwell-time histograms by the C_slow(1)-O₍₃₎-C_fast(2) scheme, accounting for the fixed dead time (Csanády, 2000), with the four rate constants r₁₃, r₃₁, r₃₂, r₂₃ as free parameters. Fitted rate constants were (A) r₁₃ = 9.75 s⁻¹, r₃₁=0.880 s⁻¹, r₃₂=1.56 s⁻¹, r₂₃=633 s⁻¹, (B) r₁₃ = 6.94 s⁻¹, r₃₁=1.14 s⁻¹, r₃₂=2.28 s⁻¹, r₂₃=633 s⁻¹, (C) r₁₃ = 3.96 s⁻¹, r₃₁=1.29 s⁻¹, r₃₂=1.14 s⁻¹, r₂₃=504 s⁻¹, and (D) r₁₃ = 9.99 s⁻¹, r₃₁=1.02 s⁻¹, r₃₂=0.969 s⁻¹, r₂₃=463 s⁻¹. Calculated bursting parameters (cf., Figure 7E–F) were (A) τ_b=1140 ms, τ_ib=103 ms, (B) τ_b=881 ms, τ_ib=144 ms, (C) τ_b=779 ms, τ_ib=253 ms, and (D) τ_b=983 ms, τ_ib=100 ms.