Properties of Heterochannels Kv(1.1-1.2)2 with Mutation T226R in the Kv1.1 Subunit

Figures

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  Figure 1. Confocal fluorescent images of intracellular distributions of K-Kv1.1m (a), K-Kv1.1m-1.2 (b), K-Kv1.1 (c), and K-Kv1.1-1.2 (d) and staining of cells with HgTx-G. Distributions of expressed protein constructs and HgTx-G are shown in red and green, respectively. Cells were incubated with 2 nM (a,c) or 0.5 nM (b,d) HgTx-G for 1 h. The bar is 10 μm. (e) Concentration-dependent binding of HgTx-G to heterochannels Kv(1.1m-1.2)2 on the membrane of Neuro-2a cells. Rav is a parameter characterizing the formation of complexes between HgTx-G and Kv(1.1m-1.2)2.

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  Figure 2. Confocal fluorescent images of cells expressing C-Kv1.2 (I), K-Kv1.1m (II), and C-Kv1.2 together with K-Kv1.1m (III,IV) at 24 h (III) or 48 h (I,II,IV) after transfection. (a,d,g,j) Images were recorded in the 465–525 nm range at λex = 458 nm. They show distribution of C-Kv1.2 (a,g,j) or absence of fluorescence in cells expressing K-Kv1.1m at these imaging parameters (d). (b,e,h,k) Images were recorded in the 590–750 nm range at λex = 458 nm and treated as described in Section 4.5 to compensate for cross-talk between fluorescence signals. They show the distribution of the FRET signal, i.e., heteromeric complexes between C-Kv1.2 and K-Kv1.1m (h,k), or the absence of a residual signal in the images of cells expressing C-Kv1.2 (b) or K-Kv1.1m (e) after the cross-talk compensation. (c,f,i,l) Images were recorded in the 590–750 nm range at λex = 561 nm. They show the distribution of K-Kv1.1m (f,i,l) or a typical distribution of intrinsic cellular fluorescence at these imaging parameters in cells expressing C-Kv1.2 (c). The scale bar is 10 µm. The intensity scale in panels (b,e,h,k) has been inverted for clarity.

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  Figure 3. Comparison of electrophysiological properties of heterochannels Kv(1.1m-1.2)2 and Kv(1.1-1.2)2 in Neuro-2a cells. (a,b) Representative traces of currents recorded in cells expressing heterochannels Kv(1.1m-1.2)2 (a) and Kv(1.1-1.2)2 (b). Currents were induced using the square-pulse depolarization protocol (inset in panel a) from the holding potential of −80 mV. (c) Steady-state activation curves of heterochannels Kv(1.1-1.2)2 and Kv(1.1m-1.2)2 formed in Neuro-2a cells. G/Gmax is the average normalized conductance of cells. (d) Densities of peak tail currents recorded after depolarization to 40 mV in cells expressing heterochannels Kv(1.1-1.2)2 and Kv(1.1m-1.2)2. (e) Half-maximum activation potentials. (f,g) Constants of channel activation (f) and deactivation (g). Significance of differences, t-test, n = (11÷13): * p < 0.05, ** p < 0.0001.