Scholl et al. 10.1073/pnas.0601631103.

Supporting Information

Files in this Data Supplement:

Supporting Figure 6
Supporting Text
Supporting Figure 7
Supporting Figure 8





Fig. 6. Subcellular distribution of barttin, rClC-K1, and hClC-Kb heterologously expressed in tsA201 cells. (A) Confocal image of a live tsA201 cell coexpressing barttin-YFP fusion protein and CFP-Mem. (B and D) Confocal images of live tsA201 cells expressing YFP-hClC-Kb (B) or YFP-rClC-K1 (D) channels alone. (C and E) Confocal images of live tsA201 cells coexpressing YFP-hClC-Kb (C) or YFP-rClC-K1 (E) together with barttin-CFP. (Scale bars: 5 mm.) CFP is shown in green, YFP in red. This color code results in an orange coloring of regions where both proteins overlap.





Fig. 7. Barttin increases the unitary current amplitude of V166E rClC-K1. (A and B) Variance analysis for V166E rClC-K1 channels, without (A) or with (B) barttin. The cell was held at 0 mV (A) or +75 mV (B), and a test step to -140 mV was applied. Shown are time dependence of the mean current obtained from 300 traces, time course of the variance, and variance versus mean current plot. The solid line is a fit of the function s2 = iI(t) - I(t)2/N + so2.





Supporting Figure 8

Fig. 8. Unitary current measurements corroborate an increased conductance of V166E rClC-K1/barttin channels. (A and C) Representative single-channel recordings from V166E rClC-K1 channels alone (A) and with (C) barttin. The patch in A contained one channel, the one in C two. (B and D) Amplitude histograms from continuous recordings at +120 mV from V166E rClC-K1 channels alone (B) and with barttin (D). The solid lines give fits with sums of Gaussian distributions. (E) Voltage dependence of unitary current amplitude determined by single channel recordings on V166E rClC-K1 channels with (Ñ) and without (l) barttin. (F) Voltage dependence of absolute open probabilities determined by single channel recordings on V166E rClC-K1 channels with (Ñ) and without (l) barttin.





Supporting Text

Nonstationary Noise Analysis. Fig. 7 shows variance analyses from one tsA201 cell expressing V166E rClC-K1 channels alone and from another cell cotransfected with the mutant channel and barttin. The time courses of the amplitudes of the mean current (I) and the variance (s2) were determined for voltage steps to a test potential of –140 mV from a holding potential of 0 mV or +75 mV, respectively, and variances were then plotted versus the corresponding mean current amplitude. These variance-mean current plots were fitted to s2 = iI – I2/N to obtain the unitary current amplitude (i) and the number of channels N at this potential (1, 2). At –140 mV, the unitary current amplitudes of V166E rClC-K1 without barttin are 0.9 ± 0.2 pA (n = 6) and 2.4 ± 0.2 pA (n = 6) with barttin, indicating that barttin increases the single-channel amplitude of V166E ClC-K1 channels about 3-fold.

Single-Channel Recordings.

 Single-channel recordings were obtained from inside-out or outside-out patches from cells transfected with mutant V166E rClC-K1 channels with and without barttin. Before changing to the outside-out configuration, the existence of a V166E rClC-K1 current was tested in the whole-cell configuration. Fig. 8 shows representative current traces from both types of channels at different potentials. In 12 of 14 patches from cells coexpressing the V166E rClC-K1 and barttin, we detected single-channel activity with 20.6 pS conductance that exhibited higher open probability at positive than at negative membrane potentials (Fig. 8C). We did not detect similar openings in any of 29 patches from cells expressing the V166E rClC-K1 alone. In nine of these patches, we recorded single-channel openings with significantly smaller single channel conductance and altered gating behavior (Fig. 8A) than in patches from cells coexpressing V166E rClC-K1 and barttin.

The single-channel amplitude and the absolute open probability were determined by fitting Gaussian distributions to amplitude histograms (Fig. 8 B and D). From linear fits of the single-channel current-voltage plots, we obtained unitary current conductances of 20.5 pS (with barttin) and 8.6 pS (without barttin), respectively (Fig. 8E). In addition, we calculated the open probability by constructing amplitude histograms from gap-free recordings of at least 3 s length. For V166E rClC-K1/barttin channels, these data correlate well with the open probability curves obtained from nonstationary noise analysis (Fig. 8F). For channels without barttin, the absolute open probabilities determined by single channel recordings are smaller than the noise analysis results. This small difference is most likely due to an underestimation of the open probability because of missed events due to the small unitary conductance of this channel.

Prokaryotic and eukaryotic ClC isoforms exhibit two ion conduction pathways, and single-channel recordings therefore show two conducting levels, of which one appears to be about twice the current of the other (3-5). However, the different unitary conductances with and without barttin are not due to a preferential occurrence of half-conductance openings in the absence and full conductance openings in the presence of barttin. For both types of channels, we observed infrequent subconductance states with a current amplitude of »50% of the 20.5 pS (with barttin) and 8.6 pS (without barttin) full conductance state.

Mutagenesis and Channel Expression.

 pRc/CMV-hClC-Kb (6) was provided by A. L. George (Vanderbilt University, Nashville, TN). rClC-K1 (7) (provided by S. Uchida, Tokyo Medical and Dental University) was subcloned into pRc/CMV using a PCR-based strategy, and human barttin (8) (provided by A. L. George) into pcDNA3.1. To enhance the percentage of bead-decorated and channel-expressing cells, we constructed a bicistronic vector containing the coding region of the respective ClC-K channel and of the CD8 antigen and a sequence encoding the viral internal ribosomal entry site (IRES) from pIRES2 EGFP (Clontech). CFP, GFP, or YFP fusion proteins of ClC-K channels and barttin were constructed as described (9). Truncated barttins were constructed by PCR-based strategies as CFP fusion proteins. The FLAG-tagged ClC-K channels were generated by insertion of a sequence encoding for DYKDDDDK at amino acid position 377 in the extracellular loop between helices L and M of the ClC-K channel by PCR-based strategies. Construction of concatamers was performed as described (10). For all constructs, PCR-amplified sequences were verified by direct sequencing, and two independent mutant clones were used for expression studies.

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