Myotonia-related mutations in the distal C-terminus of ClC-1 and ClC-0 chloride channels affect the structure of a poly-proline helix

Abstract

Myotonia is a state of hyperexcitability of skeletal-muscle fibres. Mutations in the ClC-1 Cl- channel cause recessive and dominant forms of this disease. Mutations have been described throughout the protein-coding region, including three sequence variations (A885P, R894X and P932L) in a distal C-terminal stretch of residues [CTD (C-terminal domain) region] that are not conserved between CLC proteins. We show that surface expression of these mutants is reduced in Xenopus oocytes compared with wild-type ClC-1. Functional, biochemical and NMR spectroscopy studies revealed that the CTD region encompasses a segment conserved in most voltage-dependent CLC channels that folds with a secondary structure containing a short type II poly-proline helix. We found that the myotonia-causing mutation A885P disturbs this structure by extending the poly-proline helix. We hypothesize that this structural modification results in the observed alteration of the common gate that acts on both pores of the channel. We provide the first experimental investigation of structural changes resulting from myotonia-causing mutations.

Figure 1. Surface expression analysis of ClC-1 containing mutations associated with myotonia identified in the C-terminal distal region

(A) Schematic representation of the ClC-1 channel with the cytoplasmic C-terminus that contains two CBS domains and a C-terminal distal region (CTD, in grey, residues 871–988). Arrows indicate the positions of mutations identified in the CTD region associated with myotonia (A885P, R894X and P932L). Comparison of amino acid sequences of several CLC channels after the second CBS domain indicated a conserved proline-rich amino acid segment (italics). The position of two myotonia mutations is shown in boldface. (B) Surface expression analysis of the mutants. Surface expression was quantified using antibody-mediated detection of an extracellularly inserted HA epitope and by luminometry. A representative experiment is shown. Compared with wild-type ClC-1 (100%), A885P reduced surface expression to 50% (n=43), R894X to 10% (n=114) and P932L to 50% (n=45). Inset: Western-blot analysis using the same oocytes show that the steady-state levels of the protein are similar for mutations A885P and P932L and dramatically reduced for truncation R894X. Results of a typical experiment from five different experiments are shown. Compared with wild-type ClC-1, A885P reduced protein expression to 80% (n=5), R894X to 29% (n=9) and P932L to 90% (n=5). r.u., relative units.

Figure 2. Deletion scanning mutagenesis of the C-terminal region reveals a key segment involved in gating and surface expression

(A) Conductance levels (expressed as a percentage of wild-type levels) of C-terminal deletion constructs. Oocytes were injected with 10 ng of each cRNA, and the resulting conductances (at 0 mV, in μS) was measured by two-electrode voltage clamp. Conductance levels are normalized to wild-type current. Data correspond to at least two experiments with n≥7 for each construct. (B) Surface expression (black bars) of key deletion constructs tagged with an extracellular HA epitope, determined as in Figure 1(B). The white bars indicate the conductance values in relation to wild-type ClC-1. Inset: Western-blot analysis of solubilized oocyte membranes for the same constructs. Compared with wild-type ClC-1, truncation 946X reduced protein expression to 54% (n=5), truncation 894X to 29% (n=9), truncation 871X to 102% (n=5) and truncation 854X to 39% (n=5). r.u., relative units. (C) Voltage of half-activation for some deletion constructs. Tail current analysis was used to determine these values (see the Experimental section).

Figure 3. Similar role of the conserved segment in the ClC-0 channel

Two-electrode voltage-clamp traces from oocytes expressing the indicated mutants. They were evoked by a pulse protocol consisting of a prepulse to 60 mV, followed by a series of test pulses ranging from 80 to −140 mV and a final pulse to −100 mV. Note the inverted voltage dependence of ClC-0 and mutant F785X, but not D787X.

Figure 4. Secondary structure of the conserved segment in ClC-0

(A) Typical traces from two-electrode voltage-clamp analyses of Xenopus oocytes expressing mutants from the conserved sequence (R777A, L778A, L782A and F785A). Traces were evoked by a pulse protocol as described in Figure 3. Hyperpolarization-activated currents were observed in residues Leu⁷⁷⁸, Leu⁷⁸² and Phe⁷⁸⁵, but not in the other mutants of the conserved segment (R777A and results not shown). (B) Stick (left-hand side) and surface (right-hand side) representations of the lowest-energy model derived from NMR analysis of a peptide corresponding to the CTD conserved segment of wild-type ClC-0.

Figure 5. NMR structure of a ClC-0 peptide containing a mutation identified in myotonia reveals structural differences caused by the mutation

The left panels show two-electrode voltage-clamp traces from oocytes expressing the indicated mutants evoked by the pulse protocol described in Figure 4. Note the inverted voltage dependence of wild-type compared with that of the mutant A783P. The non-overlapping currents during the 60 mV prepulse observed for mutant S784P result from an accelerated closing kinetics of the slow gate, which is otherwise very similar to wild-type ClC-0. Right panels show schematic representations of each peptide studied by NMR. Peptide models are calculated on the basis of the NOEs observed in solution (see Supplementary Figure 1). The poly-proline conformation was clearly observed in solution, resulting in a pattern of NOEs that showed all prolines in the trans conformation. As a result of the observed restraints the structure calculation displays a high convergence in the region comprising the prolines, while the remaining residues are ill defined in solution. Six structures with less energy are represented, out of ten calculated for each peptide. Figures were generated with MOLMOL [33] (http://www.molmol.org).

Figure 6. NMR structure of ClC-1 peptides reveals structural conservation between both channels

Schematic representations of each peptide studied (sequence below) by NMR. Peptide models are calculated on the basis of the NOEs observed in solution (see Supplementary Figure 1). Figures were generated with MOLMOL [33] (http://www.molmol.org). Note how the alanine to proline mutation extends the poly-proline helix.

Figure 7. Mutations in the conserved segment affect the common gate

(A) Patch–clamp traces evoked from a patch containing several ClC-0 L782A channels (average of eight recordings). After a prepulse to −140 mV the voltage was stepped up to various values ranging from +80 to −140 mV. Finally, a ‘tail’ pulse was applied to +80 mV. In (B), the normalized initial current measured during the tail pulse is plotted towards the prepulse voltage and fitted by a Boltzmann function resulting in the parameters V_0.5=−75 mV, z=1.7, p_min=0.0. Comparable values for wild-type ClC-0 obtained with a longer pulse protocol to drive the slow gate into steady state are V_0.5=−100 mV, z=1.8, p_min≈0.1 (results not shown). (C) The Figure shows recordings from a patch containing several L782A channels (−60 mV) and from a single-channel wild-type ClC-0 patch (−100 mV). Traces are at identical scales. The presence of more than one channel for the L782A patch was verified by pulses to more negative voltages (results not shown) at which the open probability increases markedly (see B). The short ‘burst’ duration of the mutant compared with wild-type is evident. Similar short burst durations were seen in all patches of mutant L782A (n=4). The comparably long burst of wild-type ClC-0 is well documented (e.g. [34]). The dotted line indicates the closed channel current level.