Single myotonia mutation strikes multiple mechanisms of a chloride channel.

Myotonia is a group of disorders affecting skeletal muscle relaxation owing to abnormal sarcolemma membrane excitability. Major symptoms include muscle stiffness and aching, sometimes accompanied by a diminished ability to terminate muscle contraction. These symptoms can be attributed to an over-activity of muscle sodium channels, which renders the membrane hyper-excitable. Alternatively, defects of a membrane stabilizing force generated by a major muscle chloride (Cl−) channel (called CLC-1) can also lead to myotonia. In this issue of The Journal of Physiology, Weinberger et al. (2012) report novel pathogenic CLC-1 mutations that convert a cysteine residue at position 277 (C277) into an arginine (C277R) or a tyrosine (C277Y). The CLC-1 Cl− channel belongs to the CLC channel/transporter family. Like other members of the CLC family, CLC-1 consists of two identical subunits, each by itself forming a Cl−-permeation pore (sometimes called protopore). Two types of gating mechanisms control the opening and closing of these two identical protopores (Chen, 2005). In one called ‘fast gating’, the two protopores are each gated independently by a pore glutamate residue (E232). It is generally held that swinging the negatively charged side-chain of E232 out of the protopore constitutes the molecular basis of the fast-gate opening (Dutzler et al. 2003). The other gating mechanism, called ‘slow gating’ or ‘common gating’, controls the opening of the two protopores simultaneously. However, the structural basis of the slow/common gating is not fully understood although subunit interactions have been suggested to play a pivotal role in this gating mechanism. Because the sarcolemmal Cl− reversal potential is near the resting membrane potential, and because CLC-1 contributes to ∼50–80% of the sarcolemmal conductance, CLC-1 provides an important membrane stabilizing force for skeletal muscles. Furthermore, both fast and slow/common gating mechanisms are voltage dependent – membrane depolarization caused by action potentials increases the open probability (Po) of CLC-1 by at least ∼5- to 10-fold. CLC-1 thus plays a critical role in bringing the membrane voltage of the excited muscle back to the resting state. A loss of CLC-1 activity due to channel mutations often severely cripples this membrane-stabilizing function, leading to myotonia. So far, more than 100 mutations of CLC-1 have been identified in patients with congenital myotonia. The mutation, C277R or C277Y, reported in Weinberger et al. (2012) affects multiple gating/permeation functions of CLC-1. Using noise analysis and single-channel recording techniques, the authors demonstrated that the C277Y mutation causes a reduction of the single protopore conductance to about two-thirds of the wild-type value. The more damaging phenotype of the mutants, however, is their abnormal gating functions. For example, it was estimated that the Po of C277Y is less than 3% of that of the wild-type channel. Furthermore, homodimeric mutant channels show a voltage dependence of channel opening opposite to that of wild-type CLC-1 – the mutant channels open in response to membrane hyperpolarization instead of depolarization! This reverse voltage dependence of channel opening results in a complete loss of CLC-1's physiological role in restoring resting state membrane potential. How do point mutations at a single amino acid residue alter channel functions so drastically? Of note, C277 of CLC-1 corresponds to C212 of a homologous CLC channel, CLC-0. It was previously shown that the C212S mutation of CLC-0 locked the slow/common gating of CLC-0 in a constitutively open state (Lin et al. 1999). The single-channel conductance and the fast-gate Po of C212S, however, were not different from those of wild-type CLC-0. Subsequent work demonstrated that the corresponding mutations in ClC-1 (C277S) (Accardi et al. 2001) or in CLC-2 (C256S) (Zuniga et al. 2004; de Santiago et al. 2005) affect the slow/common gating in these two channels in a similar manner. The observations that the C277R or C277Y mutation reduces the Po of the fast gate and that of the common gate are very different from those found in the C277S mutant (or the C212S mutant of CLC-0). What is the difference between serine versus arginine (or tyrosine) at position 277 such that the perturbations in the channel's gating functions differ? As mentioned above, the molecular mechanism for slow/common gating may involve interactions of the two subunits. Being located at a position midway between the dimer interface and the glutamate gate, C277 (or C212 of CLC-0) could relay the conformational change generated at the dimer interface to the pore region. A change of the amino acid side-chain at this position may alter this allosteric effect. Another interesting puzzle of C277 mutations is their ability to alter CLC-1's pore functions. Weinberger et al. (2012) reported that the C277Y mutation reduced the single-channel conductance and changed the anion permeability sequence of CLC-1. However, high-resolution crystal structures of bacterial CLC revealed that C277 of CLC-1 (or C212 of CLC-0) is not a pore-lining residue. What is the molecular basis underlying this alteration of pore functions? Clearly, it will require further experiments to more closely untangle the complex roles of C277 in CLC-1's gating and permeation mechanisms.