Structures of wild-type and selected CMT1X mutant connexin 32 gap junction channels and hemichannels.

In myelinating Schwann cells, connection between myelin layers is mediated by gap junction channels (GJCs) formed by docked connexin 32 (Cx32) hemichannels (HCs). Mutations in Cx32 cause the X-linked Charcot-Marie-Tooth disease (CMT1X), a degenerative neuropathy without a cure. A molecular link between Cx32 dysfunction and CMT1X pathogenesis is still missing. Here, we describe the high-resolution cryo-electron cryo-myography (cryo-EM) structures of the Cx32 GJC and HC, along with two CMT1X-linked mutants, W3S and R22G. While the structures of wild-type and mutant GJCs are virtually identical, the HCs show a major difference: In the W3S and R22G mutant HCs, the amino-terminal gating helix partially occludes the pore, consistent with a diminished HC activity. Our results suggest that HC dysfunction may be involved in the pathogenesis of CMT1X.

Probing conformational changes during activation of ASIC1a by an optical tweezer and by methanethiosulfonate-based cross-linkers.

Acid-sensing ion channels (ASICs) are neuronal, proton-gated, Na+-selective ion channels. They are involved in various physiological and pathological processes such as neurodegeneration after stroke, pain sensation, fear behavior and learning. To obtain information on the activation mechanism of ASIC1a, we attempted in this study to impose distance constraints between paired residues in different channel domains by using cross-linkers reacting with engineered Cys residues, and we measured how this affected channel function. First, the optical tweezer 4'-Bis(maleimido)azobenzene (BMA) was used, whose conformation changes depending on the wavelength of applied light. After exposure of channel mutants to BMA, an activation of the channel by light was only observed with a mutant containing a Cys mutation in the extracellular pore entry, I428C. Western blot analysis indicated that BMA did not cross-link Cys428 residues. Extracellular application of methanethiosulfonate (MTS) cross-linkers of different lengths changed the properties of several Cys mutants, in many cases likely without cross-linking two Cys residues. Our observations suggest that intersubunit cross-linking occurred in the wrist mutant A425C and intrasubunit cross-linking in the acidic pocket mutant D237C/I312C. In these mutants, exposure to cross-linkers favored a non-conducting channel conformation and induced an acidic shift of the pH dependence and a decrease of the maximal current amplitude. Overall, the cross-linking approaches appeared to be inefficient, possibly due to the geometrical requirements for successful reactions of the two ends of the cross-linking compound.

Accelerated Current Decay Kinetics of a Rare Human Acid-Sensing ion Channel 1a Variant That Is Used in Many Studies as Wild Type.

Acid-sensing ion channels (ASICs) are neuronal Na+-permeable ion channels that are activated by extracellular acidification and are involved in fear sensing, learning, neurodegeneration after ischemia, and in pain sensation. We have recently found that the human ASIC1a (hASIC1a) wild type (WT) clone which has been used by many laboratories in recombinant expression studies contains a point mutation that occurs with a very low frequency in humans. Here, we compared the function and expression of ASIC1a WT and of this rare variant, in which the highly conserved residue Gly212 is substituted by Asp. Residue 212 is located at a subunit interface that undergoes changes during channel activity. We show that the modulation of channel function by commonly used ASIC inhibitors and modulators, and the pH dependence, are the same or only slightly different between hASIC1a-G212 and -D212. hASIC1a-G212 has however a higher current amplitude per surface-expressed channel and considerably slower current decay kinetics than hASIC1a-D212, and its current decay kinetics display a higher dependency on the type of anion present in the extracellular solution. We demonstrate for a number of channel mutants previously characterized in the hASIC1a-D212 background that they have very similar effects in the hASIC1a-G212 background. Taken together, we show that the variant hASIC1a-D212 that has been used as WT in many studies is, in fact, a mutant and that the properties of hASIC1a-D212 and hASIC1a-G212 are sufficiently close that the conclusions made in previous pharmacology and structure-function studies remain valid.

Heteroarylguanidines as Allosteric Modulators of ASIC1a and ASIC3 Channels.

Acid-sensing ion channels (ASICs) are neuronal Na+-selective ion channels that open in response to extracellular acidification. They are involved in pain, fear, learning, and neurodegeneration after ischemic stroke. 2-Guanidine-4-methylquinazoline (GMQ) was recently discovered as the first nonproton activator of ASIC3. GMQ is of interest as a gating modifier and pore blocker of ASICs. It has however a low potency, and exerts opposite effects on ASIC1a and ASIC3. To further explore the molecular mechanisms of GMQ action, we have used the guanidinium moiety of GMQ as a scaffold and tested the effects of different GMQ derivatives on the ASIC pH dependence and maximal current. We report that GMQ derivatives containing quinazoline and quinoline induced, as GMQ, an alkaline shift of the pH dependence of activation in ASIC3 and an acidic shift in ASIC1a. Another group of 2-guanidinopyridines shifted the pH dependence of both ASIC1a and ASIC3 to more acidic values. Several compounds induced an alkaline shift of the pH dependence of ASIC1a/2a and ASIC2a/3 heteromers. Compared to GMQ, guanidinopyridines showed a 20-fold decrease in the IC50 for ASIC1a and ASIC3 current inhibition at pH 5. Strikingly, 2-guanidino-quinolines and -pyridines showed a concentration-dependent biphasic effect that resulted at higher concentrations in ASIC1a and ASIC3 inhibition (IC50 > 100 µM), while causing at lower concentration a potentiation of ASIC1a, but not ASIC3 currents (EC50 ≈ 10 µM). In conclusion, we describe a new family of small molecules as ASIC ligands and identify an ASIC subtype-specific potentiation by a subgroup of these compounds.