Activation of renal ClC-K chloride channels depends on an intact N terminus of their accessory subunit barttin.

ClC-K channels belong to the CLC family of chloride channels and chloride/proton antiporters. They contribute to sodium chloride reabsorption in Henle's loop of the kidney and to potassium secretion into the endolymph by the stria vascularis of the inner ear. Their accessory subunit barttin stabilizes the ClC-K/barttin complex, promotes its insertion into the surface membrane, and turns the pore-forming subunits into a conductive state. Barttin mutations cause Bartter syndrome type IV, a salt-wasting nephropathy with sensorineural deafness. Here, studying ClC-K/barttin channels heterologously expressed in MDCK-II and HEK293T cells with confocal imaging and patch-clamp recordings, we demonstrate that the eight-amino-acids-long barttin N terminus is required for channel trafficking and activation. Deletion of the complete N terminus (Δ2-8 barttin) retained barttin and human hClC-Ka channels in intracellular compartments. Partial N-terminal deletions did not compromise subcellular hClC-Ka trafficking but drastically reduced current amplitudes. Sequence deletions encompassing Thr-6, Phe-7, or Arg-8 in barttin completely failed to activate hClC-Ka. Analyses of protein expression and whole-cell current noise revealed that inactive channels reside in the plasma membrane. Substituting the deleted N terminus with a polyalanine sequence was insufficient for recovering chloride currents, and single amino acid substitutions highlighted that the correct sequence is required for proper function. Fast and slow gate activation curves obtained from rat V166E rClC-K1/barttin channels indicated that mutant barttin fails to constitutively open the slow gate. Increasing expression of barttin over that of ClC-K partially recovered this insufficiency, indicating that N-terminal modifications of barttin alter both binding affinities and gating properties.

Impaired surface membrane insertion of homo- and heterodimeric human muscle chloride channels carrying amino-terminal myotonia-causing mutations.

Mutations in the muscle chloride channel gene (CLCN1) cause myotonia congenita, an inherited condition characterized by muscle stiffness upon sudden forceful movement. We here studied the functional consequences of four disease-causing mutations that predict amino acid substitutions Q43R, S70L, Y137D and Q160H. Wild-type (WT) and mutant hClC-1 channels were heterologously expressed as YFP or CFP fusion protein in HEK293T cells and analyzed by whole-cell patch clamp and fluorescence recordings on individual cells. Q43R, Y137D and Q160H, but not S70L reduced macroscopic current amplitudes, but left channel gating and unitary current amplitudes unaffected. We developed a novel assay combining electrophysiological and fluorescence measurements at the single-cell level in order to measure the probability of ion channel surface membrane insertion. With the exception of S70L, all tested mutations significantly reduced the relative number of homodimeric hClC-1 channels in the surface membrane. The strongest effect was seen for Q43R that reduced the surface insertion probability by more than 99% in Q43R homodimeric channels and by 92 ± 3% in heterodimeric WT/Q43R channels compared to homodimeric WT channels. The new method offers a sensitive approach to investigate mutations that were reported to cause channelopathies, but display only minor changes in ion channel function.

Induced fit substrate binding to an archeal glutamate transporter homologue.

Excitatory amino acid transporters (EAATs) are a class of glutamate transporters that terminate glutamatergic synaptic transmission in the mammalian CNS. GltPh, an archeal EAAT homolog from Pyrococcus horikoshii, is currently the only member with a known 3D structure. Here, we studied the kinetics of substrate binding of a single tryptophan mutant (L130W) GltPh in detergent micelles. At low millimolar [Na(+)], the addition of L-aspartate resulted in complex time courses of W130 fluorescence changes over tens of seconds. With increasing [Na(+)], the kinetics were dominated by a fast component [k(obs,fast); K(D) (Na(+)) = 22 ± 3 mM, n(Hill )= 1.7 ± 0.3] with values of k(obs,fast) rising in a saturable manner to ≈ 500 s(-1) (at 6 °C) with increasing [L-aspartate]. The binding kinetics of L-aspartate differed from the binding kinetics of two alternative substrates: L-cysteine sulfinic acid and d-aspartate. L-cysteine sulfinic acid bound with higher affinity than L-aspartate but involved lower saturating rates, whereas the saturating rates after D-aspartate binding were higher. Thus, after the association of two Na(+) to the empty transporter, GltPh binds amino acids by induced fit. Cross-linking and proteolysis experiments suggest that the induced fit results from the closure of helical hairpin 2. This conformational change is faster for GltPh than for most mammalian homologues, whereas the amino acid association rates are similar. Our data reveal the importance of induced fit for substrate selection in EAATs and illustrate how high-affinity binding and the efficient transport of glutamate can be accomplished simultaneously by this class of transporters.

Disease-causing mutations C277R and C277Y modify gating of human ClC-1 chloride channels in myotonia congenita.

Myotonia congenita is a genetic condition that is caused by mutations in the muscle chloride channel gene CLCN1 and characterized by delayed muscle relaxation and muscle stiffness. We here investigate the functional consequences of two novel disease-causing missense mutations, C277R and C277Y, using heterologous expression in HEK293T cells and patch clamp recording. Both mutations reduce macroscopic anion currents in transfected cells. Since hClC-1 is a double-barrelled anion channel, this reduction in current amplitude might be caused by altered gating of individual protopores or of joint openings and closing of both protopores. We used non-stationary noise analysis and single channel recordings to separate the mutants' effects on individual and common gating processes. We found that C277Y inverts the voltage dependence and reduces the open probabilities of protopore and common gates resulting in decreases of absolute open probabilities of homodimeric channels to values below 3%. In heterodimeric channels, C277R and C277Y also reduce open probabilities and shift the common gate activation curve towards positive potentials. Moreover, C277Y modifies pore properties of hClC-1. It reduces single protopore current amplitudes to about two-thirds of wild-type values, and inverts the anion permeability sequence to I(-) = NO(3)(-) >Br(-)>Cl(-). Our findings predict a dramatic reduction of the muscle fibre resting chloride conductance and thus fully explain the disease-causing effects of mutations C277R and C277Y. Moreover, they provide additional insights into the function of C277, a residue recently implicated in common gating of ClC channels.