The CLC-1 Cl(-) channel is abundantly expressed on the plasma membrane of muscle cells, and the membrane potential of muscle cells is largely controlled by the activity of this Cl(-) channel. Previous studies showed that low intracellular pH increases the overall open probability of recombinant CLC-1 channels in various expression systems. Low intracellular pH, however, is known to inhibit the Cl(-) conductance on the native muscle membrane, contradicting the findings from the recombinant CLC-1 channels in expressed systems. Here we show that in the presence of physiological concentrations of ATP, reduction of the intracellular pH indeed inhibits the expressed CLC-1, mostly by decreasing the open probability of the common gate of the channel.

The effect of intracellular adenosine triphosphate (ATP) on the "common gating" of the CLC-1 chloride channel has been studied by several laboratories with controversial results. Our previous study on the channel expressed in Xenopus oocytes using excised inside-out patch-clamp methods showed a robust effect of ATP in shifting the open probability curve of the common gate toward more depolarizing voltages (Tseng, P.Y., B. Bennetts, and T.Y. Chen. 2007. J. Gen. Physiol. 130:217-221). The results were consistent with those from studying the channel expressed in mammalian cells using whole cell recording methods (Bennetts, B., M.W. Parker, and B.A. Cromer. 2007. J. Biol. Chem. 282:32780-32791). However, a recent study using excised-patch recording methods for channels expressed in Xenopus oocytes reported that ATP had no direct effect on CLC-1 (Zifarelli, G., and M. Pusch. 2008. J. Gen. Physiol. 131:109-116). Here, we report that oxidation of CLC-1 may be the culprit underlying the controversy. When patches were excised from mammalian cells, the sensitivity to ATP was lost quickly--within 2-3 min. This loss of ATP sensitivity could be prevented or reversed by reducing agents. On the other hand, CLC-1 expressed in Xenopus oocytes lost the ATP sensitivity when patches were treated with oxidizing reagents. These results suggest a novel view in muscle physiology that the mechanisms controlling muscle fatigability may include the oxidation of CLC-1.

The blockade of CLC-0 chloride channels by p-chlorophenoxy acetate (CPA) has been thought to be state dependent; the conformational change of the channel pore during the "fast gating" alters the CPA binding affinity. Here, we examine the mechanism of CPA blocking in pore-open mutants of CLC-0 in which the residue E166 was replaced by various amino acids. We find that the CPA-blocking affinities depend upon the volume and the hydrophobicity of the side chain of the introduced residue; CPA affinity can vary by three orders of magnitude in these mutants. On the other hand, mutations at the intracellular pore entrance, although affecting the association and dissociation rates of the CPA block, generate only a modest effect on the steady-state blocking affinity. In addition, various amphiphilic compounds, including fatty acids and alkyl sulfonates, can also block the pore-open mutants of CLC-0 through a similar mechanism. The blocking affinity of fatty acids and alkyl sulfonates increases with the length of these amphiphilic blockers, a phenomenon similar to the block of the Shaker K(+) channel by long-chain quaternary ammonium (QA) ions. These observations lead us to propose that the CPA block of the open pore of CLC-0 is similar to the blockade of voltage-gated K(+) channels by long-chain QAs or by the inactivation ball peptide: the blocker first uses the hydrophilic end to "dock" at the pore entrance, and the hydrophobic part of the blocker then enters the pore to interact with a more hydrophobic region of the pore. This blocking mechanism appears to be very general because the block does not require a precise structural fit between the blocker and the pore, and the blocking mechanism applies to the cation and anion channels with unrelated pore architectures.

The common gating of CLC-1 has been shown to be inhibited by intracellular adenosine triphosphate (ATP) in acidic pH conditions. Such modulation is thought to be mediated by direct binding of ATP to the cystathionine β-synthase (CBS) domains at the C-terminal cytoplasmic region of CLC-1. Guided by the crystal structure of the C-terminal domain of CLC-5, we constructed a homology model of CLC-1's C terminus and mutated critical amino acid residues lining the potential ATP-binding site. The CLC-1 mutations V634A and E865A completely abolished the ATP inhibition of CLC-1, consistent with the loss of ATP binding seen with the corresponding mutations in CLC-5. Mutating two other residues, V613 and V860, also disrupted the ATP modulation of CLC-1. However, placing aromatic amino acids at position 634 increases the apparent ATP affinity. Mutant cycle analyses showed that the modulation effects of ATP and cytidine triphosphate on wild-type CLC-1 and the V634F mutant were nonadditive, suggesting that the side chain of amino acid at position 634 interacts with the base moiety of the nucleotide. The mutation effects of V634F and V613A on the ATP modulation were also nonadditive, which is consistent with the assertion suggested from the homology model that these two residues may both interact with the bound nucleotide. These results provide evidence for a direct ATP binding for modulating the function of CLC-1 and suggest an overall conserved architecture of the ATP-binding sites in CLC-1 and CLC-5. This study also demonstrates that CLC-1 is a convenient experimental model for studying the interaction of nucleotides/nucleosides with the CBS domain.

The voltage-gated L-type Ca²⁺ channel Ca_V1.2 is crucial for initiating heartbeat and control of a number of neuronal functions such as neuronal excitability and long-term potentiation. Mutations of Ca_V1.2 subunits result in serious health problems, including arrhythmia, autism spectrum disorders, immunodeficiency, and hypoglycemia. Thus, precise control of Ca_V1.2 surface expression and localization is essential. We previously reported that α-actinin associates and colocalizes with neuronal Ca_V1.2 channels and that shRNA-mediated depletion of α-actinin significantly reduces localization of endogenous Ca_V1.2 in dendritic spines in hippocampal neurons. Here we investigated the hypothesis that direct binding of α-actinin to Ca_V1.2 supports its surface expression. Using two-hybrid screens and pull-down assays, we identified three point mutations (K1647A, Y1649A, and I1654A) in the central, pore-forming α₁1.2 subunit of Ca_V1.2 that individually impaired α-actinin binding. Surface biotinylation and flow cytometry assays revealed that Ca_V1.2 channels composed of the corresponding α-actinin-binding-deficient mutants result in a 35-40% reduction in surface expression compared to that of wild-type channels. Moreover, the mutant Ca_V1.2 channels expressed in HEK293 cells exhibit a 60-75% decrease in current density. The larger decrease in current density as compared to surface expression imparted by these α₁1.2 subunit mutations hints at the possibility that α-actinin not only stabilizes surface localization of Ca_V1.2 but also augments its ion conducting activity.

The L-type Ca²⁺ channel Ca_V 1.2 governs gene expression, cardiac contraction, and neuronal activity. Binding of α-actinin to the IQ motif of Ca_V 1.2 supports its surface localization and postsynaptic targeting in neurons. We report a bi-functional mechanism that restricts Ca_V 1.2 activity to its target sites. We solved separate NMR structures of the IQ motif (residues 1,646-1,664) bound to α-actinin-1 and to apo-calmodulin (apoCaM). The Ca_V 1.2 K1647A and Y1649A mutations, which impair α-actinin-1 but not apoCaM binding, but not the F1658A and K1662E mutations, which impair apoCaM but not α-actinin-1 binding, decreased single-channel open probability, gating charge movement, and its coupling to channel opening. Thus, α-actinin recruits Ca_V 1.2 to defined surface regions and simultaneously boosts its open probability so that Ca_V 1.2 is mostly active when appropriately localized.

The L-type Ca²⁺ channel Ca_V 1.2 governs gene expression, cardiac contraction, and neuronal activity. Binding of α-actinin to the IQ motif of Ca_V 1.2 supports its surface localization and postsynaptic targeting in neurons. We report a bi-functional mechanism that restricts Ca_V 1.2 activity to its target sites. We solved separate NMR structures of the IQ motif (residues 1,646-1,664) bound to α-actinin-1 and to apo-calmodulin (apoCaM). The Ca_V 1.2 K1647A and Y1649A mutations, which impair α-actinin-1 but not apoCaM binding, but not the F1658A and K1662E mutations, which impair apoCaM but not α-actinin-1 binding, decreased single-channel open probability, gating charge movement, and its coupling to channel opening. Thus, α-actinin recruits Ca_V 1.2 to defined surface regions and simultaneously boosts its open probability so that Ca_V 1.2 is mostly active when appropriately localized.