Local anesthetics block sodium channels in a state-dependent fashion, binding with higher affinity to open and/or inactivated states. Gating current measurements show that local anesthetics immobilize a fraction of the gating charge, suggesting that the movement of voltage sensors is modified when a local anesthetic binds to the pore of the sodium channel. Here, using voltage clamp fluorescence measurements, we provide a quantitative description of the effect of local anesthetics on the steady-state behavior of the voltage-sensing segments of a sodium channel. Lidocaine and QX-314 shifted the midpoints of the fluorescence–voltage (F-V) curves of S4 domain III in the hyperpolarizing direction by 57 and 65 mV, respectively. A single mutation in the S6 of domain IV (F1579A), a site critical for local anesthetic block, abolished the effect of QX-314 on the voltage sensor of domain III. Both local anesthetics modestly shifted the F-V relationships of S4 domain IV toward hyperpolarized potentials. In contrast, the F-V curve of the S4 domain I was shifted by 11 mV in the depolarizing direction upon QX-314 binding. These antagonistic effects of the local anesthetic indicate that the drug modifies the coupling between the voltage-sensing domains of the sodium channel. Our findings suggest a novel role of local anesthetics in modulating the gating apparatus of the sodium channel.

Regulated point modification by an RNA editing enzyme occurs at four conserved sites in the Drosophila Shaker potassium channel. Single mRNA molecules can potentially represent any of 2⁴ = 16 permutations (isoforms) of these natural variants. We generated isoform expression profiles to assess sexually dimorphic, spatial, and temporal differences. Striking tissue-specific expression was seen for particular isoforms. Moreover, isoform distributions showed evidence for coupling (linkage) of editing sites. Genetic manipulations of editing enzyme activity demonstrated that a chief determinant of Shaker editing site choice resides not in the editing enzyme, but rather, in unknown factors intrinsic to cells. Characterizing the biophysical properties of currents in nine isoforms revealed an unprecedented feature, functional epistasis; biophysical phenotypes of isoforms cannot be explained simply by the consequences of individual editing effects at the four sites. Our results unmask allosteric communication across disparate regions of the channel protein and between evolved and regulated amino acid changes introduced by RNA editing.

Changes in phosphorylation regulate the activity of various ClC anion transport proteins. However, the physiological context under which such regulation occurs and the signaling cascades that mediate phosphorylation are poorly understood. We have exploited the genetic model organism Caenorhabditis elegans to characterize ClC regulatory mechanisms and signaling networks. CLH-3b is a ClC anion channel that is expressed in the worm oocyte and excretory cell. Channel activation occurs in response to oocyte meiotic maturation and swelling via serine/threonine dephosphorylation mediated by the type I phosphatases GLC-7 and GLC-7β. A Ste20 kinase, germinal center kinase (GCK)-3, binds to the cytoplasmic C terminus of CLH-3b and inhibits channel activity in a phosphorylation-dependent manner. Analysis of hyperpolarization-induced activation kinetics suggests that phosphorylation may inhibit the ClC fast gating mechanism. GCK-3 is an ortholog of mammalian SPAK and OSR1, kinases that bind to, phosphorylate, and regulate the cell volume–dependent activity of mammalian cation-Cl^– cotransporters. Using mass spectrometry and patch clamp electrophysiology, we demonstrate here that CLH-3b is a target of regulatory phosphorylation. Concomitant phosphorylation of S742 and S747, which are located 70 and 75 amino acids downstream from the GCK-3 binding site, are required for kinase-mediated channel inhibition. In contrast, swelling-induced channel activation occurs with dephosphorylation of S747 alone. Replacement of both S742 and S747 with glutamate gives rise to kinase- and swelling-insensitive channels that exhibit activity and biophysical properties similar to those of wild-type CLH-3b inhibited by GCK-3. Our studies provide novel insights into ClC regulation and mechanisms of cell volume signaling, and provide the foundation for studies aimed at defining how conformational changes in the cytoplasmic C terminus alter ClC gating and function in response to intracellular signaling events.

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.

Intracellularly applied amphiphilic molecules, such as p-chlorophenoxy acetate (CPA) and octanoate, block various pore-open mutants of CLC-0. The voltage-dependent block of a particular pore-open mutant, E166G, was found to be multiphasic. In symmetrical 140 mM Cl^–, the apparent affinity of the blocker in this mutant increased with a negative membrane potential but, paradoxically, decreased when the negative membrane potential was greater than –80 mV, a phenomenon similar to the blocker "punch-through" shown in many blocker studies of cation channels. To provide further evidence of the punch-through of CPA and octanoate, we studied the dissociation rate of the blocker from the pore by measuring the time constant of relief from the block under various voltage and ionic conditions. Consistent with the voltage dependence of the effect on the steady-state current, the rate of CPA dissociation from the E166G pore reached a minimum at –80 mV in symmetrical 140 mM Cl^–, and the direction of current recovery suggested that the bound CPA in the pore can dissociate into both intracellular and extracellular solutions. Moreover, the CPA dissociation depends upon the Cl^– reversal potential with a minimal dissociation rate at a voltage 80 mV more negative than the Cl^– reversal potential. That the shift of the CPA-dissociation rate follows the Cl^– gradient across the membrane argues that these blockers can indeed punch through the channel pore. Furthermore, a minimal CPA-dissociation rate at a voltage 80 mV more negative than the Cl^– reversal potential suggests that the outward blocker movement through the CLC-0 pore is more difficult than the inward movement.

The canonical sequence LSGGQ, also known as the signature sequence, defines the adenosine triphosphate (ATP)-binding cassette transporter superfamily. Crystallographic studies reveal that the signature sequence, together with the Walker A and Walker B motifs, forms the ATP-binding pocket upon dimerization of the two nucleotide-binding domains (NBDs) in a head-to-tail configuration. The importance of the signature sequence is attested by the fact that a glycine to aspartate mutation (i.e., G551D) in cystic fibrosis transmembrane conductance regulator (CFTR) results in a severe phenotype of cystic fibrosis. We previously showed that the G551D mutation completely eliminates ATP-dependent gating of the CFTR chloride channel. Here, we report that micromolar [Cd²⁺] can dramatically increase the activity of G551D-CFTR in the absence of ATP. This effect of Cd²⁺ is not seen in wild-type channels or in G551A. Pretreatment of G551D-CFTR with the cysteine modification reagent 2-aminoethyl methane thiosulfonate hydrobromide protects the channel from Cd²⁺ activation, suggesting an involvement of endogenous cysteine residue(s) in mediating this effect of Cd²⁺. The mutants G551C, L548C, and S549C, all in the signature sequence of CFTR's NBD1, show robust response to Cd²⁺. On the other hand, negligible effects of Cd²⁺ were seen with T547C, Q552C, and R553C, indicating that a specific region of the signature sequence is involved in transmitting the signal of Cd²⁺ binding to the gate. Collectively, these results suggest that the effect of Cd²⁺ is mediated by a metal bridge formation between yet to be identified cysteine residue(s) and the engineered aspartate or cysteine in the signature sequence. We propose that the signature sequence serves as a switch that transduces the signal of ligand binding to the channel gate.

The term excitation-coupled Ca²⁺ entry (ECCE) designates the entry of extracellular Ca²⁺ into skeletal muscle cells, which occurs in response to prolonged depolarization or pulse trains and depends on the presence of both the 1,4-dihydropyridine receptor (DHPR) in the plasma membrane and the type 1 ryanodine receptor in the sarcoplasmic reticulum (SR) membrane. The ECCE pathway is blocked by pharmacological agents that also block store-operated Ca²⁺ entry, is inhibited by dantrolene, is relatively insensitive to the DHP antagonist nifedipine (1 µM), and is permeable to Mn²⁺. Here, we have examined the effects of these agents on the L-type Ca²⁺ current conducted via the DHPR. We found that the nonspecific cation channel antagonists (2-APB, SKF 96356, La³⁺, and Gd³⁺) and dantrolene all inhibited the L-type Ca²⁺ current. In addition, complete (>97%) block of the L-type current required concentrations of nifedipine >10 µM. Like ECCE, the L-type Ca²⁺ channel displays permeability to Mn²⁺ in the absence of external Ca²⁺ and produces a Ca²⁺ current that persists during prolonged (~10-second) depolarization. This current appears to contribute to the Ca²⁺ transient observed during prolonged KCl depolarization of intact myotubes because (1) the transients in normal myotubes decayed more rapidly in the absence of external Ca²⁺; (2) the transients in dysgenic myotubes expressing SkEIIIK (a DHPR _1S pore mutant thought to conduct only monovalent cations) had a time course like that of normal myotubes in Ca²⁺-free solution and were unaffected by Ca²⁺ removal; and (3) after block of SR Ca²⁺ release by 200 µM ryanodine, normal myotubes still displayed a large Ca²⁺ transient, whereas no transient was detectable in SkEIIIK-expressing dysgenic myotubes. Collectively, these results indicate that the skeletal muscle L-type channel is a major contributor to the Ca²⁺ entry attributed to ECCE.

P2X receptors are ligand-gated cation channels activated by extracellular adenosine triphosphate (ATP). Nonetheless, P2X₂ channel currents observed during the steady-state after ATP application are known to exhibit voltage dependence; there is a gradual increase in the inward current upon hyperpolarization. We used a Xenopus oocyte expression system and two-electrode voltage clamp to analyze this "activation" phase quantitatively. We characterized the conductance–voltage relationship in the presence of various [ATP], and observed that it shifted toward more depolarized potentials with increases in [ATP]. By analyzing the rate constants for the channel's transition between a closed and an open state, we showed that the gating of P2X₂ is determined in a complex way that involves both membrane voltage and ATP binding. The activation phase was similarly recorded in HEK293 cells expressing P2X₂ even by inside-out patch clamp after intensive perfusion, excluding a possibility that the gating is due to block/unblock by endogenous blocker(s) of oocytes. We investigated its structural basis by substituting a glycine residue (G344) in the second transmembrane (TM) helix, which may provide a kink that could mediate "gating." We found that, instead of a gradual increase, the inward current through the G344A mutant increased instantaneously upon hyperpolarization, whereas a G344P mutant retained an activation phase that was slower than the wild type (WT). Using glycine-scanning mutagenesis in the background of G344A, we could recover the activation phase by introducing a glycine residue into the middle of second TM. These results demonstrate that the flexibility of G344 contributes to the voltage-dependent gating. Finally, we assumed a three-state model consisting of a fast ATP-binding step and a following gating step and estimated the rate constants for the latter in P2X₂-WT. We then executed simulation analyses using the calculated rate constants and successfully reproduced the results observed experimentally, voltage-dependent activation that is accelerated by increases in [ATP].