Functional role of amino terminus in ClC-3 chloride channel regulation by phosphorylation and cell volume.

AIM: This study investigated the functional role of the ClC-3 amino-terminus in channel regulation in response to changes in cell volume. METHODS: Wild-type sClC-3 tagged with a green fluorescence protein (GFP) at the C-terminus was used as a template to construct a number of deletion mutants which were functionally expressed in NIH-3T3 cells. Whole cell and single channel patch-clamp electrophysiology was used to determine the functional properties of heterologously expressed channels. RESULTS: The first 100 amino acids of the ClC-3 N-terminus were removed and the truncated channel (sClC-3DeltaNT) was functionally expressed. Immunocytochemistry confirmed membrane expression of both wtsClC-3 and sClC-3DeltaNT channels in NIH/3T3 cells. sClC-3DeltaNT yielded constitutively active functional channels, which showed no response to protein kinase C or changes in cell volume. Deletion of a cluster of negatively charged amino acids 16-21 (sClC-3Delta16-21) within the N-terminus also yielded a constitutively active open channel phenotype, indicating these amino acids are involved in the N-type regulation. Intracellular delivery of a thiol-phosphorylated peptide corresponding to N-terminal residues 12-61 (NT peptide) markedly inhibited sClC-3DeltaNT whole-cell and single-channel currents, further confirming the essential role of the N-terminus in volume regulation of channel activity. CONCLUSIONS: These data strongly suggest the N-terminus of sClC-3 channels acts as a blocking particle inhibiting the flow of anions through the channel pore. This 'N-type' regulation of sClC-3 channels may be an important transducing mechanism linking changes in cell volume and channel protein phosphorylation to channel gating.

Tamoxifen activates smooth muscle BK channels through the regulatory beta 1 subunit.

Estrogen (17beta-estradiol; 17betaE) and xenoestrogens, estrogenic compounds that are not steroid hormones, have non-genomic actions at plasma membrane receptors unrelated to the nuclear estrogen receptor. The open probability (P(o)) of large conductance Ca(2+)/voltage-sensitive k(+)(BK) channels is increased by 17betaE through the regulatory beta1 subunit. The pharmacological nature of the putative membrane binding site is unclear. We probed the site by determining whether tamoxifen ((Z)-1-(p-dimethylaminoethoxy-phenyl)-1,2-diphenyl-1-butene; Tx), a chemotherapeutic xenoestrogen, increased P(o) in clinically relevant concentrations (0.1-10 microm). In whole cell patch clamp recordings on canine colonic myocytes, which express the beta1 subunit, Tx activated charybdotoxin-sensitive K(+) current. In single channel experiments, Tx increased the NP(o) (P(o) x number channels; N) and decreased the unitary conductance (gamma) of BK channels. Tx increased NP(o) (EC(50) = 0.65 microm) in excised membrane patches independent of Ca(2+) changes. The Tx mechanism of action requires the beta1 subunit, as Tx increased the NP(o) of Slo alpha expressed in human embryonic kidney cells only in the presence of the beta1 subunit. Tx decreased gamma of the alpha subunit expressed alone, without effect on NP(o). Our data indicate that Tx increases BK channel activity in therapeutic concentrations and reveal novel pharmacological properties attributable to the alpha and beta1 subunits. These data shed light on BK channel structure and function, non-genomic mechanisms of regulation, and physiologically and therapeutically relevant effects of xenoestrogens.

Functional inhibition of native volume-sensitive outwardly rectifying anion channels in muscle cells and Xenopus oocytes by anti-ClC-3 antibody.

Intracellular dialysis of NIH/3T3 cells with a commercially available anti-ClC-3 polyclonal antibody (Ab) for approximately 30 min completely inhibited expressed guinea-pig ClC-3 currents (IgpClC-3), while intracellular dialysis with antigen-preabsorbed anti-ClC-3 Ab failed to affect IgpClC-3. Anti-ClC-3 Ab was used as a selective probe to examine the relationship between endogenous ClC-3 expression and native volume-sensitive outwardly rectifying anion channels (VSOACs) in guinea-pig cardiac cells, canine pulmonary arterial smooth muscle cells (PASMCs) and Xenopus laevis oocytes. Intracellular dialysis or injection of anti-ClC-3 Ab abolished native VSOAC function in cardiac cells and PASMCs and significantly reduced VSOACs in oocytes. In contrast, native VSOAC function was unaltered by antigen-preabsorbed anti-ClC-3 Ab. It is suggested that endogenous ClC-3 represents a major molecular entity responsible for native VSOACs in cardiac and smooth muscle cells and Xenopus oocytes. Anti-ClC-3 Ab should be a useful experimental tool to directly test the relationship between endogenous ClC-3 expression and native VSOAC function, and help resolve existing controversies related to the regulation and physiological role of native VSOACs in a wide variety of different cells.

Molecular distribution of volume-regulated chloride channels (ClC-2 and ClC-3) in cardiac tissues.

The molecular identification of cardiac chloride channels has provided probes to investigate their distribution and abundance in heart. In this study, the molecular expression and distribution of volume-regulated chloride channels ClC-2 and ClC-3 in cardiac tissues were analyzed and quantified. Total RNA was isolated from atria and ventricles of several species (dog, guinea pig, and rat) and subjected to a quantitative RT-PCR strategy. ClC-2 and ClC-3 mRNA expression were calculated relative to beta-actin expression within these same tissues. The transcriptional levels of ClC-3 mRNA were between 1.8 and 10.2% of beta-actin expression in atria and between 3.4 and 8.6% of beta-actin in ventricles (n = 3 for each tissue). The levels of ClC-2 in both atria and ventricles were significantly less than those measured for ClC-3 (n = 3; P < 0.05). ClC-2 mRNA levels were between 0.04-0.08% and 0.03-0.18% of beta-actin expression in atria and ventricles, respectively (n = 3 for each tissue). Immunoblots of atrial and ventricular wall protein extracts demonstrated ClC-2- and ClC-3-specific immunoreactivity at 97 and 85 kDa, respectively. Immunohistochemical localization in guinea pig cardiac muscle demonstrates a ubiquitous distribution of ClC-2 and ClC-3 channels in the atrial and ventricular wall. Confocal analysis detected colocalization of ClC-2 and ClC-3 in sarcolemmal membranes and distinct ClC-3 immunoreactivity in cytoplasmic regions. The molecular expression of ClC-2 and ClC-3 in cardiac tissue is consistent with the proposed role of these chloride channels in the regulation of cardiac cell volume and the modulation of cardiac electrical activity.