Burton Horowitz

Molecular identification of a volume-regulated chloride channel

A volume-regulated chloride current ( I Cl.vol) is ubiquitously present in mammalian cells, and is required for the regulation of electrical activity, cell volume, intracellular pH, immunological responses, cell proliferation and differentiation. However, the molecule responsible for I Cl.vol has yet to be determined. Although three putative chloride channel proteins expressed from cloned genes (P-glycoprotein, p I Cln (ref. 5) and ClC-2 (ref. 6)) have been proposed to be the molecular equivalent of I Cl.vol, neither P-glycoprotein nor p I Cln is thought to be a chloride channel or part thereof,, and the properties of expressed ClC-2 channels differ from native I Cl.vol (refs. 3, 6). Here we report that functional expression in NIH/3T3 cells of a cardiac clone of another member of the ClC family, ClC-3, results in a large basally active chloride conductance, which is strongly modulated by cell volume and exhibits many properties identical to those of I Cl.vol in native cells,. A mutation of asparagine to lysine at position 579 at the end of the transmembrane domains of ClC-3 abolishes the outward rectification and changes the anion selectivity from I− > Cl− to Cl− > I− but leaves swelling activation intact. Because ClC-3 is a channel protein belonging to a large gene family of chloride channels,, these results indicate that ClC-3 encodes I Cl.vol in many native mammalian cells.

Cyclic GMP-dependent Protein Kinase Activates Cloned BKCa Channels Expressed in Mammalian Cells by Direct Phosphorylation at Serine 1072

NO-induced activation of cGMP-dependent protein kinase (PKG) increases the open probability of large conductance Ca2+-activated K+ channels and results in smooth muscle relaxation. However, the molecular mechanism of channel regulation by the NO-PKG pathway has not been determined on cloned channels. The present study was designed to clarify PKG-mediated modulation of channels at the molecular level. The cDNA encoding the α-subunit of the large conductance Ca2+-activated K+ channel,cslo-α, was expressed in HEK293 cells. Whole cell and single channel characteristics of cslo-α exhibited functional features of native large conductance Ca2+-activated K+ channels in smooth muscle cells. The NO-donor sodium nitroprusside increased outward current 2.3-fold in whole cell recordings. In cell-attached patches, sodium nitroprusside increased the channel open probability (NPo) ofcslo-α channels 3.3-fold without affecting unitary conductance. The stimulatory effect of sodium nitroprusside was inhibited by the PKG-inhibitor KT5823. Direct application of PKG-Iα to the cytosolic surface of inside-out patches increased NPo 3.2-fold only in the presence of ATP and cGMP without affecting unitary conductance. A point mutation of cslo-α in which Ser-1072 (the only optimal consensus sequence for PKG phosphorylation) was replaced by Ala abolished the PKG effect on NPo in inside-out patches and the effect of SNP in cell attached patches. These results indicate that PKG activates cslo-α by direct phosphorylation at serine 1072.

Kir2.1 encodes the inward rectifier potassium channel in rat arterial smooth muscle cells

1 The molecular nature of the strong inward rectifier K+ channel in vascular smooth muscle was explored by using isolated cell RT‐PCR, cDNA cloning and expression techniques. 2 RT‐PCR of RNA from single smooth muscle cells of rat cerebral (basilar), coronary and mesenteric arteries revealed transcripts for Kir2.1. Transcripts for Kir2.2 and Kir2.3 were not found. 3 Quantitative PCR analysis revealed significant differences in transcript levels of Kir2.1 between the different vascular preparations (n= 3; P < 0.05). A two‐fold difference was detected between Kir2.1 mRNA and β‐actin mRNA in coronary arteries when compared with relative levels measured in mesenteric and basilar preparations. 4 Kir2.1 was cloned from rat mesenteric vascular smooth muscle cells and expressed in Xenopus oocytes. Currents were strongly inwardly rectifying and selective for K+. 5 The effect of extracellular Ba2+, Ca2+, Mg2+ and Cs2+ ions on cloned Kir2.1 channels expressed in Xenopus oocytes was examined. Ba2+ and Cs+ block were steeply voltage dependent, whereas block by external Ca2+ and Mg2+ exhibited little voltage dependence. The apparent half‐block constants and voltage dependences for Ba2+, Cs+, Ca2+ and Mg2+ were very similar for inward rectifier K+ currents from native cells and cloned Kir2.1 channels expressed in oocytes. 6 Molecular studies demonstrate that Kir2.1 is the only member of the Kir2 channel subfamily present in vascular arterial smooth muscle cells. Expression of cloned Kir2.1 in Xenopus oocytes resulted in inward rectifier K+ currents that strongly resemble those that are observed in native vascular arterial smooth muscle cells. We conclude that Kir2.1 encodes for inward rectifier K+ channels in arterial smooth muscle.

Functional and molecular expression of volume‐regulated chloride channels in canine vascular smooth muscle cells

1 We examined the possibility of functional and molecular expression of volume‐regulated Cl− channels in vascular smooth muscle using the whole‐cell patch‐clamp technique and quantitative reverse transcriptase‐polymerase chain reaction (RT‐PCR) on cells from canine pulmonary and renal arteries. 2 Decreasing external osmolarity induced cell swelling, which was accompanied by activation of Cl−‐dependent outward‐rectifying membrane currents with an anion permeability sequence of SCN− > I− > Br− > Cl− > aspartate−. These currents were sensitive to block by DIDS, extracellular ATP and the antioestrogen compound tamoxifen. 3 Experiments were performed to determine whether the molecular form of the volume‐regulated chloride channel (ClC‐3) is expressed in pulmonary and renal arteries. Quantitative RT‐PCR confirmed expression of ClC‐3 in both types of smooth muscle. ClC‐3 expression was 76.4 % of β‐actin in renal artery and 48.0 % of β‐actin in pulmonary artery. 4 We conclude that volume‐regulated Cl− channels are expressed in vascular smooth muscle cells and exhibit functional properties similar to those found in other types of cells, presumably contributing to the regulation of cell volume, electrical activity and, possibly, myogenic tone.

Development of interstitial cells of Cajal and pacemaking in mice lacking enteric nerves

BACKGROUND & AIMS Development of interstitial cells of Cajal (ICC) requires signaling via Kit receptors. Kit is activated by stem cell factor (SCF), but the source of SCF in the bowel wall is unclear and controversy exists about whether enteric neurons express the SCF required for ICC development. METHODS Glial cell line-derived neurotrophic factor (GDNF) knockout mice, which lack enteric neurons throughout most of the gut, were used to determine whether neurons are necessary for ICC development. ICC distributions were determined with Kit immunofluorescence, and function of ICC was determined by intracellular electrical recording. RESULTS ICC were normally distributed throughout the gastrointestinal tracts of GDNF-/- mice. Intracellular recordings from aganglionic gastrointestinal muscles showed normal slow wave activity at birth in the stomach and small intestine. Slow waves developed normally in aganglionic segments of small bowel placed into organ culture at birth. Quantitative polymerase chain reaction showed similar expression of SCF in the muscles of animals with and without enteric neurons. Expression of SCF was demonstrated in isolated intestinal smooth muscle cells. CONCLUSIONS These data suggest that enteric neurons are not required for the development of functional ICC. The circular smooth muscle layer, which develops before ICC, may be the source of SCF required for ICC development.

ClC-3 is a fundamental molecular component of volume-sensitive outwardly rectifying Cl- channels and volume regulation in HeLa cells and Xenopus laevis oocytes.

Volume-sensitive osmolyte and anion channels (VSOACs) are activated upon cell swelling in most vertebrate cells. Native VSOACs are believed to be a major pathway for regulatory volume decrease (RVD) through efflux of chloride and organic osmolytes. ClC-3 has been proposed to encode native VSOACs in Xenopus laevis oocytes and in some mammalian cells, including cardiac and vascular smooth muscle cells. The relationship between the ClC-3 chloride channel, the native volume-sensitive osmolyte and anion channel (VSOAC) currents, and cell volume regulation in HeLa cells andX. laevis oocytes was investigated using ClC-3 antisense. In situ hybridization in HeLa cells, semiquantitative and real-time PCR, and immunoblot studies in HeLa cells and X. laevis oocytes demonstrated the presence of ClC-3 mRNA and protein, respectively. Exposing both cell types to hypotonic solutions induced cell swelling and activated native VSOACs. Transient transfection of HeLa cells with ClC-3 antisense oligonucleotide or X. laevis oocytes injected with antisense cRNA abolished the native ClC-3 mRNA transcript and protein and significantly reduced the density of native VSOACs activated by hypotonically induced cell swelling. In addition, antisense against native ClC-3 significantly impaired the ability of HeLa cells and X. laevis oocytes to regulate their volume. These results suggest that ClC-3 is an important molecular component underlying VSOACs and the RVD process in HeLa cells and X. laevis oocytes.

Altered properties of volume‐sensitive osmolyte and anion channels (VSOACs) and membrane protein expression in cardiac and smooth muscle myocytes from Clcn3‐/‐ mice

ClC‐3, a member of the large superfamily of ClC voltage‐dependent Cl– channels, has been proposed as a molecular candidate responsible for volume‐sensitive osmolyte and anion channels (VSOACs) in some cells, including heart and vascular smooth muscle. However, the reported presence of native VSOACs in at least two cell types from transgenic ClC‐3 disrupted (Clcn3−/−) mice casts considerable doubt on this proposed role for ClC‐3. We compared several properties of native VSOACs and examined mRNA transcripts and membrane protein expression profiles in cardiac and pulmonary arterial smooth muscle cells from Clcn3+/+ and Clcn3−/− mice to: (1) test the hypothesis that native VSOACs are unaltered in cells from Clcn3−/− mice, and (2) test the possibility that targeted inactivation of the Clcn3 gene using a conventional murine global knock‐out approach may result in compensatory changes in expression of other membrane proteins. Our experiments demonstrate that VSOAC currents in myocytes from Clcn3+/+ and Clcn3−/− mice are remarkably similar in terms of activation and inactivation kinetics, steady‐state current densities, rectification, anion selectivity (I− > Cl−≫ Asp−) and sensitivity to block by glibenclamide, niflumic acid, DIDS and extracellular ATP. However, additional experiments revealed several significant differences in other fundamental properties of native VSOACs recorded from atrial and smooth muscle cells from Clcn3−/− mice, including: differences in regulation by endogenous protein kinase C, differential sensitivity to block by anti‐ClC‐3 antibodies, and differential sensitivities to [ATP]i and free [Mg2+]i. These results suggest that in response to Clcn3 gene deletion, there may be compensatory changes in expression of other proteins that alter VSOAC channel subunit composition or associated regulatory subunits that give rise to VSOACs with different properties. Consistent with this hypothesis, in atria from Clcn3−/− mice compared to Clcn3+/+ mice, quantitative analysis of ClC mRNA expression levels revealed significant increases in transcripts for ClC‐1, ClC‐2, and ClC‐3, and protein expression profiles obtained using two‐dimensional polyacrylamide gel electrophoresis revealed complex changes in at least 35 different unidentified membrane proteins in cells from Clcn3−/− mice. These findings emphasize that caution needs to be exercised in simple attempts to interpret the phenotypic consequences of conventional global Clcn3 gene inactivation.

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

1 Intracellular dialysis of NIH/3T3 cells with a commercially available anti‐ClC‐3 polyclonal antibody (Ab) for ≈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. 2 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. 3 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 Variants of KCNQ Channels Expressed in Murine Portal Vein Myocytes A Role in Delayed Rectifier Current

Abstract— We have analyzed the expression of KCNQ genes in murine portal vein myocytes and determined that of the 5 known KCNQ channels, only KCNQ1 was expressed. In addition to the full-length KCNQ1 transcript, a novel spliced form (termed KCNQ1b) was detected that had a 63 amino acid truncation at the C-terminus. KCNQ1b was not detected in heart or brain but represented approximately half the KCNQ1 transcripts expressed in PV. Antibodies specific for KCNQ1a stained cell membranes from portal vein myocytes and HEK cells expressing the channel. However, because the antibodies were generated against an epitope in the deleted, C-terminal portion of the protein, these antibodies did not stain HEK cells expressing KCNQ1b. In murine portal vein myocytes, in the presence of 5 mmol/L 4-aminopyridine, an outwardly rectifying K+ current was recorded that was sensitive to linopirdine, a specific blocker of KCNQ channels. Currents produced by the heterologous expression of KCNQ1a or KCNQ1b were inhibited by similar concentrations of linopirdine, and linopirdine prolonged the time-course of the action potential in isolated portal vein myocytes. Our data suggest that these two KCNQ1 splice forms are expressed in murine portal vein and contribute to the delayed rectifier current in these myocytes.

Basal Activation of ATP-Sensitive Potassium Channels in Murine Colonic Smooth Muscle Cell

The function and molecular expression of ATP-sensitive potassium (KATP) channels in murine colonic smooth muscle was investigated by intracellular electrical recording from intact muscles, patch-clamp techniques on isolated smooth muscle myocytes, and reverse transcription polymerase chain reaction (RT-PCR) on isolated cells. Lemakalim (1 microM) caused hyperpolarization of intact muscles (17. 2 +/- 3 mV). The hyperpolarization was blocked by glibenclamide (1-10 microM). Addition of glibenclamide (10 microM) alone resulted in membrane depolarization (9.3 +/- 1.7 mV). Lemakalim induced an outward current of 15 +/- 3 pA in isolated myocytes bathed in 5 mM external K+ solution. Application of lemakalim to cells in symmetrical K+ solutions (140/140 mM) resulted in a 97 +/- 5 pA inward current. Both currents were blocked by glibenclamide (1 microM). Pinacidil (1 microM) also activated an inwardly rectifying current that was insensitive to 4-aminopyridine and barium. In single-channel studies, lemakalim (1 microM) and diazoxide (300 microM) increased the open probability of a 27-pS K+ channel. Openings of these channels decreased with time after patch excision. Application of ADP (1 mM) or ATP (0.1 mM) to the inner surface of the patches reactivated channel openings. The conductance and characteristics of the channels activated by lemakalim were consistent with the properties of KATP. RT-PCR demonstrated the presence of Kir 6.2 and SUR2B transcripts in colonic smooth muscle cells; transcripts for Kir 6.1, SUR1, and SUR2A were not detected. These molecular studies are the first to identify the molecular components of KATP in colonic smooth muscle cells. Together with the electrophysiological experiments, we conclude that KATP channels are expressed in murine colonic smooth muscle cells and suggest that these channels may be involved in dual regulation of resting membrane potential, excitability, and contractility.