TREK-1 currents in smooth muscle cells from pregnant human myometrium.

The mechanisms governing maintenance of quiescence during pregnancy remain largely unknown. The current study characterizes a stretch-activated, tetraethylammonium-insensitive K(+) current in smooth muscle cells isolated from pregnant human myometrium. This study hypothesizes that these K(+) currents can be attributed to TREK-1 and that upregulation of this channel during pregnancy assists with the maintenance of a negative cell membrane potential, conceivably contributing to uterine quiescence until full term. The results of this study demonstrate that, in pregnant human myometrial cells, outward currents at 80 mV increased from 4.8 ± 1.5 to 19.4 ± 7.5 pA/pF and from 3.0 ± 0.8 to 11.8 ± 2.7 pA/pF with application of arachidonic acid (AA) and NaHCO3, respectively, causing intracellular acidification. Similarly, outward currents were inhibited following application of 10 µM fluphenazine by 51.2 ± 9.8% after activation by AA and by 73.9 ± 4.2% after activation by NaHCO3. In human embryonic kidney (HEK-293) cells stably expressing TREK-1, outward currents at 80 mV increased from 91.0 ± 23.8 to 247.5 ± 73.3 pA/pF and from 34.8 ± 8.9 to 218.6 ± 45.0 pA/pF with application of AA and NaHCO3, respectively. Correspondingly, outward currents were inhibited 89.5 ± 2.3% by 10 µM fluphenazine following activation by AA and by 91.6 ± 3.4% following activation by NaHCO3. Moreover, currents in human myometrial cells were activated by stretch and were reduced by transfection with small interfering RNA or extracellular acidification. Understanding gestational regulation of expression and gating of TREK-1 channels could be important in determining appropriate maintenance of uterine quiescence during pregnancy.

Cardiac-specific, inducible ClC-3 gene deletion eliminates native volume-sensitive chloride channels and produces myocardial hypertrophy in adult mice.

Native volume-sensitive outwardly rectifying anion channels (VSOACs) play a significant role in cell volume homeostasis in mammalian cells. However, the molecular correlate of VSOACs has been elusive to identify. The short isoform of ClC-3 (sClC-3) is a member of the mammalian ClC gene family and has been proposed to be a molecular candidate for VSOACs in cardiac myocytes and vascular smooth muscle cells. To directly test this hypothesis, and assess the physiological role of ClC-3 in cardiac function, we generated a novel line of cardiac-specific inducible ClC-3 knock-out mice. These transgenic mice were maintained on a doxycycline diet to preserve ClC-3 expression; removal of doxycycline activates Cre recombinase to inactivate the Clcn3 gene. Echocardiography revealed dramatically reduced ejection fraction and fractional shortening, and severe signs of myocardial hypertrophy and heart failure in the knock-out mice at both 1.5 and 3 weeks off doxycycline. In mice off doxycycline, time-dependent inactivation of ClC-3 gene expression was confirmed in atrial and ventricular cells by qRT-PCR and Western blot analysis. Electrophysiological examination of native VSOACs in isolated atrial and ventricular myocytes 3 weeks off doxycycline revealed a complete elimination of the currents, whereas at 1.5 weeks, VSOAC current densities were significantly reduced, compared to age-matched control mice maintained on doxycycline. These results indicate that ClC-3 is a key component of native VSOACs in mammalian heart and plays a significant cardioprotective role against cardiac hypertrophy and failure.

Regulation of gap junctional charge selectivity in cells coexpressing connexin 40 and connexin 43.

Expression of connexin 40 (Cx40) and Cx43 in cardiovascular tissues varies as a function of age, injury, and development with unknown consequences on the selectivity of junctional communication and its acute regulation. We investigated the PKC-dependent regulation of charge selectivity in junctions composed of Cx43, Cx40, or both by simultaneous assessment of junctional permeance rate constants (B(dye)) for dyes of similar size but opposite charge, N,N,N-trimethyl-2-[methyl-(7-nitro-2,1,3-benzoxadiol-4-yl)amino]ethanaminium (NBD-M-TMA; +1) and Alexa 350 (-1). The ratio of dye rate constants (B(NBD-M-TMA)/B(Alexa 350)) indicated that Cx40 junctions are cation selective (10.7 +/- 0.5), whereas Cx43 junction are nonselective (1.22 +/- 0.14). In coexpressing cells, a broad range of junctional selectivities was observed with mean cation selectivity increasing as the Cx40 to Cx43 expression ratio increased. PKC activation reduced or eliminated dye permeability of Cx43 junctions without altering their charge selectivity, had no effect on either permeability or charge selectivity of Cx40 junctions, and significantly increased the cation selectivity of junctions formed by coexpressing cells (approaching charge selectivity of Cx40 junctions). Junctions composed of Cx43 truncated at residue 257 (Cx43tr) were also not charge selective, but when Cx43tr was coexpressed with Cx40, a broad range of junctional selectivities that was unaffected by PKC activation was observed. Thus, whereas the charge selectivities of homomeric/homotypic Cx43 and Cx40 junctions appear invariant, the selectivities of junctions formed by cells coexpressing Cx40 and Cx43 vary considerably, reflecting both their relative expression levels and phosphorylation-dependent regulation. Such regulation could represent a mechanism by which coexpressing cells such as vascular endothelium and atrial cells regulate acutely the selective intercellular communication mediated by their gap junctions.

Hindered diffusion through an aqueous pore describes invariant dye selectivity of Cx43 junctions.

The permselectivity (permeance/conductance) of Cx43-comprised gap junctions is a variable parameter of junctional function. To ascertain whether this variability in junctional permselectivity is explained by heterogeneous charge or size selectivity of the comprising channels, the permeance of individual Cx43 gap junctions to combinations of two dyes differing in either size or charge was determined in four cell types: Rin43, NRKe, HeLa43, and cardiac myocytes. The results show that Cx43 junctions are size- but not charge-selective and that both selectivities are constant parameters of junctional function. The consistency of dye selectivities indicates that the large continuum of measured junctional permselectivities cannot be ascribed to an equivalent continuum of individual channel selectivities. Further, the relative dye permeance sequence of NBD-M-TMA approximately Alexa 350 > Lucifer yellow > Alexa 488 >> Alexa 594 (Stokes radii of 4.3 A, 4.4 A, 4.9 A, 5.8 A, and 7.4 A, respectively) and the conductance sequence of KCl > TEACl approximately Kglutamate are well described by hindered diffusion through an aqueous pore with radius approximately 10 A and length 160 A. The permselectivity and dye selectivity data suggest the variable presence in Cx43-comprised junctions of conductive channels that are either dye-impermeable or dye-permeable.

Selectivity of connexin 43 channels is regulated through protein kinase C-dependent phosphorylation.

Coordinated contractile activation of the heart and resistance to ischemic injury depend, in part, on the intercellular communication mediated by Cx43-composed gap junctions. The function of these junctions is regulated at multiple levels (assembly to degradation) through phosphorylation at specific sites in the carboxyl terminus (CT) of the Cx43 protein. We show here that the selective permeability of Cx43 junctions is regulated through protein kinase C (PKC)-dependent phosphorylation at serine 368 (S368). Selective permeability was measured in several Cx43-expressing cell lines as the rate constant for intercellular dye diffusion relative to junctional conductance. The selective permeability of Cx43 junctions under control conditions was quite variable, as was the open-state behavior of the comprising channels. Coexpression of the CT of Cx43 as a distinct protein, treatment with a PKC inhibitor, or mutation of S368 to alanine, all reduced (or eliminated) phosphorylation at S368, reduced the incidence of 55- to 70-pS channels, and reduced by 10-fold the selective permeability of the junctions for a small cationic dye. Because PKC activation during preischemic conditioning is cardioprotective during subsequent ischemic episodes, we examined no-flow, ischemic hearts for Cx43 phosphorylated at S368 (pS368). Consistent with early activation of PKC, pS368-Cx43 was increased in ischemic hearts; despite extensive lateralization of total Cx43, pS368-Cx43 remained predominantly at intercalated disks. Our data suggest that the selectivity of gap junction channels at intercalated disks is increased early in ischemia.