SUPPLEMENTAL FILES
Electrophysiological records
The functionality of the gap junction channels between WB-F344 cell pairs was tested by using the dual whole-cell patch clamp in voltage-clamp conditions, as previously described (Valiunas et al., 2000; Formigli et al., 2005; Gonzàles et al., 2007; Meacci et al., 2010). Coverslips with adherent cells were placed in the recording chamber on the stage of an inverted microscope (Nikon Eclipse TE 2000). The cells were superfused with a normal Tyrode bath solution containing (in mM) 140 NaCl, 5.4 KCl, 1.8 CaCl2, 1.2 MgCl2, 10 d-glucose, and 5 HEPES. The patch pipettes were filled with a filling pipette solution containing (in mM) 150 CsBr, 5 MgCl2, 10 EGTA, and 10 HEPES filtered through 0.22-μm pores. The pH was set to 7.4 with NaOH and to 7.2 with tetraethylammonium-OH for bath and pipette solution, respectively. By using a micropipette vertical puller (Narishige PC-10; Narishige, Kyoto, Japan), we pulled the patch pipettes from borosilicate glass (GC 150-15; Clark, Reading, UK). Pipettes resistance was 1.3–1.7 MΩ. Each patch pipette was controlled by a micromanipulator (Narishige) and connected to the Axopatch 200B amplifier (Axon Instruments, Union City, CA). Voltage-clamp protocol generation and data acquisition were achieved by two outputs and inputs of the analog-to-digital/digital-to-analog interfaces (Digidata 1200; Axon Instruments, Union City, CA) and pClamp 9 software (Axon Instruments, Union City, CA). Currents were low-pass filtered at 1 kHz with a Bessel filter; the sampling interval was 0.6 ms.
The protocol of the stimulation and the recording procedure has already been reported in details in previous researches (Barrio et al., 1991; Valiunas et al., 2001; Formigli et al., 2005a; Gonzàles et al., 2007; Meacci et al., 2010). In brief, at the beginning of the experiment, the membrane potentials of cell 1 (V1) and cell 2 (V2) were clamped to the same value, V1 = V2. Then, V1 was modified to create a transjunctional voltage (Vj), Vj = V2 − V1. From a holding potential (HP) of 0 mV, cell 1 was stepped using a bipolar pulse protocol starting at Vj = ±10 mV and ongoing at 20-mV increments up to ±150 mV. Test pulse and interpulse lasted 5 and 0.7 s, respectively. Any current recorded from cell 1 was the result of two components: the membrane current of cell 1 and the transjunctional current (Ij). The currents recorded from cell 2 were denoted as −Ij. After the giga-seal was made, we electronically compensated (65–85%) the series resistance of electrodes 1 and 2, Rs1 and Rs2. Cm value represents the cell membrane capacitance and was used to estimate the cell membrane surface
The amplitudes of transjunctional current, Ij, was determined at the beginning (instantaneous current, Ij,inst) and at the end of each pulse (steady state current, Ij,ss). These values were used to calculate the related conductances Gj,inst and Gj,ss. The Gj,ss voltage dependence (Gj,ss−Vj plot) was best fitted by the Boltzmann function:Gj,ss=(Gmax−Gmin)/{1+exp[A(Vj−V0)]}+Gmin,
where Gmax represents the maximal Gj,ss conductance and Gmin the residual conductance at the end of the voltage steps, A is the constant that describes voltage sensitivity, Vo is the transjunctional voltage halfway between Gmax and Gmin, Gj,ss was normalized with respect to Gj,inst and plotted against Vj. For mathematical and statistical analysis of data (expressed as means ± SEM) we used pClamp9 (Axon), SigmaPlot, and SigmaStat software (Jandel Scientific). A two-sample t-test was used to compare single parameters between two independent experimental groups. ANOVA with repeated measures was used for multiple comparisons. P < 0.05 was considered significant. Experiments were made at 22°C.
Non symmetrical Ij in complexes GJ
One cells can express the same or coexpressed different connexins. Therefore, a connexson (hemichannel) may be 1) homomeric if hemichannel is composed of the same connexin and 2) heteromeric if composed by different connexin). Moreover, the type of connexons expresses in a cell pair may be the results of mixing of different connexons. Consequently, GJIC may be classified as: 1) homotypic channel if both interacting hemichannels are composed of the same connexon and 2) heterotypic if both interacting hemichannels are composed of different connexon. So, the possible GJIC expresses in a cell pair depend on the possible mixing of connexin and connexon.
Homotypic GJIC channels with different Cx isoforms showed a wide multiplicity of Vj-gating phenotypes depending on the polarity of closing, voltage sensitivity, magnitude of the residual conductance component that is Vj insensitive (Gjmin) and kinetic properties (Gonzàles et al., 2007). Moreover, homologous Cx from different species or tissues may have different Vj-gating properties (Gonzàles et al., 2007). For homotypic channels the stronger Vj-sensitive channels are those that showed a large number of gating charges (z) and a small half inactivation (V0<30 mV) or a low Gjmin value. The A parameter is an index of z, since A = zq/kT is a constant reflecting equivalent charge movement, being the charge of the electron, q, and kT constants.
In addition, the instantaneous value of Ij (Ij,ist) may be linear or non-linear with respect to voltage, especially in heterotypic GJs (Thomas et al., 1994; Vytas et al., 1991).
In real cells different kind of Cxs are expresses and these can form cluster of homotypic GJICs or can mix to form heterotypic GJICs. The total Gj, Gj,tot, is in the first case the sum of the single homotypic GJICs and the evaluated Gj,tot depend on the percentage of the type of homotypic GJICs expresses. Whereas when heterotypic GJICs are present the GJ,tot showed a further greater Vj-gating variability since the behavior of each single heterotypic GJIC change significantly; for example heterotypic channel have a reduced conductance, GJ,max, a reduced voltage-dependence of IJ and a greater Gj,min respect to homotypic GJIC (Valiunas et al., 2001).
In homotypic GJIC the Vj dependence of the steady state Gj/Vj relationships for both Vj polarities is symmetric. In contrast, for heterotypic channels composed of different Cx hemichannels, the Vj dependence of the steady state Gj,ss/Vj relationships for both Vj polarities are necessarily asymmetric. In fact, the new properties of GJIC are due either to the different unitary conductance or to the hemichannel polarity of closure of the two constituent hemichannels. In fact, the opposing Vj gates of component hemichannels may close at the same or opposite polarity of closure. Therefore, the Gj,ss/Vj curves would decrease at both polarities of Vj, and have a quasi symmeytric form, when two component hemichannels close with the same gating polarity. In contrast, when hemichannels with opposite voltage polarity of Vj gating are paired a reduction in the steady state conductance for only one polarity of Vj would be expected (Barrio et al., 1991; White et al., 1994; Brink et al., 1997; Valiunas et al., 2000; Valiunas et al., 2001;). So, these latter assume a negative or positive Vj dependence that was called VJIC of Vj(-) form and Vj(+) form, respectively. Cx have been provisionally divided in two groups according to the polarity at which they close, positive for Cx26, Cx30, Cx37, Cx40, Cx46 and Cx50 or negative for Cx32, Cx43, Cx45 or Cx31 (Gonzales et al., 2007). The more complex Ij was in heteromeric/heterotypic GJIC. In such a case, there are results also showing that the coexpression of two connexons, one voltage-sensitive (as Cx30) and one voltage-insensitive (as Cx26), may resolve with Ij Vj sensitive, insensitive, asymmetrical and symmetrical (Yum et al., 2007), depending on the junctional current profile related on the expression level of the two connexins, i.e., variations in the protein ratio of Cx26 and Cx30 in different cell pairs and thus the corresponding ratio of homotypic, heterotypic, and heteromeric channels formed (Yum et al., 2007).
Appendix
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