Pinto BI et al. (2017), Calcium binding and voltage gating in Cx46 hemi...

XB-ART-56089

Sci Rep 2017 Nov 20;71:15851. doi: 10.1038/s41598-017-15975-5.

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Calcium binding and voltage gating in Cx46 hemichannels.

Pinto BI , Pupo A , García IE , Mena-Ulecia K , Martínez AD , Latorre R , Gonzalez C .

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The opening of connexin (Cx) hemichannels in the membrane is tightly regulated by calcium (Ca2+) and membrane voltage. Electrophysiological and atomic force microscopy experiments indicate that Ca2+ stabilizes the hemichannel closed state. However, structural data show that Ca2+ binding induces an electrostatic seal preventing ion transport without significant structural rearrangements. In agreement with the closed-state stabilization hypothesis, we found that the apparent Ca2+ sensitivity is increased as the voltage is made more negative. Moreover, the voltage and Ca2+ dependence of the channel kinetics indicate that the voltage sensor movement and Ca2+ binding are allosterically coupled. An allosteric kinetic model in which the Ca2+ decreases the energy necessary to deactivate the voltage sensor reproduces the effects of Ca2+ and voltage in Cx46 hemichannels. In agreement with the model and suggesting a conformational change that narrows the pore, Ca2+ inhibits the water flux through Cx hemichannels. We conclude that Ca2+ and voltage act allosterically to stabilize the closed conformation of Cx46 hemichannels.

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Species referenced: Xenopus laevis
Genes referenced: gja3

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	Figure 1. Regulation of Cx46 hemichannels by voltage and calcium. (A) Oocytes expressing Cx46 maintained at a holding potential of +20 mV were subjected to 8 hyperpolarizing pulses from 20 mV to −60 mV in 10 mV steps. Current traces that correspond to voltage traces have the same color. (B) Graph depicting the voltage activation curve of Cx46 obtained from tail currents. The solid line represents the fitting of the data to equation (1) and using the parameters given in the inset table. (C) Oocytes at a holding potential of −70 mV were subjected to depolarizing pulses to 0 mV at different Ca2+ concentrations (from 0 to 1.1 mM). The colored line indicates the concentration of Ca2+ present in the bath. (D) Graph depicting the normalized tail currents at the different Ca2+ concentration. The solid line represents the fitting of the data using equation (2) and the parameters given in the inset table.
	Figure 2. Inhibition of Cx46 hemichannels by calcium at different voltages. Oocytes at a holding potential of −70 mV were subjected to depolarizing pulses of (A) 20 mV, (B) −30 mV and (C) −50 mV. At each voltage different Ca2+ concentrations were tested. The colored line indicates the concentration of Ca2+ present in the bath. (D) Ca2+ inhibition curves for the different voltages used. Data presented as mean ± SEM, the lines indicate the fit to the Hill equation (Table 1).
	Figure 3. Stabilization of the closed state reproduces the observed changes in inhibition. (A) Ca2+ inhibition data at different voltages was fitted using the linear Hill model. The discontinuous lines show the 95% confidence intervals of the model for the inhibition curves obtained at different voltages. (B) The points show the Log IC50 determined at different voltages. The black line indicates the prediction of the Log IC50 according to the linear Hill model, and the dotted lines show the 95% confidence intervals.
	Figure 4. Current relaxation of Cx46 shows two-time constants. (A) Activation kinetics seen after a voltage pulse to 0 mV from a holding of −40 mV shows a current that is well fitted by a sum of two exponentials. (B) The same is observed in deactivation kinetics when a pulse to −80 mV is given from a holding of 0 mV. The insets an A and B show a magnification of the activation and deactivation current trace to better appreciate the difference between fitting one or two exponentials, this difference is statistically significant. (C) At nominal Ca2+ concentration, the time constants present intrinsic voltage dependence. As can be seen in (D), Ca2+ accelerates the time constants until they reach saturation at high Ca2+ concentrations (at −100 mV).
	Figure 5. The proposed kinetic mechanism for Ca2+ and voltage regulation of Cx46 hemichannels. (A) The complete model considers three voltage-dependent transitions form O to C1 to C2 and C3, given by the equilibrium constants K1 and the rates α, β, χ and δ. Binding of Ca2+ occurs only in the closed states and is given by the association constant Ka (1/Kd), m indicates the number of binding sites. Voltage gating and Ca2+ binding are allosterically coupled by the coupling constant C and its kinetic factors c b and c f. The black arrows indicate rate-limiting steps and gray arrows indicate transitions in equilibrium.(B) The complete model can be collapsed into a simpler model in which each state contains several substates that are in equilibrium. The equations that govern this model are presented.
	Figure 6. Global fit of the data to the allosteric model with 6 Ca2+ binding sites. (A) G/Gmax vs voltage. (B) Ca2+ inhibition curves at different voltages. (C) Time constants vs voltage from activation and deactivation kinetics. (D) Time constants vs Ca2+ concentration from deactivation kinetics at −100 mV. Data is presented as mean ± SD. The lines of the fitting indicate the 95% confidence bands. To display the fitting, we draw 400 sets of parameters from the posterior distribution and the experimental results were simulated based on the proposed model with each parameter set.
	Figure 7. Calcium effects of the flux of water through Cx46 hemichannels. (A) Cx46 expressing oocytes were placed in a hypotonic solution with 5 mM Ca2+ (Cx46 + Ca2+, blue line) or without Ca2+ (Cx46, red line). (B) Oocytes injected with Cx38 antisense oligonucleotide in a hypotonic solution with 5 mM Ca2+ (CT + Ca2+, blue line) or without Ca2+ (CT46, red line). The relative volume of oocytes vs. time was plotted for both conditions; the results are shown as mean (continuous line) ± SEM (dashed lines). (C) The rate of volume change was calculated for the last 50 s of the recordings. The Cx46 condition shows a significantly higher rate of volume change than the Cx46 + Ca2+ condition and with control oocytes in the presence or absence of Ca2+. *p < 0.05 in comparison to Cx46 using Student’s t-test (n = 4 in all experimental conditions).

References [+] :

Armstrong, Interaction of tetraethylammonium ion derivatives with the potassium channels of giant axons. 1971, Pubmed