Contribution of Intracellular Calcium and pH in Ischemic Uncoupling of Cardiac Gap Junction Channels Formed of Connexins 43, 40, and 45: A Critical Function of C-Terminal Domain

<div><p>Ischemia is known to inhibit gap junction (GJ) mediated intercellular communication. However the detail mechanisms of this inhibition are largely unknown. In the present study, we determined the vulnerability of different cardiac GJ channels formed of connexins (Cxs) 43, 40, and 45 to simulated ischemia, by creating oxygen glucose deprived (OGD) condition. 5 minutes of OGD decreased the junctional conductance (G_j) of Cx43, Cx40 and Cx45 by 53±3%, 64±1% and 85±2% respectively. Reduction of G_j was prevented completely by restricting the change of both intracellular calcium ([Ca²⁺]_i) and pH (pH_i) with potassium phosphate buffer. Clamping of either [Ca²⁺]_i or pH_i, through BAPTA (2 mM) or HEPES (80 mM) respectively, offered partial resistance to ischemic uncoupling. Anti-calmodulin antibody attenuated the uncoupling of Cx43 and Cx45 significantly but not of Cx40. Furthermore, OGD could reduce only 26±2% of G_j in C-terminus (CT) truncated Cx43 (Cx43-<i>Δ</i>257). Tethering CT of Cx43 to the CT-truncated Cx40 (Cx40-Δ249), and Cx45 (Cx45-Δ272) helped to resist OGD mediated uncoupling. Moreover, CT domain played a significant role in determining the junction current density and plaque diameter. Our results suggest; OGD mediated uncoupling of GJ channels is primarily due to elevated [Ca²⁺]_i and acidic pH_i, though the latter contributes more. Among Cx43, Cx40 and Cx45, Cx43 is the most resistant to OGD while Cx45 is the most sensitive one. CT of Cx43 has major necessary elements for OGD induced uncoupling and it can complement CT of Cx40 and Cx45.</p> </div

% inhibition of junctional conductance (G_j) by oxygen glucose deprivation (OGD).

<p>OGD was performed for 5 minutes. [Ca²⁺]_i and pH_i were clamped by adding BAPTA (2 mM) and HEPES (80 mM) in pipette solution respectively. To restrict both [Ca²⁺]_i and pH_i change, 150 mM KPO₄ was used in pipette.</p

Sensitivity of Cx43, Cx40 and Cx45 to junctional Voltage (V_j).

<p>Upper panel shows the voltage step protocol. V_j steps of 20 mV increment, from −120 to +120 mV were applied for 20 sec followed by 30 sec recovery at 0 mV. A short pre pulse (100 msec) of 10 mV was applied at the beginning of each pulse to normalize G_j. Middle panel: representative I_j traces. Lower panel: normalized G_j(ss) versus V_j plot. Two states Boltzmann fitted curve, represented by the solid line was generated with pCLAMP 10 software.</p

Effect of OGD on gap junctions made of different chimeric connexions.

<p><b>A</b>, chimeric Cx43 with the CT of Cx40 (Cx43-C40) and <b>B</b>, Cx40 with the CT of Cx43 (Cx40-C43) uncoupled to a lesser extent compared to the corresponding wild type. <b>C</b>. Comparison of the uncoupling of wild type Cx45 and Cx45 chimeras containing CT of Cx40 and Cx43. <b>D</b>, statistical representation of the extent of uncoupling of different chimeras. Values are the mean±SEM of 5-7 independent experiments. ***, p<0.001.</p

Clamping of intracellular calcium ([Ca²⁺]_i) and (pH_i) during OGD.

<p><b>A</b>. Addition of 2 mM BAPTA in the pipette solution restricted [Ca²⁺]_i rise during OGD. Rhod-2 dye was used to determine [Ca²⁺]_i in presence of BAPTA. <b>B</b>. 2 mM BAPTA did not affect OGD induced acidosis, whereas 150 mM KPO₄ in the pipette, prevented pH change. <b>C</b>. OGD induced change of pH_i in presence of different concentration of HEPES in the pipette. 80 mM HEPES prevented acidosis completely. pH_i was determined ratiometrically using BCECF. <b>D</b>. 80 mM HEPES in the pipette, did not affect OGD-induced [Ca²⁺]_i rise. 150 mM KPO₄ prevented both calcium rise and acidosis (B). [Ca²⁺]_i was measured with fura-2. Values are the mean±SEM recorded from 5–10 cells.</p

Average conductance and junctional plaque diameter of different Connexins.

<p>WT: wild type; ND: not detectable; NF: not functional; data presented are mean±SEM.</p

Effect of OGD on different cardiac gap junctions.

<p><b>A</b>. representative I_j trace of Cx43 gap junction recorded from transfected N2a cells. I_j decreased at the onset of OGD. Solid line above the current trace represents duration of OGD. Voltage protocol is presented on top of the current trace. <b>B</b>. OGD reduced the G_j (normalized) of all gap junctions. Cx45 showed maximum reduction. Values are the mean±SEM. <b>C</b>. quantitative representation of the data obtained from panel B.</p

OGD induced uncoupling of CT truncated Cx43 (Cx43-<b>Δ</b><b>Δ</b>257).

<p><b>A</b>. G_j of CT truncated Cx43 reduced to a lesser extent compared to wild type Cx43, in response to OGD. <b>B</b>. quantitative representation of data generated from 5–7 independent experiments. Values are the mean±SEM. ***, p<0.001.</p

OGD inhibited Cx45-eGFP more than Cx45.

<p>OGD uncoupled eGFP tagged Cx45 almost completely, compared to 85±2% inhibition in wild type Cx45. The difference is statistically significant (p<0.001).</p