(a,b) Confocal fluorescence images of horizontal brain slices from a Gjd2-EGFP transgenic mouse showing thalamic reticular nucleus (TRN) interneurons expressing EGFP driven by Cx36 promoter. VB, ventrobasal nucleus of the Thalamus; IC, internal capsule. Scale bars correspond to 100 (a) and 10 (b) µm. (c,d) IR-DIC (c) and fluorescence (d) images of an electrically coupled pair of TRN neurons during dual whole-cell patch clamp. Scale bar correspond to 20 µm. (e) Current-clamp recordings from a TRN neuron showing typical spiking and rebound burst behavior; voltage traces were recorded during 0.5 s current steps of −100 (grey) or 200 (black) pA. (f) Voltage-clamp recordings showing averaged transjunctional current traces (10–15 averaged traces) obtained soon after patch openings (initial; left) and after ~25 min of recording (right) for low Mg²⁺ (K₂ATP, top) and high Mg²⁺ (MgATP, bottom) conditions; current traces were recorded during 0.5 s transjunctional voltage steps of −40 mV. (g) Mean percentage changes of junctional conductance (g_j) from initial values after ~25 min from patch openings with pipette solutions containing 7 mM of K₂ATP (grey) or MgATP (black). Numbers of cell pairs are indicated within columns, and error bars correspond to s.e.m.

Chimeras were generated from Cx36 (green) and Cx43 (red). Chimeras that form functional channels and junctional plaques (JPs) are shown in yellow background. Functional chimeras are named from CH1 to CH4. Chimeras that form JPs but do not exhibit electrical cell-cell coupling are shown in blue background. Chimeras that do not form JPs or exhibit electrical cell-cell coupling are shown in grey background.

Experiments were performed in pairs of RIN cells expressing Cx36 (green), Cx43 (red), Cx36/Cx43 chimeras (CH1-CH4), and amino acid substitutions (D47G or G46D). (a) Mean transjunctional conductance measured soon after patch opening (g_j,initial). (b) Mean g_j (normalized to initial g_j value) measured after ~25 min using pipette solutions containing 0.01 mM (grey) or 5 mM (black) free Mg²⁺ ([Mg²⁺]_p). The dotted line marks the value of normalized g_j equal to 1. Total numbers of cell pairs are indicated within columns, and error bars correspond to s.e.m.

(a–d) Transjunctional current (I_j) recordings of single channel events obtained at indicated V_js (top) and using [Mg²⁺]_p = 0.01 mM. Numbers attached to arrows show single channel conductances at the open state (γ_open). (e) Averaged γ_open for homotypic GJs formed by CH3, CH3*D47G, CH4, and Cx43*G46D using [Mg²⁺]_p = 0.01 (gray) or 5 (black) mM. Total numbers of cell pairs are indicated within columns, and error bars correspond to s.e.m.

(a) Changes of I_j (bottom trace) of CH3 GJs in response to repeated 30 s long V_j ramps from 0 to −90 mV and from 0 to +90 mV with intermediate small amplitude ramps (−10 mV) (top trace) using symmetric [Mg²⁺]_p = 5 mM. (b,d) Diagrams illustrating [Mg²⁺]_p in cell-1 and cell-2 and the stimulation site of the V_j protocol for experiments shown in a and c (b), or e (d). (c) g_j–V_j relations obtained from experiment shown in (a). Colors match the I_j data shown in (a). (e) Asymmetric g_j,ss–V_j dependence (normalized to g_j value at V_j zero) was measured by applying V_j ramps from 0 to −70 and from 0 to +70 mV (30 s in duration) under transjunctional Mg²⁺ asymmetry shown in (d). Negative potentials applied on the side with low [Mg²⁺]_i decreased g_j presumably by increasing Mg²⁺ occupancy of the pore through ionophoresis (n=4). (f) g_j trace (bottom) showing unitary gating events of the homotypic CH3 GJ channel obtained during V_j steps of ±55 mV (top trace) applied in cell-1 (as illustrated in d). Negative V_js facilitated closing transitions, whereas positive V_js facilitated opening transitions. (g-h) Count histograms for all g_j data obtained at negative (g) or positive (h) V_js shown in (f). Both histograms show peaks corresponding to the single channel conductance of ~115 pS.

(a–b) V_j protocols used to obtain steady-state (a) and instantaneous (b) g_j–V_j relationships. (c–e) Steady-state (grey) and instantaneous (black) g_j–V_j relationships (normalized to g_j value at V_j zero) were measured ~30 min after opening of patches under symmetric high (c), low (d), and asymmetric Mg²⁺ conditions (e) in homotypic GJs formed of Cx36 (top row), Cx36*D47G (middle row) and CH3 (bottom row). Each data point for instantaneous g_j–V_j relationships was obtained by averaging data from ~10 consecutive V_j protocols shown in (b), and error bars correspond to s.e.m. Top diagrams in each column show [Mg²⁺]_p and stimulation site. (f) Mean g_j,inst values measured at V_js equal to −85 and +85 mV (normalized to g_j,inst value at V_j zero) at symmetric high (black) and low (light gray) [Mg²⁺]_p, and asymmetric Mg²⁺ conditions (dark gray) for GJs formed of Cx36 (left) and Cx36*D47G (right). Total numbers of cell pairs are indicated within columns, and error bars correspond to s.e.m.

Among all examined heterotypic combinations between wild type Cx36 (green) or Cx43 (red) with CH1, CH2, CH3, or CH4, only those shown in yellow background formed junctional plaques and exhibit electrical cell-cell coupling, while those shown in grey background did not form junctional plaques or exhibit electrical cell-cell coupling.

(a–b) Asymmetric g_jss–V_j relationships (normalized to g_j,ss value at V_j =0, and obtained using the same V_j protocol shown in ) with pipette solutions containing symmetric high (a) or low (b) [Mg²⁺]_p in heterotypic Cx43/CH3 GJs (n=5). Top diagram in each plot shows [Mg²⁺]_p used in cell-1 and cell-2, stimulation sites and expressed Cxs. (c) I_j records of single channel events at symmetric [Mg²⁺]_p obtained during V_j steps of ±85 mV (top trace) applied in cell-1 expressing Cx43. (d) I_j records (bottom trace) under symmetric high [Mg²⁺]_p showing unitary gating events of Cx43/CH3 GJ channel obtained during V_j steps of ±60 mV (top trace) applied in cell-1 expressing Cx43. Positive V_js facilitated closing transitions, while negative V_js facilitated opening transitions. (e,f) Asymmetric g_jss–V_j relationships (normalized to g_j,ss value at V_j =0) obtained under asymmetric Mg²⁺ conditions (see diagrams) in heterotypic Cx43/CH3 GJs (n=5).

(a–b) Asymmetric steady-state (top) and instantaneous (bottom) g_j–V_j relationships shown in black (normalized to g_j value at V_j =0, and obtained using the same V_j protocol shown in , respectively) obtained under symmetric high (a) and low (b) [Mg²⁺]_p in heterotypic Cx36/CH4 GJs (n=5). The top diagram in each plot shows [Mg²⁺]_p used in cell-1 and cell-2, stimulation sites and expressed Cxs. Each data point for g_j,inst–V_j relationships was obtained by averaging data from ~10 consecutive V_j protocols, and error bars correspond to s.e.m. Simulated curves shown in grey for steady-state (top) and instantaneous (bottom) g_j–V_j relationships were obtained using the S4SM. Dotted lines show g_j,inst–V_j relationships for CH4 (purple) and Cx36 (pink) aHCs used in simulation of steady-state and instantaneous g_j–V_j relationships. (c-d) Symmetric steady-state (top) and instantaneous (bottom) g_j–V_j relationships for homotypic Cx36 GJs are shown in black (data from ). Simulated curves shown in grey were obtained using the S4SM and similar rectification parameters were obtained from Cx36 aHCs shown in (a) and (b). All parameters for simulation are reported in .

(a) Cross-sections of GJ channels formed by Cx36 (top) or Cx36*D47G (bottom) generated by sequence homology to Cx26 crystal structure. Electrostatic surface potential is displayed for both channels and was estimated with DELPHI (see Methods). The color bar at the bottom represents electrostatic surface potentials ranging from −15 (red) to 15 (blue) kTe⁻¹. (b) Side view of a Cx36 aHC indicating the pore-lining residues of only two Cx subunits represented with thin ribbon style (principal chain). Main domains of Cx subunits are depicted with different colors and the side chains of pore-lining residues are represented with sticks and spheres. (c) View of the pore from the center of the channel towards the cytoplasmic mouth. The six D47 residues are displayed with sticks and spheres to illustrate the orientation and position of their side chains with respect to the lumen of the channel. (d) Normalized g_j,inst—V_j plots for aHCs (top) and GJ channels (bottom) formed by Cx36 generated using different values for Mg²⁺-dependent coefficient (r_Mg) with the S4SM of GJ channels gating. Color bar at the top represents different [Mg²⁺]_i from 0.01 to 5 mM.