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. 2013 Mar 13;33(11):4741-53.
doi: 10.1523/JNEUROSCI.2825-12.2013.

Intracellular magnesium-dependent modulation of gap junction channels formed by neuronal connexin36

Nicolás Palacios-Prado  1 , Gregory Hoge, Alina Marandykina, Lina Rimkute, Sandrine Chapuis, Nerijus Paulauskas, Vytenis A Skeberdis, John O'Brien, Alberto E Pereda, Michael V L Bennett, Feliksas F Bukauskas
Affiliations

Intracellular magnesium-dependent modulation of gap junction channels formed by neuronal connexin36

Nicolás Palacios-Prado et al. J Neurosci. .

Abstract

Gap junction (GJ) channels composed of Connexin36 (Cx36) are widely expressed in the mammalian CNS and form electrical synapses between neurons. Here we describe a novel modulatory mechanism of Cx36 GJ channels dependent on intracellular free magnesium ([Mg(2+)]i). We examined junctional conductance (gj) and its dependence on transjunctional voltage (Vj) at different [Mg(2+)]i in cultures of HeLa or N2A cells expressing Cx36. We found that Cx36 GJs are partially inhibited at resting [Mg(2+)]i. Thus, gj can be augmented or reduced by lowering or increasing [Mg(2+)]i, respectively. Similar changes in gj and Vj-gating were observed using MgATP or K2ATP in pipette solutions, which increases or decreases [Mg(2+)]i, respectively. Changes in phosphorylation of Cx36 or in intracellular free calcium concentration were not involved in the observed Mg(2+)-dependent modulation of gj. Magnesium ions permeate the channel and transjunctional asymmetry in [Mg(2+)]i resulted in asymmetric Vj-gating. The gj of GJs formed of Cx26, Cx32, Cx43, Cx45, and Cx47 was also reduced by increasing [Mg(2+)]i, but was not increased by lowering [Mg(2+)]i; single-channel conductance did not change. We showed that [Mg(2+)]i affects both open probability and the number of functional channels, likely through binding in the channel lumen. Finally, we showed that Cx36-containing electrical synapses between neurons of the trigeminal mesencephalic nucleus in rat brain slices are similarly affected by changes in [Mg(2+)]i. Thus, this novel modulatory mechanism could underlie changes in neuronal synchronization under conditions in which ATP levels, and consequently [Mg(2+)]i, are modified.

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Figures

Figure 1.
Figure 1.
Mg2+-dependent modulation of gj and Vj-gating in HeLa cells expressing Cx36-EGFP GJs. A, C, Bottom, Dynamics of gj (normalized to initial gj value) during repeated Vj ramps (+20–−20 mV and 1.3 s in duration; top traces) at [Mg2+]p = 0.01 (A) and 5 mm (C). B, D, gj–Vj dependence (normalized to initial gj value at Vj = 0) obtained during 50-s-long Vj ramps (from 0 to −100 mV; middle traces) derived from data shown in A and C, respectively; the numbers on the gj–Vj plots correspond to numbers on gj traces in A and C. Fitted curves shown in color were obtained using the S16SM. Gray lines show fitted curves obtained from control gj–Vj plots ([Mg2+]p = 1 mm). E, Concentration–response relation of gj (normalized to initial gj value) as a function of [Mg2+]p; EC50 ≈ 0.45 mm. Averaged data are shown in colored circles. Data of individual experiments are shown in gray triangles. Roman numerals correspond to different solutions shown in Table 1. F, Averaged gj–Vj dependencies (normalized to gj value at Vj = 0) obtained at different [Mg2+]p (curves in black) were fitted using a S16SM (gj–Vj plots in colors). Roman numerals correspond to different Mg2+ concentrations shown in E. Parameters obtained after fitting are shown in Table 2. G–I, Open probabilities of fast and slow gates in α and β aHCs (PoF,α, PoS,α and PoF,β, PoS,β, respectively) depending on Vj were calculated using parameters obtained in F for [Mg2+]p = 0.01 (G), 1 (H), and 10 mm (I).
Figure 2.
Figure 2.
Differences in [Mg2+]i-dependent modulation of gj in HeLa or N2A cells expressing Cx26, Cx32, Cx36, Cx43, Cx45, or Cx47; changes in [Ca2+]i are not involved in [Mg2+]i-dependent modulation of Cx36. All data represent mean gj (normalized to initial gj value). A, Normalized gj measured in HeLa Cx36-EGFP cell pairs using pipette solutions containing 10 mm free Mg2+ and 2 mm BAPTA (free Ca2+ = 25 nm) or 10 mm BAPTA (free Ca2+ ≈ 0). B, C, Normalized gj measured using pipette solutions containing 0.01 (B) or 5 mm free Mg2+ (C) in HeLa Cx36WT, HeLa Cx36-EGFP, or N2A Cx36-EGFP cell pairs. D, E, Normalized gj measured using pipette solutions containing 0.01 (D) or 5 mm (E) of free Mg2+ in HeLa cells expressing Cx26, Cx32, Cx36, Cx43, Cx45, or Cx47. Numbers of cell pairs are indicated within columns; *p < 0.05; ns, nonsignificant p values.
Figure 3.
Figure 3.
[Mg2+]i-dependent modulation of gj and Vj-gating of GJs formed by other Cxs expressed in CNS does not affect single-channel conductance. A, C, E, Changes of Ij in response to repeated 50-s-long Vj ramps from 0 to −100 mV and intermediate small-amplitude steps (−15 mV) using [Mg2+]p = 5 mm in Novikoff (A), HeLa Cx45 (C), and HeLa Cx47-EGFP (E) cell pairs. B, D, F, First (purple) and last (cyan) gj–Vj relations (normalized to initial gj value at Vj = 0) obtained from experiments shown in A, C, and E, respectively. Last gj–Vj relation normalized to gj value at Vj = 0 (gray) is also shown for comparison. G, Concentration–response relation of gj (normalized to initial gj value) as a function of [Mg2+]p for Cx47; EC50 ≈ 2.8 mm. Averaged data are shown in colored circles; roman numerals correspond to different pipette solutions shown in Table 1. H, Averaged gj–Vj dependence (normalized to gj at Vj = 0) obtained at different [Mg2+]p for Cx47; roman numerals correspond to different [Mg2+] shown in G and Table 1. Experimental gj–Vj plots shown in black were fitted using the S16SM; calculated values of gating parameters are shown in Table 2, and the best-fitting gj–Vj plots are shown in colors that correspond to colors of circles in G. I, J, Open probabilities of fast and slow gates in α and β aHCs (PoF,α, PoS,α and PoF,β, PoS,β, respectively) depending on Vj were calculated using parameters obtained in H for [Mg2+]p = 0.01 (I) and 10 mm (J). K, Ij record of Cx43-CFP GJs obtained at Vj = −80 mV. L, Histogram from data in K shows a series of peaks separated by ∼96.5 ± 8 pS. M, Single-channel conductance for Cx47-EGFP obtained in response to a Vj step of −52 mV. The histogram shows peaks for the closed state, substate (γs = 11.2 ± 2.3 pS), and open state (γo = 49 ± 5.6 pS). The arrowhead indicates a slow transition from the closed state to open state, and the arrow indicates a fast transition from the open state to the substate. N, Ij trace from a HeLa Cx47-EGFP cell pair in response to 1.5-s-long Vj ramps from 90 to −90 mV using [Mg2+]p = 5 mm after 32, 37, and 42 min of recording.
Figure 4.
Figure 4.
Intracellular K2ATP and MgATP have opposite effects on gj and Vj-gating in HeLa cells expressing Cx36-EGFP GJ channels. A, C, Ij's recorded during repeated 35-s-long Vj ramps from 0 to −100 mV. Brief voltage steps of −10 mV were used to measure gj in between Vj ramps. Pipette solutions contained 10 mm K2ATP (A) or MgATP (C). B, D, First (purple) and last (cyan) gj–Vj relations (normalized to initial gj value at Vj = 0) measurements obtained from experiments shown in A and C, respectively. E, F, First (purple) and last (cyan) gj–Vj relations (normalized to initial gj at Vj = 0) obtained using the same Vj protocol as in A. Pipette solutions contained 1.5 mm EDTA (E) or 5 mm MgCl2 (F). In all gj–Vj plots, we assumed that the gj–Vj relation for Vj > 0 was the mirror image of that for Vj < 0 (i.e., symmetrical around Vj = 0) and the last gj–Vj plots normalized to gj at Vj = 0 (gray) are also shown for comparison. G, Changes in normalized gj for the different compositions of the pipette solutions shown at the bottom. Values were obtained from the ratios of the steady-state final gj to the initial gj. Numbers of independent experiments are shown in the histogram bars; *p < 0.05; ***p < 0.001; ns, nonsignificant p values.
Figure 5.
Figure 5.
Permeation of Cx36 or Cx47 GJ channels by Mg2+ ions. A, B, Mag-fluo-4 (MF4) fluorescence intensity measured in cell-1 (FI1) of HeLa Cx36-EGFP (A) and HeLa Cx47-EGFP (B) cell pairs increased after opening the patch in cell-1 (arrow). After FI1 reached a plateau (normalized to this value), pipette-2 was opened (arrowhead) and FI1 again increased. Pipette-1 contained 50 μm MF4 and zero MgCl2, and pipette-2 contained 10 mm MgCl2 (top diagram). The gj measurements were started after patch opening in cell-2 (top). FI1 and gj decreased after bath application of the GJ blocker octanol (1 mm); decrease in FI1 is ascribable to loss of Mg2+ into pipette-1. C, D, Rates of FI1 changes in HeLa Cx36-EGFP (gray; n = 12) and HeLa Cx47-EGFP (black; n = 15) cell pairs measured after patch opening in cell-2 and plotted over gj (C) or the calculated number of open channels (D). Gray and black lines are linear regressions for Cx36-EGFP (R2 = 0.71) and Cx47-EGFP (R2 = 0.9) data, respectively.
Figure 6.
Figure 6.
Transjunctional asymmetry of [Mg2+]i causes asymmetric Vj gating of homotypic Cx36-EGFP GJs. A, D, Transjunctional asymmetry in [Mg2+]i (see diagrams at the top of B and E for free Mg2+ concentration in pipette solutions and stimulation site) caused asymmetry in Vj-gating with decrease in gj for relative negativity on the low [Mg2+] side. Vj steps (±80 mV) of opposite polarities produced opposite effect on gj. Small-amplitude repeated Vj ramps (±20 mV, same as in Fig. 1A) were used to measured gj between Vj steps. B, E, gj–Vj relations (normalized to gj value at Vj = 0) measured by applying long (60 s) Vj ramps from 0 to +100 and −100 mV. Relative positivity on the high [Mg2+] side decreased gj. C, F, Asymmetric concentration of MgATP (C, top diagram) or K2ATP (F, top diagram) was associated with asymmetry of gj–Vj dependence (normalized to gj value at Vj = 0).
Figure 7.
Figure 7.
Fast reversal of asymmetric gj–Vj dependence by reversal of transjunctional gradient of [Mg2+]i. A, Changes in Ij during consecutive 35-s-long Vj ramps from 0 to −100 mV and from 0 to 100 mV. Initially, cell-1 was loaded with a control/standard pipette solution (MgCl2, 1 mm) and cell-2 contained MgATP (Aa). From ∼9 to 14 min after onset, pipette-2 was carefully detached and replaced with a pipette containing K2ATP (Ab) reversing the Mg2+ gradient. From ∼25 to 30 min after onset, pipette-2 was replaced with a pipette containing MgATP (Ac) reversing the Mg2+ gradient once again. B, gj–Vj plots (normalized to initial gj value at Vj = 0) from ramp pairs in (A) designated with numbers 1 and 2 (Ba), 3 and 4 (Bb), and 5 and 6 (Bc).
Figure 8.
Figure 8.
Recovery of gj after decrease induced by Vj-gating or chemical-gating depends on [Mg2+]i. A, Bottom, Dynamics of gj recovery (normalized to initial gj value) after Vj steps (−80 mV; top) at [Mg2+]p = 5 mm. Small-amplitude repeated Vj ramps (±21 mV, 1.3 s; inset) were used to measure gj before and after Vj steps. Dashed line represents averaged decay in gj in the absence of Vj steps using pipette solutions with [Mg2+]p = 5 mm. B, Dynamics of gj recovery normalized to steady-state gj value (gj,ss) before decanol (0.5 mm) application to induce uncoupling at [Mg2+]p = 0.01 mm (Ba), 1 mm (Bb), and 5 mm (Bc). Small-amplitude repeated Vj ramps (same as in A) were used to measure gj. Gray dashed lines show levels of gj recovery during washout from decanol.
Figure 9.
Figure 9.
Divalent cations decrease gj of Cx36 GJs. A, Dynamics of gj (normalized to initial gj value) changes for different divalent cations. B, Average gj (normalized to initial gj values) from experiments using pipette solution containing the following: nominally zero divalents (n = 5); 2 mm free Mg2+ (n = 3), Ca2+ (n = 5), Ba2+ (n = 5), Mn2+ (n = 6), Cd2+ (n = 4), Zn2+ (n = 3); 0.2 mm free Zn2+ (n = 4). C, Average time for gj to undergo 95% of the change from initial to virtual steady-state conductance after beginning dual whole-cell voltage clamp.
Figure 10.
Figure 10.
Mg2+-dependent modulation of Gj in pairs of MesV neurons. A, IR-DIC image of a pair of electrically coupled MesV neurons during dual whole-cell patch clamp. B, Simultaneous current-clamp recordings from a pair of electrically coupled MesV neurons; arrows indicate the direction of the spread of electrotonic potential. Voltage traces were recorded from cell-1 and cell-2 (V1 and V2, respectively) during 300 ms hyperpolarizing current steps of −300 pA injected either in cell-1 or in cell-2 (I1 and I2, respectively). C, Time course of changes in mean Gj (normalized to initial values) at [Mg2+]p = 0.01 (gray) and 5 mm (black). Each point represents an average from five independent experiments. D, Mean percentage changes of Gj from initial values after 12 min of patch openings with [Mg2+]p = 0.01 (gray) and 5 mm (black). Numbers of cell pairs are indicated within columns; *p < 0.05.
Figure 11.
Figure 11.
Diagram illustrating relation between ATP/ADP ratio and [Mg2+]i and the effect on Cx36 GJ channels. During sleep and reduced neuronal activity, ATP/ADP ratio is increased by mitochondrial metabolism of glucose and lactate coming from capillaries and surrounding glia, respectively. During wake period and enhanced neuronal activity, ATP/ADP ratio is decreased. Changes in ATP have a direct effect on [Mg2+]i, leading to changes in Cx36-dependent GJIC. Pathological conditions, such as hypoxia, ischemia, and seizures may result in decreased GJIC, while traumatic brain injury may lead to increased GJIC. GLUT, glucose transporter; MCT, monocarboxylate transporter.
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