Opening hemichannels in nonjunctional membrane stimulates gap junction formation

Abstract

We studied gap junction formation in pairs of Xenopus laevis oocytes expressing connexins that form functional hemichannels and found no correlation between junctional conductance (G(j)) and whole-cell hemichannel conductances (G(hemi)) within the first few hours of pairing. However, opening hemichannels to a threshold current stimulated a rapid G(j) increase. Moreover, cx46 hemichannel current stimulated cx40 G(j) even though cx40 and cx46 do not form heteromeric or heterotypic gap junctions. Initial growth rate and final steady-state level of stimulated G(j) were proportional to the product of hemichannel conductances. External calcium affected the growth rate of stimulated G(j) but not the final steady-state value. Time constants of formation were short in low [Ca(2+)](out) (3 min in 200 micro M Ca(2+)) and long in high [Ca(2+)](out) (15 min in 1 mM Ca(2+)), but in oocyte pairs pretreated with lectins to reduce steric hindrance imposed by large membrane glycoproteins the time constant was short and Ca(2+)-independent. We suggest that hemichannel activity stimulates G(j) by collapsing the extracellular volume between membranes to allow the end-to-end binding between hemichannels. These studies suggest the possibility that functional hemichannels could trigger or enhance junctional formation in vivo in response to appropriate stimuli.

FIGURE 1

Opening cx46 hemichannels stimulates gap junction formation. (A) The time course of gap junctional conductance between cx46-expressing oocytes clamped to −30 mV. Each point represents the junctional conductance (G_j) determined by applying a 20-mV hyperpolarizing voltage step to one oocyte and dividing the junctional current recorded in the nonstepped oocyte by the transjunctional potential. The vertical dotted arrow indicates when the external medium was changed from ND96 supplemented with 1 mM CoCl₂ to 1 mM CaCl₂ ND96. The small arrows (a and b) show when the whole-cell current traces shown below in panels a and b were taken. The hemichannel currents were excited by a 10-s depolarizing pulse to +20 mV. (B) The effects of [Ca²⁺]_out and membrane potential on stimulating gap junction formation are mediated specifically through hemichannels, not through changes in voltage or calcium concentration alone. The time courses of junctional and hemichannel conductance were obtained by first stepping both oocytes simultaneously to −10 mV to measure individual hemichannel conductances and then by stepping one oocyte −20 mV relative to the other oocyte to determine junctional conductance. Junctional conductance is shown by solid symbols and hemichannel conductance by open symbols, a convention followed throughout this paper. Changes in holding potentials and external calcium concentrations are shown on the graph. Oocyte pairs were initially clamped to −60 mV to prevent hemichannel activation during decreases in [Ca²⁺]_out. The holding potential was then elevated in 10- to 20-mV increments until a change in whole-cell conductance was elicited. After a change in junctional current was observed, the holding potential was returned to more negative potentials to close hemichannels during the course of junction development.

FIGURE 1

Opening cx46 hemichannels stimulates gap junction formation. (A) The time course of gap junctional conductance between cx46-expressing oocytes clamped to −30 mV. Each point represents the junctional conductance (G_j) determined by applying a 20-mV hyperpolarizing voltage step to one oocyte and dividing the junctional current recorded in the nonstepped oocyte by the transjunctional potential. The vertical dotted arrow indicates when the external medium was changed from ND96 supplemented with 1 mM CoCl₂ to 1 mM CaCl₂ ND96. The small arrows (a and b) show when the whole-cell current traces shown below in panels a and b were taken. The hemichannel currents were excited by a 10-s depolarizing pulse to +20 mV. (B) The effects of [Ca²⁺]_out and membrane potential on stimulating gap junction formation are mediated specifically through hemichannels, not through changes in voltage or calcium concentration alone. The time courses of junctional and hemichannel conductance were obtained by first stepping both oocytes simultaneously to −10 mV to measure individual hemichannel conductances and then by stepping one oocyte −20 mV relative to the other oocyte to determine junctional conductance. Junctional conductance is shown by solid symbols and hemichannel conductance by open symbols, a convention followed throughout this paper. Changes in holding potentials and external calcium concentrations are shown on the graph. Oocyte pairs were initially clamped to −60 mV to prevent hemichannel activation during decreases in [Ca²⁺]_out. The holding potential was then elevated in 10- to 20-mV increments until a change in whole-cell conductance was elicited. After a change in junctional current was observed, the holding potential was returned to more negative potentials to close hemichannels during the course of junction development.

FIGURE 2

The magnitude of stimulated G_j is proportional to the product of hemichannel expression levels. (A) The change in steady-state junctional conductance stimulated by hemichannel activation was plotted against the product of normalized whole-cell hemichannel conductances for each oocyte pair. To obtain a quantitative measure of expression levels, hemichannel currents elicited in 0.5 mM [Ca²⁺]_out by a 12- to 20-s voltage pulse to +30 mV were fit to a single or double exponential and the extrapolated steady-state current was divided by the driving force for hemichannels using −10 mV as the reversal potential. Data was collected from 15 different batches of oocytes. Gap junction formation was stimulated by a variety of protocols described in the text. Pairing conditions and [Ca²⁺]_out varied between experiments. All data is from vegetal-vegetal pairs where the stimulated growth in junctional conductance reached a stable steady-state level. (B) A frequency histogram of the relationship between stimulated G_j and the product of normalized whole-cell hemichannel conductances. The low conductance peak is from oocytes with low contact area and the high conductance peak is from oocytes with large contact areas, as shown schematically by drawings of oocyte pairs shown above the peaks. A Gaussian fit of the data (solid line) was used to determine the centers of the peaks.

FIGURE 3

Initial rates of G_j increase are proportional to the G_hemi product. (A) The initial rate of stimulated G_j was plotted against the product of normalized whole-cell hemichannel conductances for the oocyte pairs shown in Fig. 2. Here, only oocyte pairs where G_j was stimulated in 0–0.2 mM [Ca²⁺]_out were used. (B) A frequency histogram of the relationship between the initial rate of stimulated G_j and the product of normalized whole-cell hemichannel conductances. The low conductance peak is from oocytes with low contact area and the high conductance peak is from oocytes with large contact areas. Gaussian fits (solid line) were used to determine the centers of the peaks.

FIGURE 4

Steady-state G_j is not due to balance of formation and degradation rates. Depleting available gap junction precursors does not alter the steady-state junctional conductance for 3–4 h. Two groups of oocytes were injected with either 2.3 or 23.0 ng of cx50 cRNA and incubated in ND96-S + 1 mM CoCl₂ at 17°C for 2 days before voltage-clamp experiments. Single oocytes from each cRNA group were treated with the histidine-modifying reagent DEPC. DEPC treatment consisted of washing oocytes for 3–5 min in pH 6.0 ND96 followed by a 1-min wash in a freshly prepared solution consisting of 3.3 μL DEPC dissolved in 50 mL of pH 6.0 ND96, and then washed again in pH 6.0 ND96 for 3–5 min before being returned to ND96 + 1 mM CoCl₂. DEPC-treated and nontreated oocytes from each cRNA group were assayed for hemichannel expression by exposure to 0.1 mM CaCl₂ ND96, pH 7.6. Nine oocyte pairs were prepared from either DEPC-treated or nontreated oocytes for each cRNA group and incubated in ND96. Oocytes, 4–6 h after pairing, were voltage clamped to −40 mV and G_j was assayed by imposing 10-s transjunctional potentials ranging from 10 to 80 mV in 10-mV increments. Data is presented as the mean ± SE from n number of experiments. Pretreating cx50-expressing oocytes with DEPC completely eliminated hemichannel current and prevented the formation of functional gap junction channels, suggesting that functional hemichannels are the gap junction channel precursors. In experiments from a different batch of oocytes, G_j was stimulated in three different cx50-expressing oocyte pairs by perfusing the oocyte pair with 0-added CaCl₂ ND96, pH 8.0, to open cx50 hemichannels. The prestimulated and poststimulated G_j was assayed at 1-min intervals by imposing a 1-s, 20-mV transjunctional potential. After the stimulated G_j reached a stable level, DEPC was applied as described above and G_j was followed for an additional 20–30 min before removing the oocyte pair from the recording setup. G_j was reexamined in the same oocyte pairs 3-4 h after the initial time of DEPC treatment. The initial and steady-state G_j, and the G_j recorded at various times after DEPC treatment are shown for each of the three oocyte pairs. DEPC treatment did not alter G_j during the first 20- to 30-min recording period. The same DEPC solution was effective in eliminating hemichannel currents. G_j remained stable for up to 4 h even after rapidly depleting the pool of functional precursors (hemichannels), suggesting that the stable coupling level is not the result of an equilibrium reaction between the formation and degradation rates of gap junction channels.

FIGURE 5

Gap junction formation is stimulated by opening cx38 or cx50 hemichannels. The responses of junctional and nonjunctional conductances to changes in [Ca²⁺]_out and/or holding potential are shown for noninjected oocyte pairs (A) and cx38 antisense-treated oocytes injected with cx50 cRNA (B). Two to three oocyte pairs were examined in four different oocyte batches. Cx38 gap junction formation could be stimulated in only two of the batches. The ability to stimulate cx38 coupling was correlated with the presence of an increased whole-cell conductance generated in 0.2 mM [Ca²⁺]_out after elevating the holding potential to −20 mV. The average stimulated cx38 G_j in two different batches was 4.2 ± 1.1 μS and 8.4 ± 3.1 μS. In four different oocyte batches, gap junction formation was successfully stimulated in all oocyte pairs expressing cx50 hemichannels.

FIGURE 6

Opening cx46 hemichannels stimulates cx40 homotypic gap junction formation (A–C). Changes in junctional and nonjunctional conductances were recorded in different types of oocyte pairs subjected to conditions that open cx46 hemichannels, as described in Fig. 4 A. Cx38-antisense treated oocytes were injected with 0.1 ng cx46 cRNA, 0.4 ng cx40 cRNA, or a mix containing 0.1 ng cx46 and 0.4 ng cx40 cRNA. Oocytes, 1–2 days after cRNA injections, were paired in the following configurations: cx40/cx40, cx40/cx46, (cx40 + cx46)/cx40, (cx40 + cx46)/cx46. G_j was stimulated in (cx40 + cx46)/cx40 pairs (A) and (cx40 + cx46)/cx46 pairs (not shown) after activating cx46 hemichannel current in one or both oocytes. G_j was not stimulated in cx40/cx46 (B) or cx40/cx40 (C) pairs subjected to the same voltage and calcium conditions. However, cx40/cx40 pairs showed large coupling levels 18–24 h after pairing, whereas cx40/cx46 pairs developed no detectable coupling within 30 h of pairing. (D) Steady-state junctional conductances normalized to their values at ±10 mV and plotted against the transjunctional potential. The averaged G_j-V_j data for oocyte pairs of the same configuration were fit to a Boltzmann equation (smooth lines). The Boltzmann fit parameters were V_o = 33.4 mV, A = 0.28, G_min = 0.25 for five cx40/cx40 pairs, V_o = 28.4 mV, A = 0.30, G_min = 0.25 for four (cx40 + cx46)/cx40 pairs, and V_o = 67 mV, A = 0.09, G_min = 0.01 for two (cx40 + cx46)/cx46 pairs. A Boltzmann fit of cx46/cx46 G_j-V_j data obtained from other experiments is included for comparison.

FIGURE 7

Gap junction formation is stimulated by a threshold level of hemichannel current that is independent of hemichannel expression levels. The left-hand panels show the time courses of gap junctional conductance for two oocyte pairs prepared from the same oocyte batch. Each time point represents G_j measured as described in Fig. 1. [Ca²⁺]_out is shown in the bar just above the time scale. Lettered arrows indicate when cx46 hemichannels were simultaneously activated in both oocytes. The right-hand panels (labeled a, b, and c) show whole-cell nonjunctional current traces with labels corresponding to the lettered arrows in the G_j time course shown in the left-hand panel. Hemichannel currents were elicited by simultaneously depolarizing both oocytes to +20 mV from a holding potential of −40 mV. Incrementally larger hemichannel currents were achieved by increasing the duration of the depolarizing pulse (A) or by applying the same voltage pulse after decreasing [Ca²⁺]_out (B). Despite the large difference in expression levels (assessed by comparing hemichannel currents in 1 mM [Ca²⁺]_out), similar currents were required to initiate gap junction formation in each oocyte pair. The extent of G_j stimulated by the threshold currents was roughly proportional to expression level.

FIGURE 8

Lowering [Ca²⁺]_out stimulates gap junction formation only if the steady-state hemichannel current increases beyond a threshold level. Lowering [Ca²⁺]_out stimulates gap junction formation only when whole-cell hemichannel current increases beyond a threshold level. Paired oocytes were voltage clamped to −30 mV in ND96 supplemented with 1 mM CoCl₂. G_j was assayed periodically as described in Fig. 1. [Ca²⁺]_out was lowered every 15–20 min by perfusion with ND96 containing 1 mM, 0.5 mM, and 0.1 mM until G_j increased. The data from each oocyte pair were grouped by calcium level resulting in gap junction formation. Data in each group were averaged and are shown as mean and standard deviation for two to three pairs. Cx46 hemichannel expression level is expressed as maximum whole-cell hemichannel conductance measured in 0.5 mM external calcium (solid columns). The extent of stimulated increase in G_j is shown in the thatched columns. The cx46 hemichannel current that coincided with the initial increase in junctional conductance was measured as the difference between measured holding current and the holding current in the presence of 1 mM CoCl₂ (shaded columns).

FIGURE 9

Synchronous stimulation is more effective than sequential stimulation Activating hemichannels simultaneously in both oocytes is more effective in stimulating gap junction formation than activating hemichannels sequentially in each oocyte. Cx46-expressing oocyte pairs were voltage clamped to −30 mV in a dual two-electrode voltage clamp configuration. Junctional and nonjunctional conductances were assayed periodically as described in Fig. 1. Hemichannels were activated by applying a 9-s depolarizing pulse to +30 mV to one of the oocytes, then to the other oocyte, and then to both oocytes simultaneously, allowing 2–5 min between pulse protocols to determine if the growth rate of G_j was affected (bottom panel). Note that if hemichannels are activated in only one oocyte, a 70-mV transjunctional potential occurs and the presence of junctional coupling can be seen as current recorded in the nonstepped oocyte. In five out of six oocyte pairs from two different batches of oocytes, gap junction formation was stimulated only when hemichannels were activated in both oocytes simultaneously. Even repeated activation of hemichannels in a single oocyte sequentially failed to stimulate gap junction formation.

FIGURE 10

Increasing cytosolic [Ca²⁺] does not stimulate G_j growth. (Top) The time courses of junctional and nonjunctional conductances were recorded in oocyte pairs as described in Fig. 1. Oocyte pairs were perfused with 2 mM [Ca²⁺]_out ND96 containing 2 mM A23187 calcium ionophore (green arrow). A positive indicator that cytosolic calcium increased is the difference between the hemichannel currents activated before (bottom, a) and after (bottom, b) the addition of ionophore (green arrow). After ionophore addition, there is an increase in whole-cell conductance and a second transient component to whole-cell current traces elicited by depolarizing pulses (most evident in tail currents). This new current is very likely due to the well-known Ca²⁺-activated chloride channel, an endogenous channel expressed abundantly in most batches of oocytes (Carpenter, 1987). The activation of this current confirms the entry of a significant amount of calcium. Note that activation of hemichannels does not produce sufficient calcium entry to activate the Ca²⁺-activated chloride current in the absence of ionophore. But even when calcium entry mediated by ionophores could not stimulate G_j, opening cx46 hemichannels by lowering [Ca²⁺]_out and elevating the holding potential could.

FIGURE 11

Pairing orientation affects both threshold current and the magnitude of stimulated cx46 G_j. Oocytes were paired in four orientations to produce different contact types: vegetal-vegetal poles (V-V), vegetal pole-equatorial region (V-E), vegetal-animal poles (V-A), and animal-animal poles (A-A). An increasing fraction of total hemichannels was opened by depolarizing pulses applied in decreasing [Ca²⁺]_out until a change in the growth rate of G_j was detected. G_j was then followed over time until a new steady-state condition was reached. The whole-cell hemichannel conductance, extrapolated if necessary to 0.5 mM Ca²⁺, was used to quantify expression levels. The hemichannel expression levels and stimulated change in G_j,ss were grouped according to pairing orientation. The [Ca²⁺]_out in which gap junction formation was stimulated is presented as a measure of I_Thresh. Not only did the pairing orientation affect the stimulated increase in G_j, but it also determined the fraction of hemichannels that must be opened to initiate gap junction formation.

FIGURE 12

The time constant of stimulated gap junction formation is dependent on [Ca²⁺]_out. (Top panel) Examples of G_j growth curves stimulated under different conditions. Oocyte pairs expressing similar hemichannel levels are shown to demonstrate that the extent (as opposed to the rate) of gap junction formation was not significantly altered by [Ca²⁺]_out. Gap junction formation was stimulated in 0.2 mM [Ca²⁺]_out, 1.0 mM [Ca²⁺]_out, and 1.0 mM [Ca²⁺]_out for oocyte pairs pretreated with 20 μg/ml soybean agglutin before pairing. (Bottom panel) The time constants obtained after fitting G_j growth curves to a single exponential process. If the stimulated change in junctional conductance could be modeled by a single exponential process, data were fit to the equation G_j(t) = G_j,o + G_j,max × (1 − exp(t/τ)). Time constants were binned according to the [Ca²⁺]_out present during gap junction formation. Different symbols are used to distinguish between different expression levels to show the inverse relationship between the time constant and the total expression level seen at higher [Ca²⁺]_out.