Molecular determinants of membrane potential dependence in vertebrate gap junction channels

Results

Localization of Molecular Determinants for V_m Gating in the Cx43 Molecule.

Rat connexin 43 junctions exhibit combined membrane potential (V _m) and transjunctional voltage (V _j) dependence of junctional conductance, g _j (9, 18). The V _m dependence of g _j was explored independently of V _j dependence by applying equal displacements of the membrane potential in two cells of a coupled pair. With V _j = 0, increasing depolarizing steps applied to both cells induced progressive reduction in g _j. On returning to the holding potential of −100 mV, g _j recovered its initial value (Fig. 1 A WT). The steady state G _j/V _m curve was well described by the square of a Boltzmann relation (Fig. 1 C, Table 1), a calculation based on the assumption that there are two identical but independent V _m gates in series, one per hemichannel. Over the range of membrane potentials explored, G _jss changed ≈80% of the difference between the extrapolated values of G _jmax and G _jmin. Decay and recovery of g _j in response to application of V _m were monotonic and approximately exponential, with time constants of several seconds (Fig. 1B). We did not attempt to fit the time course data with a squared Boltzmann relation.

It has been previously reported that the g _j transitions induced by V _j in RCx43 junctions have fast and slow components, the fast component being larger at larger V _j (≈80% at V _j = −100 mV). Truncation of the CT domain by the mutation S257stop alters the transitions; there is a single component of decay in g _j with a time constant somewhat shorter than that of the slow component of WT junctions (18). V _m dependence of G _jss of S257stop–S257stop junctions was virtually identical to that of the WT channels (Fig. 1 A S257stop and 1C; Table 1), indicating that residues 257–382 of the CT domain do not participate in V _m regulation. However, shortening of the CT to the minimum length that still retains channel-forming ability (23) by the mutation G242stop almost completely abolished g _j dependence on V _m (Fig. 1 A G242stop) whereas V _j gating did not change significantly. The conductance of hybrid junctions comprised of G242stop hemichannels on one side and WT hemichannels in the other cell was V _m sensitive, but the sensitivity of G _jss was intermediate between those of the respective homotypic junctions (Fig. 1 C). The G _jss/V _m relation for G242stop-WT junctions was well fit by a single Boltzmann function with parameters close to those used in the squared Boltzmann relation to describe the behavior of homotypic WT junctions (Table 1). This result suggests that the V _m dependence of the hybrid junctions was due to the single V _m gate contributed by the WT hemichannels, a conclusion previously reported for other hybrid junctions (10, 14). Taken together, these results strongly suggest that the CT region between 242–257 contains residues required for V _m dependence of RCx43 junctions.

Interactions Between V_m Gating and V_j Gating.

Because truncation at residue 257 affects V _j sensitivity (18), and truncation at nearby residue 242 blocks V _m sensitivity with no further change in V _j sensitivity, we investigated whether V _j and V _m gating interact. At V _m values of −80 and 0 mV at which G _jss differed almost 2-fold, V _j gating was virtually the same (Fig. 3 A and B). The kinetics was not distinguishably different, and the steady state G _jss/V _j relations, normalized to the different values of g _j at V _j = 0, also were little affected. These data indicate that V _j gating operates independently of V _m gating. Previously, mouse Cx45 junctions were shown to have very similar V _j gating at different V _m values that changed g _j on depolarization by a factor of 2.5 (10).

We also studied V _m gating in the presence of a fixed, non-zero V _j. Cx43 junctions are particularly suitable for this kind of analysis because the residual conductance or V _j-insensitive component is large enough to allow estimation of V _m effects at large V _j's. For the example of Fig. 3, we defined V _m* = (V ₁ + V ₂)/2 and examined V _m* dependence for V _j = +80 mV from V _m* = −100 mV to 0 mV. A V _j of +80 mV at V _m* = −100 mV, i.e., V ₁ = −60 mV and V ₂ = −140 mV, decreased conductance to a value very close to the V _j-insensitive component (Fig. 3 B). Equal depolarization of both cells induced an additional reduction in g _j, but the sensitivity to V _m was reduced somewhat more than would result from block of a single V _m gate (Figs. 3 C WT and 3D). Thus, the presence of a large V _j appears to interfere with operation of both V _m gates. To explore this interaction further, we carried out the same experiment evaluating the effect of a maintained V _j on V _m dependence in S257stop–S257stop junctions, which lack fast V _j gating (18). Furthermore, G _jmin was markedly smaller for these mutant junctions than for wild type, 0.16 vs. 0.36 respectively for V _j < 0 and 0.13 vs. 0.24 for V _j > 0. The G _jss/V _m relation was little affected by this mutation (Figs. 1 A S257stop and 1C; Fig. 3 C S257stop left). Moreover, when g _j was decreased by a V _j of +80 mV, the fractional change in g _j was the same as in the absence of V _j gradient (Figs. 3 C S257stop right and 3D). Thus, truncation at 257, which abolished the fast V _j gating, restored V _m gating in the presence of V _j.

Discussion

Structure–Function Analysis of V_m Dependence of Cx43 Channels.

We identified two charged residues, Arg-242 and Asp-245, that are likely to be an integral part of the V _m sensor and that are located in the (cytoplasmic) CT domain close to its junction with M4, the fourth transmembrane domain. Mutations of these residues that altered V _m gating did not have any influence on V _j gating, and the S257stop truncation that greatly modified V _j gating did not affect V _m sensitivity. Taken together along with the difference in residual conductance, G _jmin, the results indicate that V _m and V _j regulation of Cx43 junctional conductance is mediated by separate but interacting domains.

Truncation by the mutation G242stop greatly reduced V _m sensitivity whereas truncation by S257stop had little effect on this property. Only two of the residues between positions 242 and 257 are charged, Arg-243 and Asp-245. When the acidic residue was replaced by Gln, a neutral amino acid, the G _jss/V _m relation was increased in steepness and V ₀ was shifted in the negative direction, suggesting that D245 is part of the V _m sensor. When the basic residue was replaced by Gln (R243Q), the G _jss/V _m relation was decreased in steepness and V ₀ was shifted in the positive direction, suggesting that R243 is also part of the V _m sensor and that it moves in the same direction as D245 during V _m gating. However, the effects of the two mutations did not summate, and the double mutation, which is electrically neutral, virtually abolished V _m dependence. Apparently the double mutation caused structural changes that prevented V _m gating. Although the anatomical symmetry of gap junctions suggests that there are two V _m gates in series, a single Boltzmann relation fit the G _jss/V _m relation about as well as the squared Boltzmann relation (with different parameters). However, the heterotypic junctions WT-G242stop would be expected to have only one V _m sensitive gate, and the single Boltzmann with the same parameters as for the squared Boltzmann applied to WT junctions gave an excellent fit. This observation is strong evidence for series V _m gates and applicability of the squared Boltzmann to WT junctions.

A Novel Gating Model for Connexin Channels with Combined V_j and V_m Dependence.

A model for the actions of V _j and V _m on Cx43 channels can be derived from data obtained in this paper and a previous analysis of the V _j dependence (18). We hypothesize that each hemichannel contains a set of three gates, one fast gate and one slow gate under V _j control and one slow gate regulated by V _m (Fig. 3 D). The three gates appear to involve different regions of the connexin molecule, because they can be modified independently by different mutations and their residual conductances are different. The distal part of CT domain is involved in the fast V _j gating process, because deletion of the 257–382 region modified the time course of g _j transitions from a two-component decay to a single-component with a slow time constant. Block of the fast component by the 257 truncation and the asymmetry of the G _jss/V _j relation in heterotypic WT-S257stop junctions suggest that there are two V _j gating mechanisms within the hemichannel, each operating with the same negative polarity of closing, defined as relative negativity in cytoplasmic side (18). The distal end of the CT may move to partially occlude the channel during fast V _j gating as has been proposed for pH gating (24). Interestingly, attachment of aequorin (25) or of enhanced green fluorescent protein (EGFP; ref. 26) to the CT of a full-length Cx43 blocks fast V _j gating. The mutations Arg-243 and Asp-245 affecting V _m gating also are located in the CT but more proximal to the region required for fast V _j gating. In the absence of V _j gradients, Cx43 conductance is exclusively dependent on V _m, which induces closure by membrane potential depolarization. When the CT domain was truncated at position 242, fast V _j and V _m gating were blocked, but the slow V _j gating seen in the S257stop mutant was little affected. In Cx43 junctions, the sensor for the slow mechanism of V _j gating may correspond to those initially proposed for Cx32 and Cx26 junctions, where the charged amino acids located in the NT domain and at the beginning of the first extracellular loop determine the polarity of closing of the channels (15, 17).

In this model, G _j should be governed by the combined action of three gating mechanisms according to their kinetic properties and their polarity of closing. Moreover, the effects on conductance will be also determined by the nature of interactions between the gating mechanisms, which will depend critically on their structural basis and where they are located with respect to each other and to the channel lumen. Interactions could be allosteric or could result from changes in the local electric field by the gating mechanisms. The fast and slow V _j gates may interact electrically. At large V _j, the fast V _j component dominates the conductance change of WT junctions, and G _jmin was larger than in S257stop junctions. This result suggests that the fast gate has a larger residual conductance and prevents closure of the slow gate. That the time constants for the 257 truncation mutant are somewhat shorter than for the slow component in the WT may result from a larger voltage across the sensor in the absence of the fast gating mechanism. Operating in a different time regime, a large value of V _j that closes most V _j gates modifies V _m gating, and it appears that the V _m gate does not operate when the V _j gate is closed (Fig. 3 C and D). (See supplemental material for further discussion of V _j–V _m interactions.)

Single channel analysis of the gating mechanisms should reveal the classes of gating events, including their probabilities, residual conductances, and intervening states. V _j induces fast transitions between fully or partially open states and residual states of low conductance (27). In addition, slow transitions to a fully closed state have been recorded at larger V _j in WT junctions (28). These two kinds of transition presumably underlie the two types of V _j sensitivity considered here. In Cx43-EGFP junctions, which lack fast V _j gating, fast transitions to a residual open state are not present, and channels close fully by slow transitions (26). The events in V _m gating have not yet been observed at the single channel level, but the ability of V _m to further reduce the V _j-insensitive component of macroscopic conductance is an indication that V _m gating mediates transitions between open and fully closed states of channels. Full closure of the channels is also suggested by the fact that the V _j gating properties of Cx43 (Fig. 3 A and B) do not change significantly at different membrane potentials although the V _m induces large variations in macroscopic junctional conductance. If V _m gates closed to a subconductance state, one would be expected that the voltage across the V _j gates would change affecting kinetics and steady state values.

The model presented here may be applicable in whole or in part to other V _m- and V _j-sensitive gap junctions of vertebrates (11, 14). The dependence on V _m in some vertebrate junctions is sufficiently great that intercellular coupling could be directly regulated by the membrane potential, a parameter that is always present and that varies, particularly in excitable cells. Dynamic changes of coupling coefficient in phase with membrane potential oscillations occur in pancreatic β-cells (29), which may indicate that the conductance of those junctions is V _m dependent. V _j gating would regulate coupling when membrane potentials differed, as in asynchronously active excitable cells.