seminars in CELL BIOLOGY, Vol 3, 1992: pp 29-47

Biophysics of gap junctions M. V.L. Bennett and V.K. Verselis Gapjunction channels, now known to beformed of connexins, connect the interiors ofapposed cells. These channels can be opened and closed by various physiological stimuli and experimental treatments. They are permeable to ions and neutral molecules up to a size of about 1 kDa or 1.5 nm diameter, includingsecond messengers and metabolites. The processes ofgating and ofpermeation are the subject of this review. Voltage is a readily applied stimulus, and transjunctionalvoltages, orthose between cytoplasm and exterior, affect mostjunctions. Single channel transitions between open and closed states arerapid and presumably involve a charge movement as occurs with channels of electrically excitable channels of nerve and muscle. Identification of gating domains and charges by domain replacement and site-directed mutagenesis is being pursued. Raising cytoplasmic H + or Ca2 + concentrations rapidly reduces junctional conductance, and this action is generally reversible, at least in part. A number of lipophilic alcohols, fatty acids and volatile anesthetics have similar actions. Phosphorylation also modulates junctional conductance, and in several cases, sites of phosphorylation are known. These gatingprocesses appear similarto those induced by voltage. Permeability measurement indicates that the channel is aqueous and that permeation is by dijfusion with only minor interactions with the channel wall. Dijferences amongjunctions are known, but further characterization of connexin and cell specificity is required.

microscopy, and fast freezing has sufficient time resolution to resolve formation or removal. A less easily quantified biophysical parameter of gap junctions than conductance is permeability, although dye fluxes can provide a convenient qualitative measure of gap junctional coupling. The advent of cloned connexins has clarified the biophysical (as well as most other) studies of gap junctions. Connexins are encoded by a gene family, and different members can have different-properties. Comparisons· among connexins, preparation of construct DNAs, and site-directed mutagenesis are permitting structure-function studies. A further area of investigation now illuminated by sequence information is posttranslational modification, which can alter the biophysical properties. A great deal of new information is about to appear in the proceedings of the gap junction meeting in Asilomar, June 1991. 1 Some of the work discussed here is also reviewed in 'Biophysics of Gap Junction Channels'v/ As a brief review, a gap junction is comprised of a group of aqueous channels connecting the cytoplasms of the coupled cells. Each channel is a dodecamer comprised of a hexamer of connexin molecules in each membrane. Each connexin has four membrane spanning regions (M1-M4), cytoplasmic amino and carboxy termini (NT, CT), two extracellular loops (E1-E2) and a cytoplasmic loop (CL). M3 is presumed to line the channel. Among connexins the CT is very divergent, the CL less so;' M1-M4 and E1-E2 are highly conserved. Gap junctions that mediate electrical coupling also permit passage of small molecules that may be involved in chemical signaling as well as metabolic buffering and cooperation.

Key words: connexin / electrotonic coupling / gating / voltage dependence / permeability

THE BIOPHYSICS of gap junctions can be construed to represent two major areas, how channels open and close, or gating, and how molecules get through the channels, or permeation. In principal, gating is a change of' the channel between open and closed conformations, and a defining characteristic is that during gating the channel remains structurally intact. In practice, gating is identified by conductance changes that are fast and reversible, but stability of junctional structure can be confirmed by electron

Measurement of (macroscopic) junctional conductance The equivalent circuit of a pair of isopotential cells coupled by gap junctions is of three conductances, a non-junctional conductance connecting each cell interior to the outside and ajunctional conductance connecting the cell interiors (Figure 1). The value of these conductances can be determined in current

From the Department ofNeuroscience, Albert Einstein College of Medicine, Bronx, NY 10461, USA ©1992 Academic Press Ltd 1043·46821921010029 + 19$5.00/0 29

M. V.L. Bennett and v.K. Verselis

30

Figure 1. Diagram of the equivalent circuit of a pair of coupled isopotential cells.

clamp by applying pulses alternately in the two cells to measure input and transfer conductance; application of the pie-tee transform then yields junctional and non-junctional conductances (gj, gnjl and gnjZ for two cells).3 Voltage clamping the two cells provides a more direct readout ofjunctional conductance. In this method, a voltage step applied in one cell results in a change in total current applied to that cell. Part goes through the non-junctional membrane of that cell and part through the junctions to the second, coupled cell. A current equal and opposite to the junctional current is applied in the second cell to maintain its potential constant. If gj changes while the voltage step is kept constant, the current in the second cell changes proportionally, and the time course of the gj change can be seen directly. The non-junctional conductance of the first cell is obtained from the total current in that cell minus the junctional current. The non-junctional conductance in the second cell is obtained similarly by applying voltage steps in that cell. Thus with AVz=O gj = gnjl =

AIz/AVI

(All + AIz)/AVI

where the subscripts stand for the two cells. Similarly with AVI = 0 gj= gnjZ =

-AII/AVz (AIz + AII)/AVz

Single channel recording from gap junctions Just as single channel recording has clarified the operation of electrically excitable and chemically activated membranes and made concrete .the

description of changes in conductance, so does the recording of single channel activity of gap junctions focus the discussion. One asks whether changes in macroscopic conductance are associated with changes in single channel conductance or in the number of channels that are open. At least under conditions where macroscopic conductance is changing, channel opening and closing are readily observed and rates should be quantifiable. Recording from single gap junction channels is possible using cells with non-junctional membranes of very low conductance. As whole cell patch clamping has shown, many cells have non-junctional conductances in the nanosiemen range and single gap junctions have a unitary conductance (y) readily detectable against that background. A simple procedure for gap junctions is to hold two coupled cells at different potentials; under these conditions opening of a junctional channel is signaled by an increase in current that is equal and opposite in the two cells (Figure 2). A single channel event in nonjunctional membrane of one of the cells is signaled by a current step in that cell alone. Single channel conductances vary over a moderate range and they appear to be connexin specific (Table 1). Cx32 generally appears to form channels with a larger y (120-150 pS) than Cx43 (50-90 pS), but y may be altered by phosphorylation.t y is expected to vary as a function of the composition of the solution in the patch pipette, and this factor should be kept in mind when comparing tissues. With some cell types ATP regenerating systems are necessary to maintain coupling.V' With others they are unnecessary. 7 Incorporation offormed channels and reconstitution of channels from solubilized protein into bilayers have been applied to gap junctions to a limited extent and permit analysis of single channels (Table 1).7-11 Channels of appropriate conductance have been obtained although some larger channels are often evident (Table 1). Permeability to large molecules is implied by the selection procedure employed by Harris. 10 An important aspect for gap junctions is that a three-compartment system is necessary to fully reconstitute the junctional relation.I? Hemichannel properties could be usefully investigated, but it is still unclear whether the observed activity in single bilayers comes from hemichannels or complete ones. If a gap junction channel had a uniform diameter of 1.5 nm over a 10 nm length and were filled with fluid of 100 {2 ern resistivity, its conductance would be about 175 pS. The access conductance for both

31

Biophysics oj gap junctions

11-,.........-~I .............u.IoIVI 12-~;'-'''''''1'''''''''''n'tI,~

I~

I

25

Figure 2. Single channel activity of gap junctions between poorly communicating cells transfected with Cx32. Upward transitions in II are simultaneous with downward transitions in 12 • Cell 1 was 46 m V positive to cell 2 so that 6.9 pA equals alSO pS channel conductance. Junctional conductance had been reduced by halothane treatment. One channel was open at a time for most of the trace, two channels were open for several brief periods (dotted lines). From ref 94. sides (channel diameter divided by medium resistivity, see ref 13) would be 1.5 nS, to give a net conductance of about 160 pS, comparable to the largest values observed. To a first approximation, junctional conductance of a tightly packed circular array of channels increases as the square of diameter while access resistance increases linearly. For a plaque of 160 pS channels about 0.3 /lm in diameter the junctional and access resistances would be equal, suggesting that making much larger junctions would Table 1.

not give much improvement. Lower conductance channels could be assembled into larger plaques without an equal loss of effectiveness.. jongsI!1a et all 4 have made more detailed calculations indicating that access resistance to larger junctions can significantly reduce the voltage across the channels and thereby affect voltage dependence, in agreement with some of their observations on cardiac cells. IS The diffusion coefficient for a hydrated K + ion about 0.3 nm in diameter would be decreased to

Gap junction single channel conductances Unitary conductance

Tissue Rat lacrimal gland Rat neonatal heart Chick embryonic heart Guinea-pig heart Hamster ovarian cells Pancreatic acinar cells Earthworm MGA septum .Astrocytes Horizontal cells Xenopus embryonic muscle cells Rat leptomeningeal cells WB cells Exogenous expression SKHepl transfected cells Cx32 Cx43 Artificial bilayers Isolated rat liver junctional membranes (Cx32, Cx26) Isolated junctional membranes and solubilized Cx32 Solubilized Cx32

110-130 pS SOpS 45pS 60-80, 165 pS 30-40 pS 22-120 pS 27, 130pS lOOpS 50·60 pS SOpS lOOpS 40-90 pS 80-90 ps 150pS 130pS

Connexin type

Refs

Cx32?, Cx26? Cx43 a Cx43 a Cx42, Cx43, Cx45 b Cx43? Cx43? Cx32 c ? Cx43 d ? Xen 43?e Cx43, Cx26 Cx43, 34 kD protein

91 94 15 92,95 96 6 6 97 98 99 100 101 102 103 94a

60pS 60, 90 pS 150pS 150pS 50-150, > 150 pS 130-160 pS 270-310 pS 50, 130 pS

9f 7 11

8 10

"Ref 105; bref 106; cref 107; drefs 98, 108; V2 , the effect of Vj = VI - V2 = SO mV is the same for VI = + 50mV, V2 = OmV and VI = 0, V2 = - SO mV). In this situation, Vjs are the same but Vi-os are not, and Vi-o sensitivity will manifest itself as a difference between the effects of equal depolarizations and hyperpolarizations. In symmetric Vj-dependent junctions equal polarizations on either side of either sign have the same effect (e.g. the effect of Vj = SO mV is the same as that of Vj = - SO mV). Junctions exhibiting Vi-o sensitivity demonstrated by polarizing both cells equally generally have also exhibited a degree of Vj sensitivity (see below). The foregoing discussion has assumed voltage dependence rather than current dependence. The decline in gj with voltage is often steep enough to generate a negative slope in the current voltage relation such that Vj in the negative slope region is a three-valued function of I while I is single valued as a function of Vj.19,20 For this reason, it is reasonable to consider gj a function of Jj rather than Ij. Also, single channel recording reveals all-or-none gating behavior like that of the classical voltage dependent channels.

The history of voltage dependence The first biophysically well analysed gap junctions were those forming the active element of the crayfish

33

Biophysics of gapJunctions

giant synapse.I! These gap junctions rectified such that they passed current more readily from the lateral and medial giant axons to the motor giant axon (orthodromically) than in the opposite direction. In the terms of voltage dependence, the conductance increased for relative positivity on the presynaptic side. Previously, septal synapses had been shown to conduct impulses in both directions, and the septal junctions of the crayfish lateral giant (septate) axon were reported not to exhibit rectification.F! Subsequent studies showed electrical linearity of these junctions over a wide range. 22 Soon thereafter, a large number of electrical synapses were found in the CNS of fishes, and then some were found in mammals as well (cf ref 23). Although it was difficult either to obtain precise measurements or to apply voltages to the junctions over a wide range, their function in synchronization, electrical linearity over a moderate range and anatomical symmetry suggested that they were electrically linear. A few additional rectifying junctions were found 24,25 and some non-linearity was seen in coupling of reticular cells in the Limulus ommatidium.e'' But for nearly 20 years it was presumed that most gap junctions were electrically linear. In 1981 we described in amphibian blastomeres a symmetric form of voltage dependence.Pv? Transjunctional voltage of either sign decreased junctional conductance (Figure 4) and the effect was essentially independent of Vi-o. Jijs of 10 m V or so caused noticeable decreases in gj, and it was still easy to believe that the other junctions were electrically linear, particularly because measurements were made under current clamp conditions when cell coupling tends to reduce Jij. Now that dual voltage clamp is routinely applied to pairs of isopotential cells, larger Jijs are being used and junctions formed of all vertebrate connexins tested so far have been shown to exhibit some degree of voltage dependence. The less sensitive gap junctions require voltages in excess of 60 m V and access resistance may reduce the voltage sufficiently to obscure what dependence there is. H Junctions between segments of the crayfish and earthworm septate axons are still electrically very linear. 22,28

these states is a linear function of voltage, then the equilibrium distribution between these two states will be given by a Boltzmann relation of the form

Pc!Po = exp(A( v- Vo) ) where Pc and Po are closed and open probabilities, V is the applied voltage, Vo is the voltage at which Po = pc, A = nq(kT) -I is a constant expressing gating charge, and n is the equivalent number of unitary positive charges, q, moving through the entire applied voltage. A more useful form of the relation is

Po= 1/(1 +exp(A(V- Vo))) If the closed state conductance is zero, gj = N'YPo where N is the number of channels and

This relation generates a sigmoidal curve with the half maximal value and inflection point at Vo (Figure 4C, inset). The AVo term is proportional to the chemical free energy difference between open and closed states. A larger gating charge results in a steeper slope; a larger Vo simply shifts the gj - V curve to the right. The assumption of constant difference in dipole moment between the two states, irrespective of the applied voltage, is consistent with rigidity of the protein in each of the conformations. For large voltages this assumption is likely to be violated, and 100 m V produces a large voltage gradient across a structure 10-15 nm thick. In most cases of voltage dependence gj does not go to zero, but instead approaches a minimum value, gjrnin, and one writes gj = (gjrnax - gjrnin)/( 1 + exp(A( V-Vo ) ) ) + gjrnin

The nature of gjrnin is unclear. It may represent a population of voltage insensitive channels in parallel, a non-zero conductance of the closed state or a nonzero Po even at large voltages. The latter possibility does not conform to the Boltzmann assumptions, but may not lead to blatant deviations from the Boltzmann sigmoid. Single channel recording should identify the basis of gjrnin; its nature may differ for different junctions.

Quantitation of voltage dependence

Jj sensitivity in homotypic junctions

If a (channel) molecule can exist in two states, say closed and open, and the energy difference between

Coupling between amphibian blastomeres exhibits pronounced Jij sensitivity as noted above (Figure 4;

.M. v.t: Bennett and

34 2.5

900

~=L

f-5

B

•/1.0

,

III Vj Ij

---



l I~ E --J'---:....-:...._ Q~ 500ms

Verselis

goo

c

A

v.x.

\

• 0.5

..,I -40 -20

v

0

l ,.... 20

v

40

(mV)

Figure 4. Voltage sensitivity of gap junctions between pairs of amphibian blastomeres. (A) During a small negative voltage pulse (T'j) applied to one of the voltage-clamped cells, junctional current (~, the current recorded in the cell coupled to the polarized cell) is constant. (B) A larger T'j causes ~ to fall from a large initial value to a level lower than that in (A). (C) The steady-state g/T'j relation for a pair of amphibian blastomeres. Steady-state junctional conductance decreased as a steep function of transjunctional voltage, reaching - 5 % of its maximum value with a V; of ± 30 mV. The conductance changes were equal for equal voltages of opposite polarity. The points are well fit by the Boltzmann relation (smooth curve in inset). From ref 27.

ref27). Conductance is decreased symmetrically for ~ of either sign, and for each polarity is well fit by a Boltzmann relation (the two relations differing in the sign of A and Va). The Va is about ± 15 mV for each polarity of ~ and the effective gating charge is about 6, comparable to that of channels of electrically excitable membrane. However, the conductance changes are relatively slow. They are well fit by exponentials with time constants of several hundred milliseconds. From time constants and steady state values of macroscopic currents one can calculate opening and closing rate constants. The rate constants are also exponential functions of voltage, but the closing rate constant is about twice as sensitive. Access resistance may distort the timecourse because the potential will be non-uniform across a large junctional plaque.tf Some workers refer to closure of gap junctions as inactivation. We would prefer that inactivation be restricted to the closing processes directly following voltage induced opening, or activation. Closing of Na channels by repolarization which is simple reversal of opening is coming to be known as deactivation. This term does not seem any better for voltage dependence of gap junctions than does closure. The ~ dependence of amphibian junctions was sensitive enough as to suggest a physiological role in compartment formation.t? It was possible to show that modest differences in resting potential could uncouple cells. Moreover, coupling could be bistable in that where two coupled cells had different

resting potentials, the cells could exist in either relatively coupled or uncoupled states. This phenomenon arises because the steepness of the g/ ~ relation leads to bistability in which uncoupling (or recoupling) are regenerative. Consider two coupled blastomeres. Application of a current to one cell increases its voltage and ~, thus decreasing gj, which causes a further increase in ~, etc. If the cells have different resting potentials over a particular range, the cells can exist in two stable states, coupled or uncoupled. Of course, if the resting potential difference between cells is great enough, the cells will be stably uncoupled with the junctions between them at low conductance. No data has yet been found confirming the existence. of voltage determined compartmentalization in embryos, although it remains an attractive possibility. Although Xenopus oocytes endogenously express mRNA for both Xenopus Cx38 and Cx43,29 oligonucleotides antisense to the Cx43 injected into oocytes have no effect on coupling between oocytes mediated by endogenous message.V' Cx43 may be involved in communication between oocytes and follicular cells, which also express Cx43 mRNA. Voltage dependence of junctions formed by Cx38 in oocyteshas different parameters from that of junctions between blastomeres. 30 ,3 1 A single Boltzmann equation. does not fit the data, and the steady state g/~ relation has a small, less sensitive component. This component may represent an additional closed state entered with a slower rate constant. Since there is no transcription in the

35

Biophysics oj gapjunctions

developing embryo until mid-blastula and DNA oligonucleotides antisense to Cx43 mRNA do not affect oocyte coupling, difference between oocytes and blastomeres is unlikely to involve connexins other than Cx38. The difference may involve a post-translational modification such as phosphorylation and is important in indicating that the same connexin can have different properties in different cells. Cx37, one of the newer rat and mouse connexins,32 is more closely related to Xenopus

Cx38 than to any other connexin and may be the direct homolog. Correspondingly, it forms very voltage dependent junctions when expressed in Xenopus oocytes. The voltage dependence is complex like that of Cx38 in oocytes. However, unlike Cx38, Cx37 is expressed widely in adult tissues, maximally in lung. Cx26 and Cx32 also form Jj dependent junctions when expressed in oocytes (Figure 5A, B). The Vos are much greater than that for Xenopus Cx38 (ca95 and 55mV compared to 15mV), and Cx32/Cx32

A 1.0

===

=

L

0.0 -120 -80 -40

V;

a

40 80 120

(mY)

Cx26/Cx26 6

1.2

0.8

&

"til L

0.4

0.0 -120 -80 -40 0

V;

40 80 120

(mY)

Cx32/Cx26

C

2.0

1.5 o

~ 1.0

e:

0.5

0.0 -120 -SO -40

V;

a

40 60 120

(mY)

Figure 5. Voltage dependence of g. in Xenopus oocytes expressing Cx32 and Cx26. Representative junctional currents and initial and steady-state conductance-voltage relations are shown for homotypic Cx32/Cx32 junctions (A) and Cx26/Cx26 junctions (B) and heterotypic Cx32/Cx26 junctions (C). The junctional currents were elicited by ± 20, 40, 60, 80 and 100 mV voltage steps applied to either oocyte for the homotypic junctions and the one expressing Cx26 for the heterotypic junctions. Junctional currents are downward for relative positivity in the stepped cell. Calibration bars are 20 nA and 1 s. The conductances are plotted as normalized to gj at ~ = 0; normalized initial gj is plotted as closed triangles and normalized steady state gj as open triangles. The smooth curves are fitted Boltzmann relations. From ref 31.

36 the junctions are unlikely to be exposed to potentials of this magnitude in cells normally expressing them. The calculated gating charge for Cx26 is about twice that for Cx32, but less than that for Cx38. Changes in conductance are slower with time constants of seconds. Where the gates are should eventually be illuminated by molecular studies. Domain exchanges. between Cx26 and Cx32 involving El, E2, CL and M3 have yet to localize the difference in gating charge. 33,34 Connexins lack an obvious analog of the S4 segment of Na +, K + and Ca2 + channels.V' Swenson et al 36 failed to see voltage dependence of rat Cx43 junctions when formed between Xenopus oocytes, but also failed to see voltage dependence of Cx32 junctions in this system. We do not understand this discrepancy. Junctions in many pairs of hepatocytes presumably formed ofCx32 (see below) exhibit voltage dependence like that in oocytes.U Transfection of a poorly coupled cell line with Cx32 also leads to voltage dependent junctions whose Vo (25 mV) is smaller than for Cx32 junctions in oocytes.? In myocytes and transfected cells, Cx43 junctions exhibit weak voltage dependence.It-P Reasons for variation-in voltage dependence with expressing system remain to be determined. Rat Cx46, when expressed in Xenopus oocytes, appears not to form junctions but rather to make large (Lucifer yellow permeable) hemichannels that are opened by depolarizationY The molecular weight of Cx46 in oocytes differs from that in lens and it is possible that oocytes do not make the mature protein .. Alternatively, Cx46 hemichannels may function as MP26 channels in the lens have been suggested to do, that is, they lead to reduction in extracellular volume by movement of extracellular fluid and solutes into the lens cells due to the osmotic pressure of the intracellular proteins.V Recently Ebihara reported formation of Cx46 junctions between oocytes in increased Ca2 + solutions. 38a A number of vertebrate connexins remained to be characterized with respect to voltage dependence.

Vi -o sensitivity in homotypic junctions The giant salivary gland cells of dipterans, because of their large size, were among the earliest cells in which coupling was studied. The closeness of coupling obscured the voltage sensitivity in current clamp, and it was not until voltage clamp was applied to isolated pairs of cells that the situation became clear. 39,40 Depolarization of both cells of a coupled pair decreased junctional conductance and hyperpolarization

Ai. V. L. Bennett and V. K. Verse/is

increased it. (Test Jij pulses too small to affect coupling were used to monitor conductancet''), The g/Vi.o relation was well fit by a Boltzmann relation, but the fit was marginally better if a squared relation was used, as would apply to two independent gates in series, each detecting Vi.o. This fact plus symmetry inclines us towards the two-gate model. In Drosophila these junctions are also sensitive to Jij. The Jij sensitivity is not a result of Vi.o sensitivity since hyperpolarization of either cell decreases gj, whereas the Vi.o sensitivity could only increase it. Also, depolarization of one cell decreases gj more than would be predicted from the Vi.o sensitivity. The Jijdependent changes are faster than the' Vi.o induced changes, and separate gating mechanisms appear to be involved, although they do not operate completely independently. Some degree of Jij dependence apparently occurs in Chironomus glands, although it was not characterized in the original publication.I? Blastomeres of squid exhibit both Vi.o and Jij sensitivity when gj is reduced by cytoplasmic acidification or treatment with alkanols (see below; ref 41). Hyperpolarization of both cells causes a small increase in conductance towards normal values, and provided there is still some conductance present, depolarization decreases it. Jij of either sign can increase gj, or in many cases, Jij of one sign increases gj while Jij of the opposite sign decreases it. Since the starting preparation is apparently symmetric, the asymmetry presumably arises as a result of differences generated by dissection and microelectrode penetration. The results suggest convergence of gj modulation by cytoplasmic acidification, alkanols and also injury (which may be due to Ca2 + in the cytoplasm) in a final common mechanism that is also voltage sensitive. Vi.o sensitivity is present to a minor extent in Cx26 homotypic junctions along with a pronounced Jij sensitivity. 30 Depolarization of either cell causes a fast increase in conductance and hyperpolarization of either cell causes a fast decrease (faster than the voltage clamp's settling time of 5-10 ms). The changes are small, ± 10 % over a range of ±100 mV. They are also observed with simultaneous polarization of both cells. The slow Jij-dependent decreases in conductance for large polarizations of either cell were noted above. These are slighly asymmetric for equal hyperpolarization and depolarization, further indicating an effect of Vi.o. In leech, some cells are coupled for depolarization of either cell, but not for hyperpolarization.O Vi.o sensitivity could account for this result, which

Biophysics of gapjunctions

appeared quite strange at the time it was described and inexplicable in terms of T-j dependent junctions. However, the actual explanation may be that the coupling pathway includes lengths of axon exhibiting inward rectification that leads to a reduced space constant and coupling for hyperpolarization.Vrf

Heterotypic junctions The heterotypic junctions to be considered here are those in which the hemichannels are homomeric, but comprised of connexins of different types in either half of the junction. Heteromeric hemichannels have not been definitively demonstrated as will be discussed below. One interesting aspect of heterotypic junctions is that they can behave as predicted from the hemichanncIs of which they are comprised or they can exhibit completely novel behavior that indicates that the hemichannels have altered properties. Heterotypic channels can also lead to rectification as considered in the next section. Before cloning, heterotypic junctions were most obvious as formed between cells from different species (cf ref 17). Junctions heterotypic in the sense of formed between different cell types were also known, but the connexins expressed may have been the same. Heterotypic junctions are now clearly seen between oocytes expressing different exogenous cRNAs. J unctions between Cx32 and Cx43 were reportedf'' but no rectification was seen (nor was there voltage dependence in their homomeric junctions). More interesting were the junctions that Cx43 formed with what was presumably Cx38. These junctions showed pronounced rectification, conductance decreasing for the Cx38 side positive with similar voltage dependence as seen for Cx38 expressed in oocytes. The sidedness of the gating confirmed our previous conclusion from effects of unilateral acidification.in blastomere pairs (see below; ref 41). This result suggested that the two hemichannels were acting in the heterotypic junctions in the same way as they did in homotypic junctions. A very different result was obtained when oocytes expressing Cx26 were paired with those expressing Cx32 (refs 30, 34; Figure 5C). One would have predicted a slow decline in conductance for a large T-j of .either sign. Instead, there is a fast T-jdependent rectification with increasing gj for T-j relatively positive on the Cx26 side and decreasing gj for T-j relatively positive on the Cx32 side. Also, for large T-j relatively positive on the Cx26 side

37 there is a slow decrease in conductance comparable to that seen in Cx26/Cx26 homotypic junctions, but no corresponding decrease for T-j relatively positive on the Cx32 side. In these heterotypic junctions the resultant properties would not have been predicted from the behavior of the homotypic junctions. It appears that interactions between hemichannels, presumably mediated by the extracellular loops, E1 and E2, alter channel properties. The relevant parts of the connexin molecules are under investigation using replacement of domains between them. 33 ,34 The situation remains not entirely clear, and two examples are illustrative. Consider the chimeric connexin, Cx32 with its E1 replaced by that of Cx26, which we term Cx32*26EL.Pairing oocytes expressing this and the native connexins forms Cx32/Cx32*26E1 and Cx32*26El/Cx26 junctions. These heterotypicjunctions are both similar to Cx26/Cx32 junctions; they show fast T-j-dependent rectification and reduced or absent slow T-j dependence on the corresponding side. In the first case only the E1 domains have sequence differences between the hemichannels, in the second case only the E1 domains are the same. This result is further evidence that hemichanncI interactions can profoundly affect hemichannel behavior. Do heteromeric hemichannels form in cells expressing more than one connexin such as hepatocytes, which express both Cx26 and Cx32? Although the two connexins are found in the same plaques, anatomical resolution to the single hemichannel level has not been achieved. 43a ,44 Oocytes injected with cRNAs for both Cx26 and Cx32 form junctions with cells expressing Cx32 whose voltage dependence is intermediate between that of Cx32/Cx32 and Cx26/Cx32 junctions.s'' From macroscopic data one could not distinguish whether the intermediate properties were due to single channels with intermediate properties as might be found with heteromeric hemichannels or to the parallel activity of channels with the properties of homomeric hemichannels. The result did indicate that homotypic junctions were not formed highly preferentially and that heterotypic junctions would be likely to form in cells expressing both Cx26 and Cx32. In agreement, some rat hepatocyte pairs have junctions with a component suggesting that Cx26/ Cx32 junctions or a heterornericintermediate are formed in situ, although most cell pairs appear to have only Cx32/Cx32 junctions.U The gradient ofCx26 along the rat hepatic acini makes it likely that

38 macroscopic asymmetry between hepatocytes will result. 44 Junctions formed of other connexins have more distinctive parameters of voltage dependence, and intermediate properties indicative of heteromeric hemichannels might be more readily detected. Oocytes expressing endogenous Cx32 and exogenous Cx43 form junctions with two separable components of voltage dependence corresponding to Cx38 and Cx4-3.45 It appears that these two connexins do not aggregate side by side to form heteromeric hemichannels, although they can join head to head to form heterotypic channels comprised of homome ric hemichannels. Single - channel recording also could identify heteromeric channels. However, two connexins permit 14 possible hemichannel types, which allows for 196 full channels, without considering differences in head to head alignments. Obviously, electrophysiological characterization could distinguish little more than the existence of intermediate channel types.

Rectification Coupling of cells expressing dissimilar connexins is an obvious way in which to build rectifying junctions, and the Cx38/Cx43 junctions provide a concrete example. However, these junctions are quite slow in their operation, much too slow to transmit neural signals in the way in which rectifying synapses do. The fast Vj dependent rectification of Cx26/Cx32 junctions is very rapid compared to the Vj-dependent changes in homotypic junctions formed by these connexins. Whether it is fast enough to account for the < 0.1 ms changes at rectifying synapses has not been determined. The speed of rectifying synapses is not yet accounted for. If the energy barrier between open and closed states were small, the transitions could be rapid as they are in Na + channels, for example. Alternatively, there could be a difference in cytoplasmic constituents with a blocking ion in higher concentration on one side, comparable to the situation hypothesized for the inward K + rectifier where M g2 + may block from the inside. The fast rectification of Cx26/Cx32 junctions between oocytes could not have this mechanism. Also, the large channel size of the crayfish rcctifying synapses indicated by dye permeability makes the blocking hypothesis less likely.

M. V. L. Bennett and V.K. Verselis

»'here are the gates? Vj dependence with no dependence on Vi-o and symmetry suggested that in amphibian blastomercs each hemichannel had a gating mechanism or voltage sensor near its cytoplasmic rnouth.J? Moreover, reversal of Vj suggested that there were two gates in series and that the one closed by a given polarity of Vj had to open before the opposite polarity of Vj could close the other one. However, symmetry prevented us from knowing which gate was closed by which polarity of Vj. An answer was subsequently provided by injecting acid into one cell of a coupled pair.t! ThegTVj relation was altered for Vj positive on the acidified side, but not for Vj negative on that side. This effect was similar to that seen for both polarities of Vj during acidification on both sides. Thus, we could conclude that Vj closed the gate on the positive side. This identification of sidedness was confirmed by behavior of Cx38/Cx43 heterotypic junctions as noted above. This polarity may not hold for all voltage dependent junctions in vertebrates (and does not for junctions between squid blastomeres, also shown by unilateral acidificationt-). The putative hemichannels formed by Cx46 in Xenopus oocytes-? and by an unidentified connexin in horizontal cells (in low Ca2 + solutionjt'' are in both cases opened by depolarization, which is relative positivity at the channel mouth. If Vj sensors are located at the channel mouth, one might be inclined to put Vi-o voltage sensors near to the center of the channel, perhaps in the wall formed by E1 and E2. However, comparison to voltage dependent cation channels suggests that the Vi-o sensors could just as well be in the connexin structure spanning the membrane from cytoplasm to intercellular gap along side the channel.

Cytoplasmic cations and uncoupling More than 10 years ago Turin and Warner'? reported that cytoplasmic acidification uncoupled amphibian blastomeres. We extended their observations and reported a simple titration curve for decrease in gj.48 Subsequently, pH effects have become more complex, although the initial observation of rapid and (often largely) reversible decrease in gj remains valid.U It remains uncertain whether .there is a direct H + action on channels as the titration curve would suggest. Perfused preparations

39

Biophysics of gapjunctions

have suggested in some cases direct actions? and in others indirect action, i.e. involving a cytoplasmic intermediate. 50 Reconstituted or incorporated preparations have? and have notl? shown sensitivity to acidification. Problems include need for rapid changes in H + concentration to avoid slow secondary changes while making the changes slow enough that the pH sensing electrode has responded and conductance changes and diffusion have virtually reached equilibrium. In any case, the apparent pKs for block vary over a moderate range, from low pH6 to low pH7. Optical methods of pH determination should improve the data. In amphibian blastomeres, lowering cytoplasmic pH affects voltage dependence, as noted above. For modest acidification the gr fj relation is shifted slightly towards larger fj. We did not test the effect when acidification significantly lowered gj because of difficulties in keeping pH constant. For large voltages, low pH did not obviously change the rate of junctional closing by voltage, and it was suggested that the gating mechanisms did not interact. 51 In Chironomus salivary gland, low pH simply shifts the gr Vi,o relation along the voltage axis towards more hyperpolarized levels.V Rise in cytoplasmic Ca 2 + has also been implicated in block of junctional conductance. 53 ,54 It has been difficult to quantitate sensitivity, and use of Ca2 + indicators shows that at low concentration Ca2 + quite freely permeates gap junctions, as would be predicted from its molecular size (e.g. ref 55). An earlier study demonstrated permeability to C o2 + at low concentration, but that high concentrations of C o2 + blocked the junctions. 56 In cardiac myocytes Ca2 + and H + ions interact such that the H + blocking effect is shifted along the concentration axis towards lower concentrations at higher intracellular Ca2 + .57 Further complications include release from internal Ca2 + stores evoked by Ca2 + , IP3 or other second messenger, interaction between Ca2 + and H + buffering systems such that raising one tends to raise the other, and activation of Ca2 + dependent proteases. Millimolar Ca2 + does not affect Cx32 channels reconstituted in bilayers.I'' In the economy of coupled cells, physiological changes in internal Ca2 + even in the ItM range probably do not affect the junctions between them. Ca2 + and H + dependent uncoupling may be important in pathological conditions, such as anoxia induced acidification and rupture of the cell membrane. Most who work with coupled cells are

aware that injury to one cell decreases the junctional conductance between it and healthy cells (presumably of protective value), and in the short term this effect is likely to be Ca2 + mediated since low Ca2 + solutions can prevent it. Over the longer term coupling can disappear even at low Ca2 + , which may be associated with loss of ATP or other maintaining system or loss of junctions through turnover as by internalization by the intact cell. Whatever the mechanism, bathing in weak acids (which acidify the cytoplasm after the uncharged, undissociated form crosses the cell membrane) remains a convenient experimental test of whether intercellular communication is mediated by gap junctions rather than cytoplasmic continuity. Neither weak acids nor the lipophilic agents described in the next section are specific in their action and are not good agents for identifying the physiological role of gap junctions by rendering them non-functional.

Lipophilic blockers A number of agents that reduce gap junctional conductance are uncharged and presumably act on membrane lipid or a hydrophobic region of the connexin molecule. 58 These agents may be the most useful uncoupling agents because of their rapid action and reversibility, although they affect other channels as well. Lipophilic blockers include n-heptanol and n-octanol, longer and shorter hydrocarbon chains being less effective, and volatile anesthetics, such as halothane. Relative potencies of these and other lipophils capable of intercalating into lipid bilayers suggest that they act by perturbing the membrane toward the middle of the bilayer. 58 Single channel recording indicates that transitions between closed and open states are rapid in the presence of these agents. Thus, it appears that they decrease the stability of the open state with respect to the closed state but do not affect the nature of the transition between them.

Other blocking agents Gap junctions in hepatocytes are rapidly and reversibly uncoupled by CC14 .59 This action appears to depend on cytochrome P450 and may be mediated by increased oxidation potential. Arachidonic acid uncouples cardiac myocytes which also may depend on oxidation. 58

40 Phosphorylation The actions of kinases to phosphorylate connexins and of phosphatases to dephosphorylate them comprise a rapidly developing story (cf ref 17). The bottom line is that different kinases affect junctional conductance differently in different tissues expressing the same or different connexins. In hepatocytes, we identified a serine in Cx32 which is phosphorylated by A kinase, and cAMP increasing agents increase junctional conductance sufficiently rapid to suggest (but not prove) a gating phenomenon rather than an effect on formation or removal. 60 Serine phosphorylation of Cx43 appears necessary for formation of junctions in some cells''! but probably not in others,62 and truncation mutants lacking much of the C terminus where the phosphorylation sites are presumed to be located still form functional junctions.C' In a cell line transfected with Cx43, treatment with C kinase reduces single channel conductance of Cx43. 4 Steady state voltage dependence is unaffected by phosphorylation, but transition rates are increased. This finding suggests that the energy difference between open and closed states is unaffected but that the energy barrier between states is lower in the phosphorylated form. Phosphorylation of a specific tyrosine of Cx43 blocks junctional conductance.s" The time scale is minutes and a gating action is likely. Perfused and reconstituted preparations should permit direct analysis of the action ofkinases (and other enzymes) on gating.

Antibodies and antisense A few antibodies act intercellularly to block junctional conductance. 44,65,66 The action is likely to be quite specific, although cross reactivity with other antigens is being seen. 67 Antibodies to extracellular epitopes block development of coupling, but as they are too large to get into the intercellular gap they do not affect formed junctions.f" Extracellular antibodies may be useful in blocking coupling where turnover is rapid; they are also more readily applied to larger numbers of small cells, but at the same time their application is less localized. The mode of action of intracellular (and extracellular) antibodies may be simply stearic hindrance. Alternatively, an intracellular antibody may bind preferentially with the. closed conformation.

M. V. L. Bennett and V. K. Verselis

Immunization with isolated gap junctions, which should be closed under the experimental conditions, should be a more favorable method of raising blocking antibodies than immunizing with specific oligopeptide sequences. Antisense DNA oligonucleotides are an effective way of blocking endogenous coupling in oocytes. 30,3! They may prove useful in studies of the role of coupling in development.

Permeation Gap junctions are quite non-selective in their permeability to molecules up to a diameter of 1-1.5 nm, neutral or with charge of either sign. Some of the earliest reports of gap junctional permeability to relatively large molecules did contain errors of fact (permeation by proteinsf") or procedure (failure to exclude permeation through non-junctional membrane.Z'' or through regions of cytoplasmic conrinuity/J). However, the development of highly fluorescent probes/- to which the non-junctional membrane was impermeable73,74 as well as large labeled probes to which the gap junctions were impermeable (but which could cross cytoplasmic bridges) established the relatively large size of the gap junction channel,75,76 Use of radioactive tracers and cultured cells also demonstrated permeation by a wide range of molecules of intermediary metabolism, and firmly established the gap junction as a mediator of metabolic cooperation and potential pathway for chemical signaling,77,78 Areas to be investigated now include more precise definition of the size limit of permeation for different junctions or connexins and whether cells can alter the permeability of their gap junctions to larger molecules independently of smaller ones. Quantitation analogous to measurement of electrical coupling coefficients may become important in consideration of chemical signaling and metabolic coupling. Observation of transfer of probe or dye to which the non-junctional membrane is impermeant and which is not metabolized (or bleached) is described by different equations than electrotonic coupling. In the case of dyes, the steady state coupling coefficient is zero for junction impermeable molecules, and unity for junction permeable molecules, although equilibration time may be very long. In experiments purporting to show impermeability, one needs to question the observation time and sensitivity. Dye fluxes do provide an important qualitative indicator

41

Biophysics of gap junctions

of the presence of coupling and a not very sensitive measure of modulation. In analysing intercellular communication, the lack of complete correspondence between electrical and chemical coupling should be kept in mind.

Channel size To estimate channel diameter by determining the largest permeable molecule, most studies have used fluorescent tracers such as the fluorescein derivatives (e.g. carboxy-, dichloro-, etc.) and the sulphonated 4-aminonapthalimides (e.g. Lucifer yellow 72 ) . Estimates of an upper size limit for permeation differ among preparations. Mammalian cell lines probed with a series of linear dye-conjugated peptides showed diminishing permeabilities with increases in the size of the peptide backbone and/or the number of charges on the peptide backbone. 75 (The expressed connexin is likely to be Cx43.) A similar finding was obtained in the earthworm median giant septate axon using a series of halogen derivatives of fluorescein.Zf Both these studies showed channel diameter to be in the range of 1.5 nm with diminished permeability with increased negative charge of the tracer. Examination of insect salivary glands with the same probes suggested a larger channel size of ca 2.0 nm.7 6 ,80

Impermeability When pairs or small aggregates of cells are compared under constant conditions, relative permeabilities are easy to measure. Absolute impermeability and comparison to other junctions requires some basis on which to normalize and an indication of sensitivity. 'Failure to observe dye coupling may not mean that the junctions are less permeable to the dye, but that there are fewer of them, that the geometry is less favorable or that there is a higher background or other source of optical interference. Many observations are suggestive of dye impermeability at specific junctions (e.g. refs 81-83; border cells), but in none has a lower specific permeability compared to other junctions been shown. Permeation by smaller or more readily detectable tracers (such as biocytin) reveals more extensive connectivity, 84 but does not address the issue of specific permeability. Similarly, when treatments appear to affect permeability to larger molecules preferentially,

it is necessary to establish that the changes are not a matter of changes in number of channels superimposed on measurement with a threshold. Although failure to observe permeation by a large molecule in a system in which permeation by a smaller molecule is readily observed may suggest that channel diameter has been bracketed, the relation between the effective diffusion coefficient of a probe as a function of diameter falls off rapidly well before channel diameter is reached. For two molecules of diameter 0.5 and 0.75 times the channel diameter the permeability ratio is about 10 (cf ref 15). With ordinary measurements, the channel diameter might easily be underestimated, and since molecular weight increases nominally as the cube of diameter, the size limit for slow permeation could be significantly larger.

Quantitation To quantitate a conventional coefficient of permeability, Ij*, requires a knowledge of junctional membrane area. Although area can be assessed by electron microscopy, it is a potentially large source of error, and the fraction of channels open may be variable.v'' Values of Ij * for K +, [HC] TEA and Procion Yellow at the intercalated disc of calf and sheep ventricular muscle were calculated as 7.7x10- 3 , 1.3 x 10- 3 and 1.4x10- 6cm s'-I, respectively inversely related to their size. 86 For TEA, permeability of the surface membrane was less by a factor of 10 - 4 and Procein yellow was virtually impermeable. Ij* to fluorescein and its derivatives quantified in the earthworm median giant septate axon ranged from 5.4 x 10- 5 to 4 x 1O- 6cm s also inversely related to their molecular weights and, as typical for large tracers, the surface membrane was virtually impermeant to them. 79 ,87 r

' ,

Permeability as related to conductance Given the uncertainties in junctional area, a more useful measure of permeability than Ij* is the Ij/gj ratio, where Ij is the total permeability, conveniently defined as the mean single channel permeability times the number of open channels. By this definition Ij is analogous to gj, the mean single channel conductance times the number of open channels. Ij in units of cm 3s - 1 and gj in p.S both represent fluxes normalized to electrochemical driving forces.

42

Neither term takes into consideration the membrane area. Given that there was a single permeant ion A:!: and no electric field, gj would be related to 1j by gj = Ijz2F2[A :!: ]/RT.13 However, when gj and lj represent fluxes of different ions, a change in lj need not be accompanied by proportional change in gj' In practice, gj is largely determined by K + ions which are significantly smaller than the larger permeant species being tested. Thus, the lj/gj ratio should provide a more reliable measure of relative lj than the permeability calculated per unit junctional area. Differences in the lj/gj ratio would indicate differences in channel selectivity or size. Allor-none gating, as well as channel turnover, would change diffusional and electrical fluxes proportionately, i.e, the lj/gj ratio would remain constant. For gating, in which channels were closed in a graded manner the passage of larger molecules would be preferentially restricted as the channels constricted, and the lj/gj ratios would decrease. The time course of dye flux permits calculation of lj/gj ratios (cf ref 88). However, the use of fluorescent dyes for junctional flux studies can be hampered by cell autofluorescence, pigmentation, bleaching and binding to intracellular components. Also, the dyes are generally so large'(l1-14A) that intercellular fluxes in many preparations are slow and difficult to quantitate. An alternative approach employed the tetraalkylammonium ions, a series of cations whose intracellular activities can be measured with ion-selective microelectrodes constructed with a liquid ion-exchanger (Figure 6A; ref 89). These ions possess a single positive charge and their diameters vary over a range for which there is little quantitation of permeability. For blastomeres of a frog and a teleost, measurement of lj/gj ratios indicated an effective channel diameter of about 1.5 nm (Figure 6B; ref 15). For squid blastomeres a larger channel of about 2.0 nm was indicated. Diameters of gap junction channels in insects, e.g. salivary gland cells from Chironomus 76 ,90 and epidermal cells from larval beetles, Tenebrio molitor, 82 are in the same range. In respect to gating, the lj/gj ratio for tetraethylammonium ions (TEA) remained constant as channels between amphibian blastomeres were closed by voltage.s? (The electrical driving force on TEA altered the fluxes and had to be included in the lj calculation.) The constancy of the lj/gj ratio implies that voltage gating is all-or-none, i.e. that voltage alters the mean open time rather than the channel conductance (see also ref90). However, there could

M. V. L. Bennett and V. K. Verselis

be subconductance states that did not affect relative permeability, as might occur if the selectivity filter were distinct from a large nonselective gating region, or if the lowered conductance resulted from fast unresolved flicker. Single gap junction channel measurements in some instances appear to exhibit substates or slow transitions between open and closed states.6,91,92 The constancy of the lj/gj ratio has not been tested under conditions where gating might not be all-or-none, such as altered phosphorylation state.

Frictional model of gap junction channel permeability The ability oflarge hydrophilic dyes to permeate gap junction channels together with their high ionic conductance and lack of selectivity suggested that the gap junction is a large aqueous filled pore. QIos of changes in gj where H 20 had been replaced with deuterium oxide (D 20 ) showed that solvent effects predominantly govern mobility of ions through gap junction channels.P'' This result indicates that gap junction channels are filled with water and that viscosity, which dictates ion mobility in bulk solution, is maintained within the channels. Permeation by fluorescent dyes in D 20 solutions is more reduced than gj, particularly with cooling when dye fluxes can be blocked at low enough temperature. Permeation block is attributable to solvation of the dye molecules, as well as the channel wall, to make the dye-solvent complex larger than the channel. This interpretation is consistent with the higher blocking temperatures observed for dyes with a larger number of ionizable groups. lj/gj ratios for the tetraalkylammonium ions decrease with increasing size to a degree quantitatively consistent with permeation through aqueous channels (Figure 6B; ref 15). Although charges at the channel mouth or lining the channel pore are likely to affect permeation, permeant species considerably smaller than the channel are less likely to encounter charged groups. The tetraalkylammonium ions, by nature of their structure, have a reduced surface charge density with increasing size so that mobilities of the larger members of the series are less likely to be influenced by charge groups. For large fluorescent dyes, which contain exposed charged groups, deviations from predictions based on frictional models are evident suggesting the existence of fixed negative charges in the channel wall.7 5,79

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