Accepted Manuscript

Regulation of connexin hemichannel activity by membrane potential and theextracellular calcium in health and disease

Ilaria Fasciani, Ana Temperán, Leonel F. Pérez-Atencio, Adela Escudero, PalomaMartínez-Montero, Jesús Molano, Juan M. Gómez-Hernández, Carlos L. Paino,Daniel González-Nieto, Luis C. Barrio

PII: S0028-3908(13)00136-6

DOI: 10.1016/j.neuropharm.2013.03.040

Reference: NP 5018

To appear in: Neuropharmacology

Received Date: 17 December 2012

Revised Date: 26 March 2013

Accepted Date: 27 March 2013

Please cite this article as: Fasciani, I., Temperán, A., Pérez-Atencio, L.F., Escudero, A., Martínez-Montero, P., Molano, J., Gómez-Hernández, J.M., Paino, C.L., González-Nieto, D., Barrio, L.C.,Regulation of connexin hemichannel activity by membrane potential and the extracellular calcium inhealth and disease, Neuropharmacology (2013), doi: 10.1016/j.neuropharm.2013.03.040.

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Highlights:

1. Ubiquitous presence of functional hemichannels at the cell surface as a normal

phase in the connexin life cycle

2. Strict regulation of hemichannel activity by membrane potential and extracellular

calcium

3. Update of mechanisms and molecular basis of hemichannel voltage-gating and of

regulation by calcium

4. Hemichannel dysfunction in “connexinopathies”

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3/25/2013

REGULATION OF CONNEXIN HEMICHANNEL ACTIVITY BY

MEMBRANE POTENTIAL AND THE EXTRACELLULAR CALCIUM

IN HEALTH AND DISEASE

Review article for Neuropharmacology: special issue on connexin-based channels

Ilaria Fasciani 1, Ana Temperán 1, Leonel F. Pérez-Atencio 1, Adela Escudero 1,2,

Paloma Martínez-Montero 2, Jesús Molano 2, Juan M. Gómez-Hernández 1,

Carlos L. Paino 1, Daniel González-Nieto 1,3 and Luis C. Barrio 1

1Unit of Experimental Neurology-Neurobiology, "Ramón y Cajal" Hospital (IRYCIS),

Madrid, Spain 2 Unit of Molecular Genetics-INGEM, Hospital La Paz (IDIPAZ), Madrid, Spain 3Center for Biomedical Technology, Universidad Politécnica de Madrid, Spain

Corresponding author: Luis C. Barrio, MD PhD, Unidad de Neurología Experimental, Departamento de Investigación, Hospital "Ramón y Cajal", Carretera de Colmenar km 9, 28034-Madrid, Spain. Phone: +34-91-336-8320; Fax: +34-91-336-9816; e-mail: luis.c.barrio@hrc.es Manuscript information:

• Text pages: 31 • Figures: 4 • Tables: 1 • Abstract words: 146 • Total number of words: 8.977

Running title: Normal and aberrant connexin hemichannels

Key words: connexins, hemichannels, voltage-gating, calcium regulation, genetic

diseases, leaky hemichannels

Abbreviations: Cx, connexin; HC, hemichannel, GJC, gap junction channel, Vm,

membrane potential; Vj, transjunctional voltage; Ghj, hemichannel

conductance

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ABSTRACT

Connexins are thought to solely mediate cell-to-cell communication by forming gap

junction channels composed of two membrane-spanning hemichannels positioned end-to-

end. However, many if not all connexin isoforms also form functional hemichannels (i.e.,

the precursors of complete channels) that mediate the rapid exchange of ions, second

messengers and metabolites between the cell interior and the interstitial space. Electrical

and molecular signaling via connexin hemichannels is now widely recognized to be

important in many physiological scenarios and pathological conditions. Indeed, mutations

in connexins that alter hemichannel function have been implicated in several diseases.

Here, we present a comprehensive overview of how hemichannel activity is tightly

regulated by membrane potential and the external calcium concentration. In addition, we

discuss the genetic mutations known to alter hemichannel function and their deleterious

effects, of which a better understanding is necessary to develop novel therapeutic

approaches for diseases caused by hemichannel dysfunction.

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Contents

1. The ubiquitous presence of functional hemichannels at the cell surface is a normal

phase in the connexin life cycle

2. Regulation of hemichannel activity by membrane potential and extracellular calcium

2.1. Mechanisms and molecular basis and of hemichannel voltage-gating

2.2. Mechanisms and molecular basis of hemichannel regulation by calcium

3. Hemichannel dysfunction in “connexinopathies”

4. Concluding remarks and perspectives

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1. The ubiquitous presence of functional hemichannels at the cell surface is a

normal phase in the connexin life cycle

Connexins (Cx) are proteins encoded by a multigene family that typically form cell-

cell channels. To date, 21 Cx genes have been identified in the human genome and most

of their Cx orthologues have been described in other vertebrate species. The assembly of

connexins into gap junction channels (GJC) occurs in two main stages. Connexins first

oligomerize into hexameric hemichannels (HC) in the Golgi apparatus, from where they

are transported to the cell surface in vesicles and they fuse to the plasma membrane in an

exocytotic process (1). The amount of HCs delivered to the plasma membrane can be

represent only the 4-15% of total Cx protein (2,3). Subsequently, hemichannels presented

by adjacent cells make contact and dock to form a complete intercellular channel. During

the docking process, the channel interior becomes isolated from the extracellular

environment and the newly formed channel opens, allowing the direct exchange of ions,

second messengers and metabolites between the interior of the two cells. New intercellular

channels aggregate to form plaque-like structures, while older channels are endocytosed

as double-membrane gap junction structures by one of the two contributing cells, indicating

that the hemichannels remain docked during gap junction degradation. It was previously

thought that hemichannels remained closed until docking during channel formation.

However, the opening of undocked hemichannels has been demonstrated in solitary

Xenopus oocytes expressing the lens Cx46 (4) and in isolated horizontal cells of the

catfish retina (5). Twenty years later, there is now evidence that HCs composed of different

connexins can be electrically and chemically activated (reviewed in 6), and in native cells

these HCs can mediate the rapid flow of ions across the cell membrane to regulate ionic

homeostasis, and to facilitate the release of ATP, NAD+, glutamate or prostaglandins

involved in autocrine/paracrine signaling (reviewed in ref. 7), as well as the transfer of nitric

oxide (8).

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The relative number of solitary HCs and GJCs at the cell surface is determined by

the speed and efficiency of HC incorporation into GJCs. The efficiency of incorporation can

be estimated by comparing the total number of solitary HCs expressed at the cellular

surface of isolated oocytes with the number of undocked HCs and GJCs on paired oocytes

(9). The size of the HC and GJC pools can be determined electrophysiologically by

simultaneously measuring the transmembrane and transjunctional currents, respectively.

In the case of cells expressing human Cx32, this calculation reveals a rather inefficient

docking process, as only 30-60% of solitary HCs delivered to the plasma membrane are

incorporated into GJCs in a time-dependent manner. Moreover, a significant proportion of

solitary HCs (~50%) undergo internalization before they can dock (Fig. 1). These data

clearly indicate that sufficient solitary HCs can reside at the cell surface to act primarily as

functional hemichannels, and that the intercellular and transmembrane exchange of ions

and small molecules mediated by GJCs and HCs can occur simultaneously.

The relative amounts of HCs and GJCs can vary depending on the cell type and Cx

isoform. Human polarized intestinal cells, which express high levels of Cx26, Cx43 and

Cx32, predominantly form functional HCs located mainly in the basolateral plasma

membrane domain and they form few GJCs (10). By contrast, the structural analysis of

lens fibers revealed that Cx50-GJCs are at least one order of magnitude more numerous

than HCs (11). Connexins such as rat Cx29 / human Cx31.3 do not form functional GJCs

but they can form open HCs, suggesting that HCs fulfill their main biological role (12,13).

However, for many Cxs the ability to form open HCs has not been fully documented or

remains unclear. It has been reported that rat Cx36 expressed in Xenopus oocytes cannot

form functional voltage-activated HCs, even in the absence of extracellular Ca2+ (14), in

contrast to the orthologous Cx35 of perch, shake or zebrafish (15,16). However, ATP

release via rat Cx36-HCs has been demonstrated during depolarization of cerebellar

granule neurons in vitro (17). The ability to form open HCs may also be species specific as

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human and sheep but not rat Cx26-HC can form functional HCs due to a single

evolutionary amino acid change (D159N) in the rodent protein (18).

2. Regulation of hemichannel activity by membrane potential and extracellular

calcium

The electrophysiological properties of Cx HCs have been extensively

characterized. Most HCs are activated by depolarization of the membrane potential (Vm),

and channel opening is critically dependent on the concentration of extracellular Ca2+ ions

([Ca2+]e) and other divalent cations. Also, HCs are regulated by intracellular Ca2+

concentration but in opposite direction, increasing [Ca2+]i from its resting value induces a

significant opening of HCs, as measured by ATP release experiments (19,20). HCs formed

by different Cx isoforms can differ significantly in their regulation by voltage and external

calcium. A subset of HCs, including those formed by human Cx32 and Cx26 (18,21), can

be readily detected using electrophysiological approaches and by dye-uptake experiments

under “resting” conditions (i.e., at the membrane potential of unclamped cells and at

physiological millimolar extracellular [Ca2+]). Following cRNA injection, isolated Cx26

oocytes typically undergo progressive depolarization of the membrane potential, which is

accompanied by a reduction in input resistance as the number of open HC increases (Fig.

2A, i-ii). Because the membrane potential is now governed by open Cx26 HCs with poor

ion selectivity, the resting potential shifts towards the equilibrium potential of HCs close to

zero millivolts (18). This increment in ionic membrane conductivity correlates well with the

observed increase in permeability to larger molecules as they are added to the external

solution (e.g. propidium iodide, MW 668, +2 charges; Fig. 2C). At this depolarized resting

potential, large activated inward currents can be detected upon switching to voltage-clamp

mode. These currents diminish slowly, are fully deactivated by hyperpolarization (at -80

mV) and they can be reactivated by the application of pulses of more positive voltages (≥ -

50 mV), indicating a strong influence of membrane potential on HC activity (Fig. 3). The

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interplay between depolarization and HC opening creates a regenerative, circular

sequence of events: depolarization promotes HC opening, increasing ionic fluxes and

further depolarizing membrane potential. Thus, in the presence of strong HC activity, the

resting potential and input resistance of the plasma membrane can be completely run

down. Another subset of HCs, such as those containing human Cx43, exhibits a low

opening probability in “resting conditions” (in agreement with the poor dye uptake observed

in these cells), which is insufficient to significantly alter the resting potential or input

resistance (Fig. 2A and B; unpublished data). Because reduced extracellular Ca2+ is

essential for Cx43 HC opening (22), the regenerative cycle of opening-depolarization can

be activated in Cx43 oocytes at micromolar [Ca2+]e, which produces a large increase in

ionic conductivity and dye uptake, and a rapid depolarization of the resting potential. These

effects are rapidly reversed upon returning to normal millimolar [Ca2+]e (Fig. 2B and C).

Interestingly, a recent study using hippocampal slices reported that astrocyte Cx43 HCs

act as sensors of external Ca2+ depletion in the signaling between neurons and glia (23).

2.1. Mechanisms and molecular basis of hemichannel voltage-gating

Studies of the voltage dependence of Cx HCs have demonstrated that the

membrane potential, a stimulus that is always present and that can vary depending on the

cell type and functional state, is a potent regulator of HC activity. Although all HCs are

activated upon depolarization and are effectively closed by hyperpolarization, voltage

dependency, in terms of voltage sensitivity, kinetic properties and the polarity of closure,

varies significantly between HCs containing different Cxs. The complexity of HCs voltage

behavior relies on the fact that there are two distinct hemichannel gates, which mediate

transitions between different conductance states and possess separate voltage sensors

closing either for the same polarity or opposite polarity. Thus, the voltage behavior of HCs

can be unipolar or bipolar. HCs with unipolar voltage behavior include those formed by

human Cx32 (9), skate and zebrafish Cx35 (15,16), mouse and chicken Cx45 (24), and

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chicken Cx56 (25). The activation of these HCs progresses monotonically with increasing

depolarization across the entire negative and positive voltage range (Fig. 3A). At normal

millimolar [Ca2+]e, the slowly activating macroscopic currents of human Cx32 HC recorded

upon depolarization correspond to the transition of single HCs from the fully closed state to

the main open state of low conductance (~18 pS), with a progressive increase in the open

duration and the number of open channels as the depolarizing pulses augment (Fig. 3C).

When the holding potential returns to negative values, inward unitary currents flicker, and

HCs undergo frequent and recurrent transitions between the open and fully closed states

before finally closing.

The subset of HCs exhibiting bipolar voltage behavior includes those formed by the

rat Cx46 (26), mouse Cx30 (27), rat Cx43 (28, 29), Xenopus Cx38 (16), rat Cx50 (30) and

its chicken counterpart Cx45.6 (25), and human Cx26 (18) and Cx37 (31). The

macroscopic conductance of these HCs follows a bell-shaped curve (e.g., human Cx26-

HCs: Fig. 3D and E), with currents initially increasing as depolarization progresses and

then decreasing following polarization to higher positive potentials due to the inactivation of

the hemichannels. Single channel recordings reveal that the opening of Cx26 HCs upon

depolarization at negative potentials involves a transition from the fully closed state to a

main open state of ~430 pS (approximately double that of the corresponding GJC).

Moreover, as depolarization progresses, HCs remain stable in this high conductance state

until polarization reaches greater positive potentials, whereupon they inactivate by closing

partially to a subconductance or residual open state of ~35 pS (Fig. 3F). Activation and

deactivation normally refers to processes of the same gate while inactivation occurs when

one gate closes while another is still open. This inactivation mechanism prevents the

complete activation of HCs in response to high and prolonged depolarizations, and they

are maintained in a residual open state of reduced conductance. The voltage range and

sensitivity of inactivation varies among Cx HCs, whereby it is strong in mouse Cx30-HCs

(27) but in rat Cx43-HCs inactivation is only observed at very high positive potentials

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(28,29). This bipolar voltage behavior is consistent with a model of two gates in series with

opposing polarities that mediate transitions between different conductance states. Based

on the differences in the time course of unitary transitions, these two voltage gates are

provisionally referred to in the literature as the “slow” gate (i.e., the mechanism of

activation that mediates opening from the fully closed state upon depolarization and the

return to the fully closed state upon hyperpolarization) and the “fast” gate (which mediates

transitions between the main fully open and residual open states). In the case of human

Cx32 HC, the characteristic transitions attributable to the “fast” gate between a main open

state of high conductance (~90 pS) and the residual open state (~18 pS) only occur at low

[Ca2+]e (see Section 2.2 below).

Because the “fast” and “slow” gates appear to mediate similar transitions between

equivalent conductance states in HCs and GJCs, they are also referred to as mechanisms

of “fast” transjunctional voltage (Vj) gating and “slow” Vj-gating, respectively. Moreover, the

“slow” gating is also termed “loop” gating since it is thought to mediate the opening of

newly formed GJCs during the docking process, in which the extracellular loops are

involved. Functional and structural analyses, mainly of Cx26 and Cx32, indicate that the

first positions of the cytoplasmic NT-domain contain charged residues that determine the

magnitude and polarity of “fast” Vj-gating, and that they influence ion permeation (reviewed

in 32). The “fast” gate of Cx26 channels, with an Asp residue at position 2, closes each HC

in response to a relative inside positive potential, while in Cx32 channels with an Asn

residue at position 2 the HC closes in response to a relative negative inside potential.

Based on NMR studies of the NT-domain of human Cx26, which revealed an α-helix

structure at the NT end, it was initially proposed that a glycine hinge positions the NT-helix

deeper within the channel pore (33). Subsequent determination of the crystal structure of

the Cx26 channel at a resolution of 3.4 Å confirmed the presence of 6 NT-helices attached

to the inner wall of the channel pore via hydrophobic interactions between Trp3 and Met34

that maintain the channel open (34). This 3-D model predicts that in conditions of a

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positive inside electrical potential, the 6 α-helices in which the voltage sensor is thought to

reside detach from the wall and form a plug structure that is stabilized by a circular network

of hydrogen bonds between Asp2 and the main chain of Thr5, resulting in physical

occlusion of the channel pore (34). This prediction is supported by electron

crystallographic analysis of mutated Met34AlaCx26 channels at 10 Å resolution,

demonstrating the presence of a plug structure in the center of the pore that is probably

due to disruption of the hydrophobic interactions between TM1 and Trp 3 of the NT helices

(35). Such plug density was dramatically reduced by the NT deletion of Cx26M34A missing

amino acids 2-7 (36). Further studies are required to determine whether the “plug” gating

model can be extended to other connexins, and whether other cytoplasmic domains, the

cytoplasmic loop or the CT domain also contribute to or interact with the plug structure.

Truncation of the CT-domain in some but not all Cxs eliminates the “fast” Vj-gating

transitions to a residual open state without altering “slow” gating (reviewed in 32).

Functional studies of Cx43 channels and NMR structural analysis of its CT-domain led to

the proposal that “fast” Vj-gating follows a "particle-receptor" model. In this model the long

and flexible CT-domain acts as a "gating particle", and it binds to a specific region ("L2") in

the CL-domain (acting as a "receptor") located near the internal vestibule of the pore,

ultimately occluding the channel lumen.

The basic mechanisms of “slow” or “loop” gating are elusive and it remains to be

determined whether the components of “fast” and “slow” gating mechanisms fundamentally

differ. The negative polarity of closure of “slow” gating is unaffected by mutations that

reverse the polarity of “fast” gating. Similarly, “slow” gating remains intact in the absence of

“fast” Vj-gating (reviewed in 32). To date, it has not been possible to selectively eliminate

“slow” gating, probably because HCs in which this mechanism is abolished remain fully

closed in a non-conductive state. Perhaps this is the case of the rat Cx26 HCs that cannot

form open voltage-gated hemichannels and such functionality can be restored by

substituting the Asn residue at position 159 in the second extracellular loop Cx26 with a

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negatively charged Asp as in human and sheep Cx26, thereby conferring rat Cx26 with

similar, if not identical, voltage gating properties to those seen in HCs formed by its human

orthologue (18). To this respect, it is interesting to mention the studies that using

cysteines-scanning mutagenesis have revealed conformational rearrangements of several

residues in Cx46 (Ala39, Glu43, Gly46 and Asp51) and Cx50 (Phe43, Gly46 and Asp51)

HCs in response to membrane hyperpolarization, suggesting that they are associated with

the closure of “loop” gate (37,38). This finding is in agreement with the crystal structure of

Cx26 channel since those residues of TM1/E1 boundary and N-terminal half of E1 that

react with the thiol modification reagents form the inner wall of the extracellular entrance of

the pore (29).

2.2. Mechanisms molecular basis of hemichannel regulation by external calcium

Because HC activity is critically dependent on the extracellular concentration of

Ca2+ ions, HCs may act as sensors of the changes in external Ca2+. The inhibition of HC

activity at resting [Ca2+]e may serve as a regulatory mechanism to protect the cell from the

potentially adverse effects of leaky hemichannels, while the opening of HCs induced by

decreases in [Ca2+]e to micromolar levels may be required to initiate cell signaling

processes (23). Substitution of Ca2+ in the external solution with other divalent cations

inhibits HC activation and human Cx32 HCs for example, whose regulation by Ca2+ has

been studied in detail (39), are inhibited by divalent cations as follows:

Cd2+>Co2+≈Ca2+>Mg2+>Ba2+. The dose-response curve of hCx32 HC ranges from a

maximum at 0.5 mM to a minimum at 3-5 mM [Ca2+]o, with an EC50 of ~1.3 mM. The

concentration of Ca2+ required to block HC currents by 50% varies significantly among

different Cx HCs; the EC50 of Cx46 HCs is in the millimolar range (40), while those formed

by Cx43 and Cx50 in rat and mouse, Xenopus Cx38 and human Cx26 have EC50’s in the

micromolar range (22, 41-43).

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Variations in external divalent cation concentration regulate HC currents at both

macroscopic and unitary levels in a complex manner. External [Ca2+] is known to affect the

voltage gating of HCs (44,45). Indeed, the removal of external Ca2+ typically causes a

marked increase in the amplitude of HC currents, shifting activation to more negative

potentials and altering the kinetics of activation and deactivation. Conversely, increasing

external Ca2+ has the opposite effect. These changes may be due to screening of the

membrane surface charge by divalent cations, to serial access resistance errors due to the

large conductance induced in the absence of divalent cations, or to the direct action of

Ca2+ on HCs.

Single HC recordings have furthered our understanding of the mechanisms by

which Ca2+ regulates HC activity, as this approach permits the relationship between the

concentration of divalent cations and the complexity of HC voltage gating to be analyzed.

Two distinct blocking actions of millimolar concentrations of external Ca2+ have been

reported for unipolar human-Cx32 HCs (Fig. 4A and B; ref. 39). External calcium blocks

the opening of Cx32 HCs to the fully open state (~90 pS), as activation and deactivation of

Cx32 HCs at normal [Ca2+]e only results in transitions from the fully closed state to a

subconductance state (~18 pS) upon depolarization, and in closure in response to

hyperpolarization. Activation at lower [Ca2+]e (≤0.5 mM) involves transitions to both the 18

pS subconductance state and the fully open state of ~90 pS, while deactivation typically

follows a two-step sequence, whereby HCs in the main open state (~90 pS) undergo a

transition to the 18 pS state before closing fully. Thus, the two-gate model proposed for

bipolar HCs may also be applicable to hemichannels with unipolar voltage behavior,

assuming that both gates operate at the same voltage polarity (i.e., both gates open upon

depolarization and close in response to hyperpolarization).

External Ca2+ also blocks the activity of Cx32 HC in the residual 18 pS open state, a

process thought to involve narrowing of the cytoplasmic vestibule, in a voltage- and Ca2+

concentration-dependent manner. Inward but not outward unitary currents exhibit a

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characteristic flickering activity that is interrupted by frequent and recurrent transitions

between the conductive and non-conductive states, which results in an inward rectification of

macroscopic currents, indicating that external Ca2+ blocks ion conduction (Fig. 4B).

These two blocking actions of external Ca2+ on hCx32 H are mediated by an unique

and specific Ca2+ binding site located within the extracellular vestibule of the pore (39).

This binding site may be formed by the carboxylate oxygens contributed by two acidic

residues, D169 and D178, flanking the first and third Cys within the second extracellular

loop. Substitution of Asn for Asp at either of these two positions abolishes completely the

Ca2+ regulation, while the formation of heteromeric D169N and D178N hemichannels can

reverse both the Ca2+-mediated blockage of voltage-gating and of ionic conduction. Hence,

it would appear that each Ca2+-binding site is composed of two Asp residues belonging to

adjacent subunits, and that a ring of 12 Asp residues able to bind up to six Ca2+ ions can

account for the open channel block. The same motif is present in some but not all

connexins, suggesting the existence of more than one molecular mechanism for Ca2+

binding. For Ca2+ regulation of human Cx37 HCs, it has been also proposed a voltage-

dependent mechanism of open channel block (46) and in the case of Cx46 HCs, by

contrast, divalent cations can act as modifiers of intrinsic voltage-gating by stabilizing the

fully closed conformation (47). Mutational analysis of negative charged residues within

second extracellular loop of these two connexins will be necessary to determine the

possibility that these residues constitute the site for Ca2+ binding.

3. Hemichannel dysfunction in “connexinopathies”

Mutations in connexin genes have been linked to several human hereditary

diseases, known as “connexinopathies” (reviewed in 48), with evidence from functional

assays implicating hemichannel dysfunction in the pathogenesis of some such diseases

(Table 1). Many of these mutations interfere with HC regulation by voltage and calcium.

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Mutations in Cx32 and Cx47 expressed in Schwann cells and oligodendrocytes

provoke disorders in peripheral and central myelin, respectively. Cx32 mutations give rise

to a common peripheral neuropathy, the X-linked form of Charcot-Marie-Tooth disease

(CTMX; ref. 49). In the Schwann cells that ensheath peripheral nerves the expression of

Cx32 is mainly concentrated in non-compact regions of myelin, the paranodal zone and

Schmidt-Lanterman incisures. At these locations, Cx32 forms hemichannels and reflexive

gap junctions across the myelin sheath, speeding up communication through the myelin

layers that separate the adaxonal and abaxonal cytoplasm (50). This radial pathway may

be involved in the spatial buffering of K+, which permits the renewal of action potential

propagation along the axon. Moreover, the release of ATP by Cx32 HCs (19) suggests that

HCs are also involved in the myelination and survival of Schwann cells. To date, almost

400 different CMTX mutations have been identified, situated throughout all the topological

domains of Cx32. The effects of many CMTX mutations have been investigated at the

cellular level, revealing a wide range of pathological mechanisms that include hemichannel

anomalies. Many of these mutations result in partial or complete loss of HC function and

less frequently, in a gain-of-function that affects HC permeation or their regulation by

voltage and calcium (Table 1). Nonsense CTMX mutations that truncate the Cx32 protein

to the minimum length required for HC expression at the cell surface, which must include

up to Arg 215 (i.e., the Y211stop; ref. 9), can provoke the complete loss of a functional HC.

Missense CMTX mutations resulting in an entirely cytoplasmic cellular localization (e.g.,

R142W, E186K, E208K, or R215W; ref. 9, 51, 52) can also prevent the formation of

functional HCs. By contrast, other mutations can increase HC activity, and CT-truncated

CMTX mutants (e.g., the C217stop, R220 stop, R265stop and S281stop; ref. 9) that are

fully capable of forming open HCs, cannot assemble into complete GJCs, enhancing the

number of solitary HCs at the plasma membrane. Although HC activity is enhanced in

these mutants, it is lower than expected due to the increases in the voltage threshold of

truncated HCs that parallel decreases in CT-domain length (Table 1). Anyhow this

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enhanced HC activity is associated with severe neuropathy, supporting the hypothesis that

an excess of functional HCs aggravates the clinical phenotype. Other CMTX mutations

within the CT-domain also alter voltage gating but in the opposite direction, such as the

F235C mutation that is associated with an unusually severe neuropathy due to modified

voltage-gating properties of unipolar Cx32 HCs. This mutation causes HCs to open at less

depolarized potentials and close at more negative potentials, resulting in marked increases

in HC activity (21). Such aberrant HC activity (which is also observed in coupled cell pairs)

has a deleterious effect on cell survival as it runs down the resting potential, it causes a

loss of intracellular ion and metabolite homeostasis, and it results in cell swelling and

death. These toxic effects in Schwann cells are well correlated with the severe

demyelination observed in nerve biopsies from patients (21). Certain CMTX mutations also

interfere with channel regulation by Ca2+, as is the case for the D178Y mutation that

destroys the Ca2+ binding site (39). At normal [Ca2+]e, D178YCx32 HCs behave like wild-

type HCs in the absence of divalent cations: opening to the high conductive state (90 pS)

is no longer prevented and nor is ion conduction blocked. Cx47 mutations cause a severe

autosomal recessive disorder, a hypomyelinating leukodystrophy known as Pelizeaus-

Merzbacher-like disease (PMLD; ref. 53). Some PMLD mutants result in loss-of-function,

preventing the formation of oligodendrocytic Cx47/Cx47 GJCs and oligodendrocyte-

astrocyte Cx47/Cx43 GJCs (54), but their HC function was not studied. Other PMLD

mutations may result in the impairment or loss of HC function but in this case it is not

known how they can affect to GJC function (Table 1; ref. 55).

The most common connexin-related diseases are caused by mutations in the

Cx26 gene, which account for half of all congenital and autosomal recessive non-

syndromic forms of hearing loss. Dominant Cx26 mutations have also been linked to

syndromic hearing loss, which is accompanied by a variety of mild to severe skin disorders

(reviewed in 56). Mutations in 3 other Cx genes (Cx30, Cx30.3 and Cx31) have also been

associated with various forms of syndromic and non-syndromic deafness, and skin

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disorders not associated with hearing loss. To date the only recessive mutation in which

HC function has been tested is the E47K mutant, which results in non-functional HCs (57).

The effects of dominant Cx26 and Cx30 mutations in HC function have been studied more

extensively. Functional analysis of the R75WCx26 mutation, which causes hearing loss

accompanied by palmoplantar keratoderma, reveals the absence of gap junction

communication and of functional HCs, as well as cell lysis (42,58,59). R75WCx26 HCs

display unitary conductance and Ca2+ regulation similar to that of wild type HCs, but

inactivation at positive potential is enhanced by the increased voltage sensitivity of the

“fast” gating mechanism (42). Electron-microscopy studies indicate that gap junction

formation is reduced (58), suggesting that cell lysis by leaky HCs is due to an increase in

the pool of solitary mutant HCs, which in turn is caused by defective docking.

With the exception of S17F, the dominant mutations in Cx26 (G12R, N14K, A40V,

G45E and D50N), linked to keratitis-ichthyosis-deafness (KID) and hystrixlike-ichthyosis-

deafness (HID), and the Cx30 mutations (G11R and A88V), causing the Clouston

syndrome without hearing loss, result in leaky hemichannels and cell death (57,60-63).

These deleterious effects correlate well with the pathognomonic atrophy of stratum

granulosum observed in the epidermis of KID/HID patients. Interestingly, these mutations

are grouped at the pore-lining NT, TM1, E1 and TM2 domains involved in voltage-gating

and pore permeability (reviewed in 32). Indeed, G45ECx26 HCs promote increased

permeability to Ca2+ influx, while the A40V mutation is associated with a partial reduction in

sensitivity to external Ca2+, thereby increasing the likelihood of HC opening at normal

[Ca2+]e (43). The contribution of increased HC activity to the epidermal manifestations

associated with this group of diseases was confirmed by generating an inducible

transgenic mouse that expresses G45ECx26 in keratinocytes (64). This transgenic mouse

displays skin abnormalities that correlate with KIDS pathology and increased HC current in

keratinocytes, which undergo swelling, apoptosis and abnormal proliferation.

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The lens is an avascular organ whose homeostasis depends critically on the

function of an extensive network of GJCs and HCs. Indeed, mutations in Cx46 and Cx50

result in hereditary congenital cataracts (65,66). Defects in the trafficking, formation of

functional GJCs and hemichannel dysfunction have been linked to several mutations. The

N63S mutation in hCx46 that impaired GJCs formation induced much smaller HC currents

than the wild type but they increased however dramatically when the concentration of

divalent cations was reduced, suggesting an increased sensitivity to divalent cations

(45,67). In the case of the Cx50 mutants, normal HC currents in the absence of cell-cell

communication have been reported for the E48K mutation (68), while the W45S mutation

gives rise to non-functional HCs and GJCs despite the normal formation of gap junction

plaques (69). Expression of the G46V mutant, which reaches the plasma membrane and

promotes normal electrical coupling, provokes cell death due to strongly enhanced HC

currents compared to the wild-type Cx50 at physiological calcium concentrations, given the

increased voltage sensitivity of the mutant HCs (69,70). This abnormal HC activity persists

when the G46V mutant is co-expressed with wild-type Cx50 and Cx46, consistent with a

dominant gain-of-function at the HC level (69).

Oculodentodigital dysplasia (ODDD) is a rare and predominantly autosomal

dominant syndrome caused by mutations in Cx43 (71), a connexin expressed in multiple

cell types and that plays essential roles during embryonic development. ODDD mutations

are highly penetrant with intra- and interfamilial phenotypic variability and the spectrum of

phenotypic anomalies includes: ophthalmological, craniofacial, dental, digital and cardiac

malformations, arrhythmias, neurological affectation and skin disorders (reviewed in 72).

Non-functional HCs have been associated with six ODDD mutations, some of which are

linked with neurological abnormalities (Y17S, L90V, I130T, G21R, A40V and F52dup; ref.

73). By contrast, another four ODDD mutants that impair intercellular communication

(I31M, G138R, G143S and H194P) can form functional HCs and, with the exception of

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H194P, they enhance ATP release into the extracellular space (74). Such increased HC

activity correlates well with the longer half-life of the mutated Cx43 protein, as observed for

the G138R and G143S but not the I31M mutations (74). Furthermore, the increased HC

activity induced by the G138R mutation persists in cells derived from conditional

Cx43+/floxG138R heterozygous mice (embryonic stem cells, cardiomyocytes and astrocytes)

that co-express the mutant with wild type Cx43 (23,75). These mutations correspond to

those of patients exhibiting a phenotype characteristic of ODDD (the I31M and G138R

mutations), as well as those without facial abnormalities (the G143S mutation) or

syndactylies (the H194P mutation). Accordingly, it is difficult to infer clear genotype-

phenotype correlations from these studies of ODDD hemichannels.

In the cardiovascular system, ATP release via Cx37 HCs is associated with

reduced adhesion and decreased recruitment of monocytes to the vascular wall

endothelium, as well as the subsequent transmigration and progressive conversion to

macrophage foam cells, suggesting that HCs protect against atherosclerosis (76). The

S319 allelic variant is perhaps a polymorphic prognostic marker for atherosclerotic plaque

development (77), and it results in decreased HC currents and reduced ATP release by

HCs, probably due to a decrease in HC unitary conductance and increased cell

adhesiveness (31).

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CONCLUDING REMARKS AND PERSPECTIVES

There is growing evidence that the ubiquitous presence of open hemichannels in the

non-junctional plasma membrane is a normal phase of the connexin life cycle. These HCs

fulfill physiologically important roles and they are also activated during pathological

processes. The transfer of ions and other small molecules between the cell interior and the

interstitial space is dependent on the expression of solitary HCs at the cell surface, as well

as their open probability, unitary conductance and permeability, phenomena that are

regulated by multiple stimuli including the membrane potential and external calcium

concentration. In turn, the pool size of solitary HCs is the end result of the balance

between the total number of HCs delivered to the plasma membrane and the proportion of

HCs that undergo internalization or are incorporated in a non-reversible manner into GJCs.

HC activity is strictly controlled by the membrane potential. Two distinct voltage

gating mechanisms operating in series regulate the transition of HCs between different

conductive states. A gating mechanism inherent to all functional HCs (referred to as “slow”

or “loop” gating) mediates the transitions to and from the fully closed state, and it controls

the opening and closing of HCs in response to depolarization and hyperpolarization. A

second voltage-gating mechanism (referred to as “fast” or “Vj” gating) mediates transitions

from the fully open state to a subconductance or residual state in response to negative or

high positive polarizations, depending on the connexin isoform, resulting in unipolar or

bipolar voltage behavior, respectively. Voltage-gated HCs are also regulated by the

external calcium concentration. The combination of single HC recordings and mutagenesis

has demonstrated that Ca2+ regulation in the Cx32-HC is more complex that initially

thought, and that it is mediated by a specific Ca2+-binding site formed by two aspartate

residues located within the extracellular vestibule of the pore. The binding of Ca2+ inhibits

HC opening to the fully open state and blocks ion conduction in a voltage-dependent

manner. The molecular determinants of this complex regulatory mechanism remain poorly

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understood, and it remains to be determined whether fundamental differences in voltage

gating and Ca2+ regulation exist between different connexin HCs.

Mutated HCs may play an important role in the pathogenesis of genetic diseases

caused by mutations in Cx genes or “connexinopathies”. As discussed here, disease-

related mutations can impair the expression of HCs in the plasma membrane, modulate

the number of solitary HCs, or alter the voltage-gating, Ca2+-regulation, or permeation of

HCs. Mutants that cause cellular toxicity due to enhanced hemichannel activity are

particularly relevant, and mutations that give rise to leaky HCs are generally associated

with severe clinical phenotypes. Further studies are required to better characterize

disease-related HCs and to develop specific HC-targeting drugs for therapeutic

intervention.

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ACKNOWLEDGEMENTS

This work was supported by grants from the Spanish Ministry of Science and

Technology (SAF-2009/1164 and Consolider CSD2008-00005 to LCB) and the Community

of Madrid (Neurotec-P2010/BMD-2460 to JM, LCB and DGN). J.M.G. is an investigator for

FIBio-HRC supported by the Comunidad Autónoma de Madrid.

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(GJA1) mutations cause the pleotropic phenotype of oculodentodigital dysplasia. Am. J.

Hum. Genet. 72, 408-418.

72. Paznekas, W.A., Karczeski, B., Vermeer, S., Lowry, R.B., Delatycki, M., Laurence, F.,

Koivisto, P.A., Van Maldergem, L., Boyadjiev, S.A., Bodurtha, J.N., Jabs, E.W., 2009.

GJA1 mutations, variants, and connexin 43 dysfunction as it relates to the

oculodentodigital dysplasia phenotype. Hum. Mutat. 30, 724-733.

73. Lai, A., Le, D.N., Paznekas, W.A., Gifford, W.D., Jabs, E.W., Charles, A.C., 2005.

Oculodentodigital dysplasia connexin43 mutations result in non-functional connexin

hemichannels and gap junctions in C6 glioma cells. J. Cell Sci. 119, 532-541.

74. Dobrowolski, R., Sommershof, A., Willecke, K., 2007. Some oculodentodigital

dysplasia-associated Cx43 mutations cause increased hemichannel activity in addition

to deficient gap junction channels. J. Membrane Biol. 219, 9-17.

75. Dobrowolski, R., Sasse, P., Schrickel, J.W., Watkins, M., Kim, J-S., Rackauskas, M.,

Troatz, C., Ghanem, A., Tiemann, K., Degen, J., Bukauskas, F.F., Civitelli, R., Lewalter,

T., Fleischmann, B.K., Willecke, K., 2008. The conditional connexin43G138R mouse

mutant represents a new model of hereditary oculodentodigital dysplasia in humans.

Hum. Mol. Genet. 17, 539-554.

76. Wong, C.W., Christen, T., Roth, I., Chadjichristos, C.E., Derouette, J.P., Foglia, B.F.,

Chanson, M., Goodenough, D.A., Kwak, B.R. 2006. Connexin37 protects against

atherosclerosis by regulating monocyte adhesion. Nat Med. 12, 950-954.

77. Boerma, M., Forsberg, L., Van Zeijl, L., Morgenstern, R., De Faire, U., Lemne, C.,

Erlinge, D., Thulin, T., Hong, Y., Cotgreave, I.A. 1999. A genetic polymorphism in

connexin 37 as a prognostic marker for atherosclerotic plaque development. J Intern

Med. 246, 211-218.

78. Barrio, L.C., Suchyna, T., Bargiello, T.A., Xu, L.X., Roginski, R. S., Bennett, M.V.L.,

Nicholson, B.J., 1991. Gap junctions formed by connexin 26 and 32 alone and in

combination are differently affected by applied voltage. Proc. Natl. Acad. Sci. USA 8,

8410-8414.

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FIGURE LEGENDS

FIGURE 1. Life cycle of connexin hemichannels at the cell surface. Experimental

design to estimate the expression of solitary HCs at the cell surface and their incorporation

into complete GJCs (modified from 9). After injection of hCx32 cRNA, a group of oocytes is

maintained in isolation (A) while another group is permitted to form cell-to-cell contacts in

pairs to promote the formation of GJCs (B). Control oocytes are injected with an antisense

oligonucleotide to block endogenous Cx38 expression (78). The HC pool size is estimated

from the amplitude of the slowly activating currents (Im) induced by depolarizations (Vm,

from -40 to +120 mV, 10 sec duration) in both isolated and paired oocytes (oocytes 1 and

2). The pool size of newly formed GJCs is determined by measuring junctional currents (Ij)

evoked by transjunctional voltages (Vj; from 0 to +160 mV, 10 sec duration). (C) Time

course of HC expression at the cell surface estimated through the macroscopic

hemichannel conductance (gjh). The presence of functional HCs in isolated cells is

detectable after a short latency, and it increases rapidly before slowly declining over time

after injection (red). The pool size of solitary HCs in the uncoupled pairs formed between

Cx32 and control oocytes (green) exhibits a similar profile. (D) Time course of Cx32 GJC

formation. Junctional conductance (gj) increases progressively in parallel with the

decrease in solitary HCs (in C, orange). Note that a significant proportion of HCs can still

be detected as solitary HCs at the plasma membrane of each paired cell throughout the

process of GJC formation.

FIGURE 2. Differential effects of human Cx26 and Cx43 hemichannel expression on

plasma membrane properties in unclamped conditions. (A) Human Cx26, but not

Cx43 or control oocytes, incubated in normal medium (ND96 solution containing 1.8 mM

Ca2+ and 1 mM Mg2+) undergo a progressive depolarization of their resting potential (i) and

a reduction in the input membrane resistance (ii) over time following cRNA injection

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(modified from 18). (B) Lowering the external divalent cation concentration (free-Ca2+ and

Mg2+) available to Cx43 oocytes rapidly depolarizes the resting potential and decreases

input resistance (arrows), and both these membrane parameters recover their initial values

on returning to normal millimolar [Ca2+]e (unpublished data). (C) Propidium iodide (1 mM)

uptake after a 30 min of incubation. Cx26 oocytes are very permeable to the dye in normal

medium, while little dye uptake is observed into Cx43 oocytes, although uptake increases

significantly at low divalent cation concentrations. Scale bars = 200 µm.

FIGURE 3. Voltage dependency of connexin hemichannels. Unipolar voltage behavior

of human Cx32 HCs (A-C) and bipolar behavior of human Cx26 HCs (D-F) in normal

medium (1.8 mM Ca2+ and 1 mM Mg2+: modified from 9 and 18). (A and D) Macroscopic

HC currents (Im) activated by depolarization of the membrane potential (Vm). (B and E)

Graphs showing HC conductance vs. voltage (Ghj/Vm). (C and F) Unitary HC currents.

Activation of both Cx32 and Cx26 HCs initially increases with the degree of depolarization.

However, while Cx32 currents continue to increase monotonically across the entire

negative and positive voltage range due to the continuous recruitment of single open HCs,

Cx26 HCs are inactivated at higher positive potentials due to the transition from the main

open state to a subconductance or residual open state (arrows in D-F). The bipolar voltage

behavior of Cx26 HCs is consistent with a model of two gates in series operating with

opposing polarities of closure that mediate transitions between different conductance

states.

FIGURE 4. Ca2+ regulation of human Cx32 hemichannels. (A and B) Single HC

recordings in cells attached of oocytes expressing Cx32, using a pipette containing ND96

solution containing: (i) 1.8 mM Ca2+ and 1 mM Mg2+; or (ii) free-Ca2+ and Mg2+. HC opening

upon depolarization (+80 mV) at normal Ca2+ and Mg2+ concentrations is mediated by

transition to a conductance substate of ~18 pS (γ18). At low divalent cation levels, Cx32

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HCs open to conductance states of ~18 pS and ~90 pS (γ90), indicating that external

divalent cations block HC transitions to the fully open state (in A). Deactivation upon

hyperpolarization in normal external solution (i) involves frequent and recurrent transitions

between the fully closed state (c) and the 18 pS open state (γ18), as well as brief opening

times with flickering unitary currents (in B, i). By contrast, at low divalent cation

concentrations (ii), HCs in the very stable (90 pS) open state first undergo a transition to a

long-lasting open 18 pS state before closing completely. (C) The Ca2+-binding site of Cx32

HCs is formed by residues D169 and D178 within the second extracellular loop. HCs in

which Asp is substituted by Asn at either position (i and ii) behave like wild-type HCs at

physiological divalent cation concentrations in the absence of divalent cations (modified

from 35).

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5 s

2µA

100mV

5 s

2µA

100mV

2µA

2µA

24 h.a.i. 48 h.a.i. 72 h.a.i. 144 h.a.i.

48 h.a.i. 72 h.a.i. 144 h.a.i.

24 h.a.p. 48 h.a.p. 120 h.a.p.

oocyte 1

oocyte 2

Vm

Im

Ij

Vm/Vj

Im

Im

time after cRNA injection (hours)

0 24 48 72 96 120 144

ghj

(µS)

0

10

20

30

40

50

60

0 24 48 72 96 120 144

0

10

20

30

40

50

60

70

time after cRNA injection (hours)

gj

(µS)pairing time

isolatedH32-H32H32-control

Figure 1

A

B

C

D

Isolated oocytes

Paired oocytes

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A B

C

Figure 2

0 24 48 72

-60

-50

-40

-30

-20

-10

0

Mem

bra

ne

Po

ten

tial

(mV

)

Time after injection (hours)

hCx26hCx43control

0 24 48 72

0.00.10.20.30.40.50.60.70.80.91.01.11.2

Inp

ut

Res

ista

nce

(M

Ohm

)

Time after injection (hours)

i) ii)ND96

0 Ca2+ 0 Mg2+ND96

1.8 Ca2+ 1 Mg2+ND96

1.8 Ca2+ 1 Mg2+

hCx43

10 mV

20 s

control

- 51 mV0.5 MOhm

- 18 mV0.1 MOhm

hCx43

hCx43

0 Ca2+ 0 Mg2+

hCx431.8 Ca2+ 1 Mg2+

hCx261.8 Ca2+ 1 Mg2+

control

1.8 Ca2+ 1 Mg2+

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Figure 3

Ghj

Vm

-40 -20 0 20 40 60 80 100120

0.0

0.2

0.4

0.6

0.8

1.0

1.2

-40 mV

+120 mV

5 s

µA

mV

100

5

Vm

Im2 s

5pA

+80

+100

+40+20 mV

+120

-40 mV

+60

-40 mV

A B C

hCx32

-80 mV

+80 mV 100mV

400nA

5 s

+60+80

+60

+80

Vm

Im

hCx26

D E FV(mV)

I (pA)

0

0

+ 80

- 80

- 20

- 10

+10

+20

+30

γ1 = 432 pS

γ2 = 877 pS

-80 -60-40-20 0 20 40 60 80

0,0

0,2

0,4

0,6

0,8

1,0

Ghj

Vm

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c

γ18

1.8 Ca2+ + 1 Mg2+

A

2 s

4 pA

0 Ca2+ + 0 Mg2+

γ18

γ90

c2 s

Figure 4

C

5 pA

c

90γD169N D178N

+60 mV

5 pA

2 s

-40 mV

2 s

18γ

90γ18γ c

B1.8 Ca2++ 1 Mg2+

10 s

5 pA

c

0 Ca2+ + 0 Mg2+

γ18

cγ18

γ90

i) ii)

i)

ii)

i) ii)

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TABLE 1. Hemichannel dysfunctions in “connexinopathi es”

Connexin

HEMICHANNELS (HC) PATHOLOGY Inheritance

pattern Gene Protein Mutation Domain

HC activity Voltage sensitivity

External Ca2+sensitivity

GAP JUNCTION CHANNELS

(GJC) References

Myelin disorders

D178Y E2 � increased HC currents

� rectifying unitary conductance

slightly increased

abolished (fully open conductance at normal Ca2+)

very low electrical coupling due to defective docking

35

E208K CT no HC currents -- -- no electrical coupling 9 Y211stop CT no HC currents -- -- no electrical coupling 9 R215W CT no HC currents -- -- no electrical coupling 9 C217stop CT

increased HC currents

greatly reduced nd

� very low electricalcoupling due to defective docking

� greatly increased Vj sensitivity

9

R220 stop CT increased HC currents

reduced nd � lower electrical coupling

due to defective docking � increased Vj sensitivity

9

F235C CT � increased HC currents

� normal unitary conductance

� cell death

greatly increased

nd � higher electrical coupling

due to enhanced docking � normal Vj sensitivity

21

R238H CT normal HC currents

normal nd � normal electrical coupling � normal Vj sensitivity

9

R265stop CT increased HC currents

slightly reduced nd

lower electrical coupling due to defective docking 9

C280G CT normal HC currents normal nd normal electrical coupling 9

X-linked Charcot-Marie-Tooth disease (CMTX)

X

GJB1 Cx32

S281stop CT normal HC currents

normal nd slightly lower electrical

coupling due to defective docking

9

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Connexin HEMICHANNELS

(HC) PATHOLOGY

Inheritance pattern

Gene Protein Mutation Domain

HC activity Voltage sensitivity

External Ca2+

sensitivity

GAP JUNCTION CHANNELS

(GJC) References

A98G_ V99insT

CL greatly reduced HC currents nd nd no GJ plaques 55

G149S CL greatly reduced HC currents

nd nd GJ plaques 55

G236S E2 greatly reduced HC currents nd nd no GJ plaques 55

T265A M4 greatly reduced HC currents

nd nd no GJ plaques 55

Pelizaeus-Merzbacher-like Disease (PMLD)

AR GJC2

Cx47

T398I CT reduced HC currents

nd nd GJ plaques 55

Cataracts AD GJA3 Cx46 N63S E1 reduced HC

currents normal increased � no GJ plaques � no electrical coupling 45,67

W45S E1 no HC currents nd nd � GJ plaques � no electrical coupling

69

E48K* E1 normal HC currents

nd nd no electrical and dye

coupling 68

Zonular pulverulent cataracts AD GJA8 Cx50

G46V* E1 � increased HC currents

� normal unitary conductance

� cell apoptosis

increased normal � GJ plaques of � normal electrical coupling

69, 70

Deafness and/or skin disorders

Non-syndromic deafness

AR GJB2 Cx26 E47K* E1 no dye uptake nd nd no intercellular Ca2+ transfer 57

Deafness and palmoplantar keratoderma (PPK)

AD GJB2 Cx26 R75W E1 � reduced HC currents

� normal unitary properties

� cell lysis

increased normal � normal/reduced GJ

plaques � no electrical coupling

42,58,59

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Connexin HEMICHANNELS

(HC) PATHOLOGY Inheritance

pattern Gene Protein Mutation Domain HC activity Voltage sensitivity

External Ca2+

Sensitivity

GAP JUNCTION CHANNELS

(GJC) References

G12R NT � increased HC currents

� cell death nd nd no electrical coupling 62

S17F NT � no HC currents � no cell death

-- -- � no dye coupling � no electrical coupling

62

A40V M1 � increased HC currents

� cell death

slighly increased

reduced � electrical coupling � normal Vj sensitivity

43,60,62

� increased dye uptake

� increased HC currents

� increased Ca2+ permeation

� higher unitary conductance

� apoptosis, cell death

increased reduced

� dye coupling and Ca2+ transfer

� electrical coupling � increased voltage

sensitivity

43,57,62

G45E* E1

Cx26G45E ketatinocytes: � increased

currents � swolling,

apoptosis, cell death and proliferation

nd nd nd 64

Keratitis-ichthyosis-deafness syndrome (KID) and Hystrix-like ichthyosis deafness syndrome (HID)

AD GJB2 Cx26

D50N E1 � increased HC currents

� cell death nd reduced no electrical coupling 62

G11R NT increased HC currents and ATP release

abnormal nd � dye coupling � electrical coupling � abnormal Vj sensitivity

61 Clouston syndrome: hidrotic ectodermal dysplasia without hearing loss

AD GJB6 Cx30

A88V M2 Increased HC currents and ATP release

nd nd � dye coupling � lower electrical coupling � increased Vj sensitivity

61

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Connexin HEMICHANNELS

(HC) PATHOLOGY Inheritance

pattern Gene Protein Mutation Domain HC activity Voltage sensitivity

External Ca2+

Sensitivity

GAP JUNCTION CHANNELS

(GJC) References

Syndromic oculodendrodigital dysplasia

Y17S NT no dye uptake -- -- � reduced GJ plaques � no dye transfer

73

G21R M1 no dye uptake -- --

� reduced GJ plaques � no dye transfer 73

I31M M1 increased ATP release

nd nd � GJ plaques � no dye transfer

74

A40V* M1 no dye uptake -- --

� reduced GJ plaques � no dye transfer 73

F52dup E1 no dye uptake -- -- � very reduced GJ plaques � no dye transfer

73

L90V M2 no dye uptake -- -- � reduced GJ plaques

� no dye transfer 73

I130T CL no dye uptake -- -- � reduced GJ plaques � no dye transfer

73

increased ATP release

nd nd

� GJ plaques � no dye transfer 74 G138R CL

Cx43/G138R cells: increased ATP release

nd nd

� low dye coupling � Cx43-G138R heterotypic

channels: asymmetric Vj sensitivity

23,75

G143S CL increased ATP release nd nd � GJ plaques

� no dye transfer 74

Oculodendrodigital dysplasia (ODDD)

AD GJA1 Cx43

H194P E2 normal ATP release

nd nd � no GJ plaques � no dye transfer

74

Cardiovascular diseases

Atherosclerosis

allelic variant GJA4 Cx37 S319 CT � reduced HC

currents � reduced ATP

release

as P319 allelic variant

nd

� electrical coupling � increased Vj sensitivity � smaller unitary

conductance than P319 variant

31

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Abbreviations: AD, autosomal dominant; AR, autosomal recessive; X, X-linked; NT, amino terminus; M1-4, transmembrane domains; E1, E2, extracellular loops; CL, cytoplasmic loop; CT, carboxy terminus; Vj, transjunctional voltage; nd, not determined; * different mutations in an equivalent position (e.g., Cx26G45E and Cx50G46V); * same mutation in an equivalent position (e.g., Cx26E47K and Cx50E48K).