Review

Next-Generation Connexin
and Pannexin Cell Biology
Jessica L. Esseltine and Dale W. Laird*

Connexins and pannexins are two families of large-pore channel forming pro-
Trends
teins that are capable of passing small signaling molecules. While connexins
Connexins and pannexins routinely
serve the seminal task of direct gap junctional intercellular communication, localize and function at the plasma
pannexins are far less understood but function primarily as single membrane membrane but may also have func-
tional roles in the mitochondria,
channels in autocrine and paracrine signaling. Advancements in connexin and nucleus, and endoplasmic reticulum.
pannexin biology in recent years has revealed that in addition to well-described
classical functions at the plasma membrane, exciting new evidence suggests Truncated polypeptides of connexins
and pannexins have been identified
that connexins and pannexins participate in alternative pathways involving that alter channel function and cellular
multiple intracellular compartments. Here we briefly highlight classical functions localization.
of connexins and pannexins but focus our attention mostly on the transforma-
Common members of the metabolome
tive findings that suggest that these channel-forming proteins may serve roles pass through both connexin and pan-
far beyond our current understandings. nexin channels.

Pannexins and connexins have been

Introduction convincingly connected to a multitude
Communication between adjacent cells as well as between cells and their environment is a of diseases and inherited connexin-
crucial aspect of development and perturbations of these processes are associated with multiple linked diseases can be both common
and rare affecting nearly every major
pathological states. Major mechanisms of cellular communication involve connexin-based gap
organ.
junctional intercellular communication (GJIC), as well as pannexin- and connexin-linked para-
crine signaling. The 21-member family of connexin proteins share a similar topology consisting Connexins are attractive therapeutic
of cytosolic amino and carboxyl termini, four membrane-spanning domains, two extracellular targets for a variety of pathologies
including chronic wounds and cardiac
loops, and one intracellular loop [1]. Connexin proteins assemble into hexameric arrangements reperfusion injury, while pannexin ther-
in the endoplasmic reticulum or Golgi apparatus, which are then transported as connexons, or apeutics have yet to emerge.
hemichannels, to the cell surface (Figure 1, upper panel) [2]. Typically, connexons from one cell
seek to partner and dock with connexons from adjacent cells to form gap junctional intercellular CRISPR/Cas9-mediated gene editing
and patient-derived iPSCs will enhance
channels. Gap junctions allow the passage of ions, metabolites, small molecules, and second the understanding of connexin-linked
messengers directly between cells, without exposure to the extracellular environment [3]. These diseases.
channels tend to cluster into large crystalline-like structures between cells termed gap junction
plaques. Many of the connexin family members are short lived and undergo constant renewal
by being internalized into connexosomes (also called annular junctions) prior to lysosomal
degradation. This rapid turnover mechanism predicts that the entire gap junctional content
of the heart, as an example, is renewed on a daily basis [4]. The life cycle of connexins is further
guided by an elaborate interactome and connexin post-translational modifications that are
reviewed elsewhere [5].

The pannexin family of large-pore channels consists of only three members (Panx1, Panx2, and
Department of Anatomy and Cell
Panx3) that share similar topology to connexins including four transmembrane domains with Biology, Schulich School of Medicine
cytoplasmic amino and carboxyl termini [6]. Like connexins, pannexins undergo hexameric and Dentistry, The University of
oligomerization early in the secretory pathway, although some evidence suggests that Panx2 Western Ontario, London, ON, Canada

may form an octamer [6,7]. Pannexins appear to follow the classical secretory pathway prior to
appearing as functional channels at the plasma membrane (Figure 2, upper panel) [8]. Pannexin *Correspondence:
channels have been well documented to release ATP but likely serve more generic roles in small dale.laird@schulich.uwo.ca (D.W. Laird).

944 Trends in Cell Biology, December 2016, Vol. 26, No. 12 http://dx.doi.org/10.1016/j.tcb.2016.06.003
© 2016 Elsevier Ltd. All rights reserved.
 Classical connexin biology

(A)

(C) (B)

(G)

(D)
(E)

(D) (F)

Alternave connexin biology

Figure 1. Traditional Paradigms Make Room for New Theories in Connexin Biology. Upper cell: classical
connexin biology. (A) Connexins are cotranslationally inserted into the endoplasmic reticulum and assemble into hexameric
connexons or hemichannels in the endoplasmic reticulum or Golgi apparatus. (B) Connexons traffic to the cell surface where
they pair to form gap junctional intercellular channels, which tend to cluster into large gap junction plaques. (C) Members of
the metabolome pass through intercellular gap junction channels without exposure to the extracellular environment. Lower
cell: alternative connexin biology. (D) In addition to full-length connexins, truncated connexin fragments may be generated
through the use of internal translation initiation sites. These regulatory connexin fragments may be found within the
cytoplasm, while other connexin fragments have been reported in the nucleus. (E) Undocked hemichannels are found at
the cell surface where they participate in small molecule release or uptake. (F) Cx43 localized to the inner membrane of
the mitochondria has been reported. (G) In addition to members of the metabolome, much larger noncoding RNAs have
been shown to pass through connexin channels. Pink depicts closed/inactive connexons while blue denotes open/active
channels.

Trends in Cell Biology, December 2016, Vol. 26, No. 12 945

 Classical pannexin biology

((B))
(A)

((C))

(E)

(D)

Alternave pannexin biology

Figure 2. Established and Emerging Theories in Pannexin Biology. Upper cell: classical pannexin biology. (A)
Pannexins are cotranslationally inserted, glycosylated, edited, and oligomerized into hexameric channels while in the
endoplasmic reticulum, prior to further editing upon delivery to the Golgi apparatus. (B) Pannexin channels are trafficked to
the cell surface where they act in molecular release and uptake. Lower cell: alternative pannexin biology. (C) Several Panx1
spice variants have been described some of which appear to regulate Panx1 assembly and function. (D) Panx3 has been
shown to have a functional role in releasing Ca2+ from the endoplasmic reticulum. (E) All pannexins have been found in
intracellular compartment (in addition to the plasma membrane) where they serve poorly understood functions. Pink depicts
closed/inactive pannexin channels while blue represents open/active channels; Channels with black dots denote splice
variants of pannexins.

946 Trends in Cell Biology, December 2016, Vol. 26, No. 12

molecule release and even small molecule uptake as revealed by dye uptake assays [6,9,10].
Unlike most connexins, pannexin channels appear to be long-lived and remain in a closed state
until induced by voltage changes, pH, mechanical stretch, high potassium levels, or other stimuli
to transiently open [9,11,12].

Multiple recent reviews cover connexin and/or pannexin structure, trafficking, post-translational
modifications, and channel functions [5,6,13–16]. Here, we endeavor to focus on emerging, and
potentially transformative, aspects of connexin and pannexin biology that have drawn consid-
erable attention in the last decade. These topics include the in vivo impact of connexin hemi-
channels and their discernable functions from pannexin channels; the complexity of the
metabolome that passes through these channels; the atypical distribution patterns of connexins
and pannexins as it may relate to function; and the role of shorter polypeptide variants of these
channel proteins. Finally, we turn our attention to evidence linking connexins and pannexins to a
wide spectrum of diseases that involve the majority of human organs making these channel
proteins compelling targets for preclinical studies and for future therapeutics.

Connexins Function as Both Classical Gap Junctions and Hemichannels

Up until the 1990s, the classical function of connexins was limited to their role in GJIC. Evidence
then began to emerge suggesting that this definition was too restrictive and the physiological
functions of connexins should include undocked connexons at the cell surface, known now as
connexin hemichannels (Figure 1, lower panel) [17,18]. There remains little argument that
functional connexin hemichannels exist on the surface of cells as revealed by hundreds of
papers that have addressed their functional role, mostly in cultured cell models [17–21].
However, few studies have examined hemichannels in native tissues. In one study that com-
pared cells overexpressing connexins to native cells, measureable hemichannel activity was
reported only in the former [22]. This example highlights a long-standing question: where and
when do connexin hemichannels function in vivo? Some have reasoned that the functional
importance of connexin hemichannels may be more relevant in pathology [19,20]. This notion is
supported by the fact that most studies gate hemichannels open by bathing cells in low calcium,
which is a condition not routinely seen in healthy tissue environments. Conclusive visualization of
cell surface hemichannels in either primary cell cultures or in situ has been challenging, leaving
some to argue that their disperse nature on the cell surface makes them difficult to optically
detect. Virtually all imaging modalities have reported that the bulk of cell surface connexins are
localized to gap junction plaques, which appears to be the more energetically favorable, steady-
state localization site. Nevertheless, evidence supports a role of Cx46 and Cx50 hemichannels in
the lens while other in vivo studies suggest that astrocytic Cx43 hemichannels are active in brain
slice preparations [23–29]. Thus, there is a role for hemichannels in connexin biology that must
be fully understood. Going forward, the connexin community needs to uncover where, when,
and how connexin hemichannels operate in vivo. The field must also wrestle with the possibility
that only a subset of connexins form functional hemichannels in vivo, while others only transition
through a closed connexon state en route to gap junction channel and plaque formation.
Additionally, it will be important for the community to dissect and understand the overlapping
and independent functions of connexin hemichannels versus pannexin channels.

Pannexins Are an Independent Family of Cell Surface Large-Pore Channels

Given their 25% sequence homology to the invertebrate innexins, pannexins were originally
proposed to be a new family of gap junction proteins; a notion that was supported early by
studies that suggested pannexins made intercellular channels in Xenopus oocytes [30]. Sur-
prisingly, pannexin channels are blocked by many of the same reagents that inhibit connexin
hemichannels and connexin channels (e.g., carbenoxolone) suggesting that they may share
common gating mechanisms [31]. Overwhelming evidence now supports the position that
pannexin channels are remarkably different from connexin intercellular gap junction channels in

Trends in Cell Biology, December 2016, Vol. 26, No. 12 947

that they almost always form single membrane channels [32]. Despite our knowledge that
pannexin channels are conduits for single membrane passage, they have been referred to as
hemichannels, which infers an incomplete assembly state of half a channel. It is notable that
pannexins are extensively glycosylated on one of the extracellular domains, which would be
repulsive to the close apposition needed to seal docked pannexin channels across the extra-
cellular space [33,34]. Moreover, pannexins are frequently visualized at cell membranes where
there are no opposing cells, and they are rarely seen to cluster at the surface, further separating
them from the connexin family of gap junction proteins. An argument could also be made that
pannexins should not be lumped together with connexins at all. For example, CALHM1 channels
are also four transmembrane hexameric channels, which share some functional characteristics
with connexins and pannexins, but are considered a distinct channel family [35]. Going forward
we encourage the community to use the term channels and not hemichannels when referring to
pannexins. As a result of the overlap in the members of the metabolome that can pass through
both pannexin channels and connexin hemichannels, the community will be challenged to
dissect cellular functions linked to each channel type.

Molecules That Pass through Connexin and Pannexin Channels

Both connexin and pannexin channels have large pores with exclusion limits that may exceed
1 kDa depending on the physical shape, charge, and size of the molecule. Analysis of The
Human Metabolome Database reveals that there are over 35 000 biological molecules that
could theoretically pass through a pannexin channel, connexin channel, or connexin hemi-
channel (http://www.hmdb.ca/structures/search/metabolites/mass). There has been a large
push in the last 20 years to determine at least some of the signaling molecules, ions, and
metabolites that pass through specific gap junction channels and hemichannels [3]. It has
been well established that selectivity based on size and charge are determined by the
connexin composition of the channels and post-translational modifications of the constituent
subunits [3]. The pore size of different connexin channels was initially established by micro-
injecting fluorescent molecules of varying size into cells and observing the transfer of the dye
to adjacent cells. Additionally, passage of specific second messengers including cAMP, Ca2+,
and IP3 was monitored via fluorescent sensors, fluorescence resonance energy transfer
(FRET), biosensors, and radiolabelling [36–42]. Meanwhile, current assays for investigating
connexin hemichannels or pannexin channels involve cellular dye uptake, dye release, or ATP
release under conditions that presumably activate only one channel type. Caveats of these
assays include the report that the P2X7 receptor can facilitate dye uptake independently of
Panx1 in an agonist-dependent manner and that the permeability of connexin hemichannels
to dyes such as ethidium do not necessarily reflect channel permeability to physiologically
relevant molecules [22,43]. In addition to ATP metabolites, a wide variety of signaling
molecules like UTP and glutamate are also expected to pass through Panx1 channels, raising
the profile of how active Panx1 channels may regulate cellular functions [6,15]. Determining
the biological significance of small molecules that pass through homomeric, heteromeric, and
heterotypic connexin channels and pannexin channels is a daunting task that will require new
and innovative approaches that discern between these two distinct channel types [44–51].
Moreover, assigning function to one specific molecule when thousands of others may also be
involved is challenging.

Perhaps one of the most surprising discoveries in recent years was the finding that at least some
small, noncoding RNAs (siRNAs and miRNAs) can pass through gap junction channels
(Figure 1, lower panel) even though their molecular size exceeds the expected cutoff limit
of the connexin channel pore [52–65]. One can only speculate that miRNAs must linearize
and insert into the channel much like threading a needle. This was a revolutionary finding as
miRNAs serve to regulate gene expression during crucial cellular events including proliferation,
differentiation, and malignant transformation [53–65]. Given the many examples of miRNAs

948 Trends in Cell Biology, December 2016, Vol. 26, No. 12

passing through gap junction channels it will be interesting to determine if these same miRNAs
are excluded from pannexin channels that would serve to deliver these gene-regulating factors
to the extracellular milieu.

Connexin and Pannexin Localization in Unexpected Organelles and

Compartments
Classically, connexins follow a traditional secretory pathway prior to reaching the plasma
membrane and functioning as gap junction channels, but this may not always be the case.
In addition to dysregulation of connexin expression, connexin mislocalization or relocalization is
associated with numerous disease states, including various cancers and heart failure [66,67].
Aside from disease- or mutation-associated mislocalization, the possibility that connexins
could be localized to and function at cellular locations other than the plasma membrane is
an exciting concept (Figure 1, lower panel). Cx43 has been localized to the inner membrane
of myocardial subsarcolemmal mitochondria (SSM) of cardiomyocytes where it plays a role in
ischemic preconditioning during ischemia–reperfusion injury [21,68–81]. Ischemic precondi-
tioning of cardiac myocytes in rodent hearts increases Cx43 occupancy, phosphorylation and
S-nitrosylation at the inner SSM, and knocking out or blocking Cx43 in the myocardium
decreased the effectiveness of pharmacologically protective agents of ischemia–reperfusion
injury [21,68–81].

Cx43 fragments also localize to the nucleus, where they may serve to regulate gene transcription
or cell growth (Figure 1, lower panel) [82,83]. One study suggested that Cx43 translocated to the
nucleus during anaphase via its interaction with A-kinase anchoring protein 95, where it
regulated cell cycle progression [83]. While the notion that Cx43 fragments or even full-length
Cx43 may enter the nucleus has been contemplated for over a decade, the field awaits more
insights into the mechanisms involved as well as clarification as to the scope of cellular conditions
when this nuclear localization occurs.

In analogous studies, the cellular sites of pannexin function may be more complex than originally
anticipated (Figure 2, lower panel). Overexpressed and endogenous Panx1 or Panx3 are
generally localized at the cell surface regardless of whether the cell is physically in contact with
an adjacent cell [15,33,84,85]. However, antibody labeling for Panx1 and Panx3 has also
revealed their presence in poorly understood intracellular compartments [15,33,84,85]. In fact,
Panx3 has been proposed to play a role in calcium release from the endoplasmic reticulum [86].
Panx2 has been particularly difficult to consistently localize in vitro and in vivo because it has
frequently been found within intracellular compartments, although it appears that at least a
subpopulation of this unique family member does reach the cell surface [34,87,88]. Clearly, the
field has yet to fully understand the scope of compartments where pannexins may exhibit their
function. Moreover, we have little knowledge on how alternative splice variants or internal
translation initiation sites play a role in directing the localization of both connexin and pannexin
species.

Internal Translation Initiation Sites within Connexin-Encoding Genes and

Alternative Splice Variants of Pannexins
The classical immunoblot profile of the most studied connexin family member, Cx43, includes a
series of three protein bands ranging in molecular weight from 40 to 47 kDa, representing its
differential phosphorylation states. Occasionally, lower molecular weight Cx43 fragments have
been detected with antibodies specific to the C-terminal domain of Cx43. Originally thought to
be Cx43 degradation products, several groups have now proposed that these high-mobility
bands correspond to independent translation products produced via internal ribosomal entry
sites (IRESs) of inframe start codons within the connexin mRNA [89–96]. These Cx43 fragments
of varying lengths, including the predominant 20-kDa fragment found in mouse and human

Trends in Cell Biology, December 2016, Vol. 26, No. 12 949

cells, are sensitive to Cx43 knockdown strategies and are absent in Cx43 knockout animals
[90–96]. Meanwhile, an 11-kDa fragment of Cx55.5 has also been described in zebrafish
horizontal cells, generated from an inframe AUG within the larger Cx55.5 transcript and initiated
via an IRES element [94,96]. Cx55.5 fragments localize to the nuclei of zebrafish horizontal cells,
while Cx43 fragments are Triton soluble, residing mainly in the cytoplasm or in some association
with the endoplasmic reticulum [94,96,97]. However, the function of these Cx43 fragments
remains poorly understood. The Cx43 20-kDa fragment has been proposed to function as a
chaperone of full-length Cx43 protein, guiding its parent connexin to the cell membrane to
facilitate gap junction plaque formation (Figure 1, lower panel) [92]. These studies have the
potential to revolutionize the way we understand the genes that encode connexins as poly-
peptides that represent connexin fragments have real potential to be regulatory through
both dominant-negative and gain-of-function mechanisms [82,98–100] by competing for mem-
bers of the Cx43 interactome. For instance, the C-terminal fragment of Cx43 can regulate pH
gating of Cx43 channels [101,102]. That being said, caution is needed in interpreting the
importance of truncated Cx43 fragments as the C-terminal region of Cx43 can be particularly
unstable, and was even excluded from initial Cx43 protein isolations [103]. Additionally, numer-
ous immunoblots of many Cx43-positive primary cells, cell lines, and tissues do not reveal
these Cx43 fragments even when some of the best antibodies to the C-terminal tail domains
of Cx43 are used.

At present, there is no evidence that pannexin genes have IRES elements, but these genes
are encoded on four or five exons, and alternative splice variants may exist for one or more of
the pannexin family members (Figure 2, lower panel). Two isoforms of human Panx1, termed
Panx1b and Panx1bv, were found to result from alternative splicing of exon 5 but all of these
variants appeared to traffic normally to the cell surface [104]. Recently, two novel shorter
isoforms of Panx1, termed Panx1c and Panx1d, lacking sections of exon 2 and 4, respec-
tively, were identified in the pituitary gland [105]. These unique isoforms were found within
cytoplasmic compartments, which may help to explain why Panx1 is not always found at the
cell surface when localized with anti-Panx1 antibodies (Figure 2, lower panel). Whether splice
variants exists for Panx2 and/or Panx3 has yet to be determined, but such events may
provide insight into the variable spatial localization profiles of pannexins. When considering
the multiple links that connexins and pannexins have to diseases, it will be important to
determine if any of the clinical presentations of these diseases are due to the existence of
shorter polypeptide species of connexins and/or pannexins, and whether they are amenable
to therapeutics.

Inherited Connexin- and Pannexin-Linked Diseases

Collectively, mutations in over half of the connexin gene family cause a variety of diseases that
can affect nearly every human organ. The mechanisms linking connexin gene mutations to
syndromic or nonsyndromic disease are complicated and have been well reviewed elsewhere
[13,14,106]. There are currently 15 known inheritable disorders involving connexin gene muta-
tions, including a variety of skin diseases, X-linked Charcot Marie Tooth disease, congenital
cataracts, and other rare conditions [13,14,106–113]. In contrast to these rare disorders,
inherited mutations in GJB2 (Cx26) are directly responsible for congenital sensorineural hearing
loss; one of the most prevalent inherited diseases in the world [114–118]. In some populations,
nearly half of the children born with congenital hearing loss harbor mutations in GJB2 and/or
GJB6 (encoding Cx30) [13,116]. While reports of GJB2 gene mutations vary, several meta-
analyses have estimated that nonsyndromic GJB2-related hearing loss accounts for 1:5000 of
all births, with some regions reaching as high as 1:2000 [114–117]. Overall, the estimated
worldwide carrier rate of regionally prevalent GJB2 mutations is as high as one in 22 individuals
[114,117]. To put this into context, the carrier rates of CFTR mutations in the United States is
estimated to be one in 25 individuals [118].

950 Trends in Cell Biology, December 2016, Vol. 26, No. 12

The first and only human patient with an inherited autosomal recessive variant in PANX1 has
recently been identified and was found to suffer from multisystem pathologies [119]. Notably,
this PANX1 gene variant was only identified after whole-exome sequencing, which is now
emerging as standard practice in genetic clinics for patients with suspected hereditary diseases
or syndromes. With the onset of revolutionary strategies using clustered regularly interspaced
short palindromic repeats in conjunction with an RNA-guided DNA endonuclease (CRISPR-
Cas9) to edit genes and gene activation strategies, it is now possible to foresee a day when a
subset of these genetic deficiencies can be targeted for therapies.

Connexin and Pannexin Therapeutics

Most current pharmacological agents for connexin and pannexin inhibition are notoriously
nonspecific, making it difficult to ascribe causal relationships to a particular channel type or to
consider them in therapeutic designs. Thus, there is an undeniable need for more selective connexin
and pannexin inhibitors and activators with defined mechanisms of action. To that end, both
connexin antisense and peptide mimetics have emerged as specific regulators of connexins with
enormous therapeutic potential. Zealand Pharma has been a leader in connexin peptide mimetics
and have taken the gap junction channel activator, Danegaptide, into a Phase II proof-of-concept
clinical trial with the primary endpoint of assessing whether this mimetic can protect against cardiac
reperfusion injury in heart attack patients (http://www.fiercebiotech.com/press-releases/
zealand-announces-results-phase-ii-proof-concept-trial-danegaptide-cardiac). A new startup
company, FirstString Research, is currently engaged in clinical trials using a small peptide that
mimics the C-terminal of Cx43 as a therapeutic in tissue regeneration and chronic wound healing
(http://firststringresearch.com). These studies are further complemented by novel studies by CoDa
Therapeutics Inc. where an antisense oligonucleotide targeting Cx43 was found to improve wound
healing in phase 2b trials in patients with chronic venous leg ulcers (http://www.codatherapeutics.
com/news-nexagon-8-1-13.html) [120]. Still another company, Theranexus, is investigating the
value of drug-targeting connexins in narcolepsy (http://www.theranexus.com/news). The fact that
several companies have dedicated resources to blocking or enhancing gap junction channel or
hemichannel activity has raised the profile of connexins as viable therapeutic targets.

Panx1 has also been implicated in prevalent human pathologies including ischemia, metabolic
disease, and inflammation, making it a promising novel target for intervention [121]. However,
the targeting of pannexins in therapeutics remains in its infancy, owing in large part to their more
recent discovery. In two promising new studies, inhibition of Panx1 channels with carbenoxolone
reduced the efficiency of triple-negative breast cancer metastasis to the lungs in a preclinical
mouse model, while disrupting the association between NMDA-Src-Panx1 via a TAT-tagged
peptide protected against ischemic stroke in adult rats [12,122]. Given that Panx1 phosphor-
ylation by Src has recently been reported to induce channel opening, this particular peptide may
also be a useful Panx1 channel inhibitor [122–124]. In another study, Panx3 ablation inhibited the
onset of surgically induced osteoarthritis, raising the possibility that a peptide therapeutic
specifically inhibiting Panx3 channels may provide treatment for a poorly controlled disease
[125,126]. While these studies are encouraging, new and more specific inhibitors of pannexin
channels are necessary. Importantly, these novel therapeutics should serve the research
community well as the field continues to also develop an arsenal of connexin- and pan-
nexin-specific reagents.

Human Models to Investigate Connexin Diseases

Insights into the functional importance and mechanisms governing the regulation of con-
nexins has been obtained through the use of cultured cell models and the use of genetically
modified mice engineered to lack a specific connexin or to express a disease-causing
connexin mutant. While these approaches have advantages, they often do not adequately
represent the human condition and are not necessarily well designed for drug screening. To

Trends in Cell Biology, December 2016, Vol. 26, No. 12 951

 Directed differenaon
disease modeling
Bone Neural

Drug
discovery
Cardio Carlage

Rx
Connexin-linked
disease

Primary paent Paent-derived iPSCs

Cells
Reprogramming
hOct4, hKlf4, hSox2, hc-Myc

Regenerave
medicine
Gene eding
CRISPR – + – +

GJA1 Uncut Cx43

GJA1 Cut GAPDH

PCR WB

Figure 3. Human Models to Investigate Connexin-Linked Disease. Mutations in the connexin gene family contribute
to 15 inherited diseases that include oculodentodigital dysplasia, which is caused by mutations in the GJA1 gene encoding
Cx43. Fluorescent micrographs highlight Cx43 expression (green; blue = nuclei) in human dermal fibroblasts as well as in
human iPSCs. iPSCs from patients and familial controls can be differentiated into a variety of cell types including osteoblasts
(Alizarin red staining denotes calcification as seen in bone), chondrocytes (Alcian blue staining denotes proteoglycan
deposition as seen in cartilage), cardiomyocytes (/-actinin, red) and neurons (unstained phase contrast image). CRISPR-
Cas9-mediated gene editing can be used for gene deletion (shown is GJA1/Cx43 ablation in AD293 cells; green bands
denote Cx43, red bands denote GAPDH) as well as fine genome editing to either repair or insert specific disease-related
mutations. Together with novel strategies for drug screening, these powerful new technologies provide the platform for
personalized regenerative medicine targeting connexin-linked diseases. Scale bar = 100 mm. Abbreviations: PCR, poly-
merase chain reaction; WB, western blotting.

overcome these limitations, it is now possible to obtain primary cells from patients that
harbor connexin-linked diseases and assess how differential changes in connexin function
lead to changes in gene expression profiles and cellular phenotypes (Figure 3). A comple-
ment to these approaches involves iPSCs, wherein commercially available kits enable the
reprogramming of connexin-linked patient cells into iPSCs. The signaling pathways that
regulate the differentiation of iPSCs into neurons, osteoblasts, chondrocytes, cardiomyo-
cytes, or any specialized cell type can be mechanistically studied and novel drugs can be
used in an attempt to rescue defective cell differentiation pathways caused by the cells
harboring connexin gene mutations. Combined with the ability to use CRISPR-Cas9 to repair
connexin-disease-linked gene mutations, this approach should revolutionize the way we
examine connexin-linked diseases that affect millions of people (Figure 3). Already it has
become clear that Cx43 is a master gene highly expressed in iPSCs that is differentially
regulated depending on the differentiation pathway activated [127–131]. In the event that a
population of inherited pannexin gene mutations are found to exist in society, a similar
approach could prove invaluable in the quest for novel treatments.

952 Trends in Cell Biology, December 2016, Vol. 26, No. 12

Concluding Remarks Outstanding Questions
Fifty years of gap junction and connexin research has resulted in a remarkable number of How and when do hemichannels
discoveries that have demonstrated their importance in virtually every human organ. The fact that selectively open in vivo and does
this occur primarily in pathologies
mutations in the genes that encode these large-pore channels inevitability lead to disease further or states of cell injury or does it also
emphasizes their importance in human health. Our knowledge of pannexins, in contrast, is less occur routinely in healthy tissues?
advanced, but they are already known to be linked to over a dozen human diseases by
mechanisms that have not been fully elucidated. The existence of pannexins has caused Probably one of the most fundamental
questions in the field relates to under-
connexin biologists to systematically consider which cellular functions linked to large-pore
standing when and where connexin
channels are due to connexins or pannexins; a process that will continue to improve with hemichannels function in situ and what
the advent of new and more specific pharmacology (see Outstanding Questions). fully distinguishes them functionally
from pannexin channels.
Acknowledgments
The authors thank Sara Bober for her artistic help in generating Figures 1 and 2. This study was supported by grants What biological molecules pass
through connexin and pannexin
(130530 and 123228) from the Canadian Institutes of Health Research to DWL.
channels in vivo?

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