1. Introduction 3261.1 What? Terminology and general properties 3271.2 Why? Reasons for biophysical study 3291.3 How? Special issues for study of connexin channels 3302. Molecular and structural context 3312.1 Biochemical features 3312.2 Structures 3342.2.1 Junctional channels 3352.2.2 Hemichannels 3382.2.3 Heteromeric channels 3422.2.4 Junctional plaques 3473. Experimental approaches and issues specific to study of connexin channel physiology 3493.1 Macroscopic currents 3493.1.1 Junctional channels 3493.1.2 Hemichannels 3543.2 Single-channel currents 3553.2.1 Junctional channels 3553.2.2 Hemichannels 3583.3 Molecular permeability 3613.3.1 A selection of tracers 3613.3.2 Junctional channels 3623.3.3 Hemichannels 3663.4 Other 3674. Structural issues 3684.1 What lines the pore? 3684.2 Docking between hemichannels 3734.2.1 Structural and molecular basis 3744.2.2 Determinants of specificity of interaction 3805. Permeability and selectivity 3815.1 Among the usual ions 3835.1.1 Unitary conductance 3835.1.2 Selectivity 3845.1.3 Nonlinear single-channel I–V relations and their molecular determinants 3865.2 Among large permeants 3915.2.1 Uncharged molecules 3925.2.2 Charged molecules 3935.2.3 Cytoplasmic/signaling molecules 3966. Voltage sensitivity 3996.1 Macroscopic transjunctional voltage sensitivity 4046.2 Microscopic voltage sensitivity – Vj-gating 4076.2.1 Molecular basis – voltage sensor 4076.2.2 Molecular basis – transduction and/or state stability 4096.3 Microscopic voltage sensitivity – loop gating 4126.4 Vm-gating 4147. Direct chemical modulation 4157.1 Phosphorylation 4177.2 Cytoplasmic pH and aminosulfonates 4197.3 Calcium ion 4247.4 Lipophiles 4247.4.1 Long chain n-alkyl alcohols 4257.4.2 Fatty acids and fatty acid amides 4267.4.3 Halothane 4267.5 Glycyrrhetinic acid and derivatives 4277.6 Cyclic nucleotides 4287.7 Other candidates 4308. Connexinopathies 4319. Summary 43510. Acknowledgements 43811. References 438Connexins are the proteins that form the intercellular channels that compose gap junctions in vertebrates. Connexin channels mediate electrotonic coupling between cells and serve important functions as mediators of intercellular molecular signaling. Convincing demonstration of the latter function has been elusive, as have the experimental tools required for detailed functional study of the channels. Recently, substantial progress has been made on both fronts. Connexin channels are now known to be dynamic, multifunctional channels intimately involved in development, physiology and pathology, and amenable to study by state-of-the-art approaches. A host of developmental and physiological defects are caused by defects in connexin channels, and therefore in the intercellular molecular movement they mediate. The channel structure has been determined to 7·5 Å resolution within the plane of the membrane. Experimental paradigms have been developed that enable application of the tools of modern channel biophysics to study connexin channel structure–function. As a result, the biophysical mechanisms and biological functions of connexin channels now enjoy a vigorous and expanding experimental interest. This article focuses on the former, but with attention to issues likely to have biological consequences.

Connexin channels are known to be permeable to a variety of cytoplasmic molecules. The first observation of second messenger junctional permeability, made ∼30 years ago, sparked broad interest in gap junction channels as mediators of intercellular molecular signaling. Since then, much has been learned about the diversity of connexin channels with regard to isoform diversity, tissue and developmental distribution, modes of channel regulation, assembly and expression, biochemical modification and permeability, all of which appear to be dynamically regulated. This information has expanded the potential roles of connexin channels in development, physiology and disease, and made their elucidation much more complex -30 years ago such an orchestra of junctional dynamics was unanticipated. Only recently, however, have investigators been able to directly address, in this more complex framework, the key issue: What specific biological molecules, second messengers and others, are able to permeate the various types of connexin channels, and how well? An important related issue, given the ever-growing list of connexin-related pathologies, is how these permeabilities are altered by disease-causing connexin mutations. Together, many studies show that a variety of cytoplasmic molecules can permeate the different types of connexin channels. A few studies reveal differences in permeation by different molecules through a particular type of connexin channel, and differences in permeation by a particular molecule through different types of connexin channels. This article describes and evaluates the various methods used to obtain these data, presents an annotated compilation of the results, and discusses the findings in the context of what can be inferred about mechanism of selectivity and potential relevance to signaling. The data strongly suggest that highly specific interactions take place between connexin pores and specific biological molecular permeants, and that those interactions determine which cytoplasmic molecules can permeate and how well. At this time, the nature of those interactions is unclear. One hopes that with more detailed permeability and structural information, the specific molecular mechanisms of the selectivity can be elucidated.

Intercellular connexin channels (gap junction channels) have long been thought to mediate molecular signaling between cells, but the nature of the signaling has been unclear. This study shows that connexin channels from native tissue have selective permeabilities, partially based on pore diameter, that discriminate among cytoplasmic second messenger molecules. Permeability was assessed by measurement of selective loss/retention of tracers from liposomes containing reconstituted connexin channels. The tracers employed were tritiated cyclic nucleotides and a series of oligomaltosaccharides derivatized with a small uncharged fluorescent moiety. The data define different size cut-off limits for permeability through homomeric connexin-32 channels and through heteromeric connexin-32/connexin-26 channels. Connexin-26 contributes to a narrowed pore. Both cAMP and cGMP were permeable through the homomeric connexin-32 channels. cAMP was permeable through only a fraction of the heteromeric channels. Surprisingly, cGMP was permeable through a substantially greater fraction of the heteromeric channels than was cAMP. The data suggest that isoform stoichiometry and/or arrangement within a connexin channel determines whether cyclic nucleotides can permeate, and which ones. This is the first evidence for connexin-specific selectivity among biological signaling molecules.

The pH of the cytoplasm (pHt) measured with pH-sensitive microelectrodes in cleavage-stage blastomeres of amphibian (Ambystoma) and teleost (Fundulus) embryos is about 7.7. In electrotonically coupled cell pairs, junctional conductance is rapidly and reversibly reduced by acidification of the cytoplasm. The relation between junctional conductance and pHi is the same for increasing and decreasing pH and is independent of the rate of change over a wide range. The relation is well fitted by a Hill curve with K = 50 nM (pK = 7.3) and n = 4 to 5. The closure of gap junction channels at low pHi appears to be a cooperative process involving several charged sites. The absence of hysteresis and identity of effects for fast and slow pHi changes implies that protons act directly on the channel macromolecules and not through an intermediate in the cytoplasm.