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 – V_j-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 V_m-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.

The p H of the cytoplasm ( p H _i ) measured with p H-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 p H _i is the same for increasing and decreasing p H and is independent of the rate of change over a wide range. The relation is well fitted by a Hill curve with K = 50 n M (p K = 7.3) and n = 4 to 5. The closure of gap junction channels at low p H _i appears to be a cooperative process involving several charged sites. The absence of hysteresis and identity of effects for fast and slow p H _i changes implies that protons act directly on the channel macromolecules and not through an intermediate in the cytoplasm.

The conductance of junctions between amphibian blastomeres is strongly voltage dependent. Isolated pairs of blastomeres from embryos of Ambystoma mexicanum, Xenopus laevis, and Rana pipiens were voltage clamped, and junctional current was measured during transjunctional voltage steps. The steady-state junctional conductance decreases as a steep function of transjunctional voltage of either polarity. A voltage-insensitive conductance less than 5% of the maximum remains at large transjunctional voltages. Equal transjunctional voltages of opposite polarities produce equal conductance changes. The conductance is half maximal at a transjunctional voltage of approximately 15 mV. The junctional conductance is insensitive to the potential between the inside and outside of the cells. The changes in steady-state junctional conductance may be accurately modeled for voltages of each polarity as arising from a reversible two-state system in which voltage linearly affects the energy difference between states. The voltage sensitivity can be accounted for by the movement of about six electron charges through the transjunctional voltage. The changes in junctional conductance are not consistent with a current-controlled or ionic accumulation mechanism. We propose that the intramembrane particles that comprise gap junctions in early amphibian embryos are voltage-sensitive channels.

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.

We have proposed that the gap junctions between amphibian blastomeres are comprised of voltage-sensitive channels. The kinetic properties of the junctional conductance are here studied under voltage clamp. When the transjunctional voltage is stepped to a new voltage of the same polarity, the junctional conductance changes as a single exponential to a steady-state level. The time constant of the conductance change is determined by the existing transjunctional voltage and is independent of the previous voltage. For each voltage polarity, the relations between voltage, time constant, and steady-state conductance are well modeled by a reversible two-state reaction scheme in which the calculated rate constants for the transitions between the states are exponential functions of voltage. The calculated rate constant for the transition to the low-conductance state is approximately twice as voltage dependent as that for the transition to the high-conductance state. When the transjunctional voltage polarity is reversed, the junctional conductance undergoes a transient recovery. The polarity reversal data are well modeled by a reaction scheme in which the junctional channel has two gates, each with opposite voltage sensitivity, and in which an open gate may close only if the gate in series with it is open. A simple explanation for this contingent gating is a mechanism in which each gate senses only the local voltage drop within the channel.