Connexin40 (Cx40) contains a specific binding site for spermine (affinity ∼100 μm) whereas connexin43 (Cx43) is unaffected by identical concentrations of intracellular spermine. Replacement of two unique glutamate residues, E9 and E13, from the cytoplasmic amino terminal domain of Cx40 with the corresponding lysine residues from Cx43 eliminated the block by 2 mm spermine, reduced the transjunctional voltage (V_j) gating sensitivity, and reduced the unitary conductance of this Cx40E9,13K gap junction channel protein. The single point mutations, Cx40E9K and Cx40E13K, predominantly affected the residual conductance state (G_min) and V_j gating properties, respectively. Heterotypic pairing of Cx40E9,13K with wild‐type Cx40 in murine neuro2A (N2A) cells produced a strongly rectifying gap junction reminiscent of the inward rectification properties of the Kir (e.g. Kir2.x) family of potassium channels. The reciprocal Cx43K9,13E mutant protein exhibited reduced V_j sensitivity, but displayed much less rectification in heterotypic pairings with wtCx43, negligible changes in the unitary channel conductance, and remained insensitive to spermine block. These data indicate that the connexin40 amino terminus may form a critical cytoplasmic pore‐forming domain that serves as the receptor for V_j‐dependent closure and block by intracellular polyamines. Functional reciprocity between Cx40 and Cx43 gap junctions involves other amino acid residues in addition to the E or K 9 and 13 loci located on the amino terminal domain of these two connexins.

Calmodulin (CaM) binding sites were recently identified on the cytoplasmic loop (CL) of at least three α-subfamily connexins (Cx43, Cx44, Cx50), while Cx40 does not have this putative CaM binding domain. The purpose of this study was to examine the functional relevance of the putative Cx43 CaM binding site on the Ca²⁺-dependent regulation of gap junction proteins formed by Cx43 and Cx40. Dual whole cell patch-clamp experiments were performed on stable murine Neuro-2a cells expressing Cx43 or Cx40. Addition of ionomycin to increase external Ca²⁺ influx reduced Cx43 gap junction conductance (G_j) by 95%, while increasing cytosolic Ca²⁺ concentration threefold. By contrast, Cx40 G_j declined by <20%. The Ca²⁺-induced decline in Cx43 G_j was prevented by pretreatment with calmidazolium or reversed by the addition of 10 mM EGTA to Ca²⁺-free extracellular solution, if Ca²⁺ chelation was commenced before complete uncoupling, after which g_j was only 60% recoverable. The Cx43 CL_136–158 mimetic peptide, but not the scrambled control peptide, or Ca²⁺/CaM-dependent kinase II 290–309 inhibitory peptide also prevented the Ca²⁺/CaM-dependent decline of Cx43 G_j. Cx43 gap junction channel open probability decreased to zero without reductions in the current amplitudes during external Ca²⁺/ionomycin perfusion. We conclude that Cx43 gap junctions are gated closed by a Ca²⁺/CaM-dependent mechanism involving the carboxyl-terminal quarter of the connexin CL domain. This study provides the first evidence of intrinsic differences in the Ca²⁺ regulatory properties of Cx43 and Cx40.

Connexin hemichannels are postulated to form a cell permeabilization pore for the uptake of fluorescent dyes and release of cellular ATP. Connexin hemichannel activity is enhanced by low external [Ca²⁺]_o, membrane depolarization, metabolic inhibition, and some disease‐causing gain‐of‐function connexin mutations. This paper briefly reviews the electrophysiological channel conductance, permeability, and pharmacology properties of connexin hemichannels, pannexin 1 channels, and purinergic P2X₇receptor channels as studied in exogenous expression systems includingXenopusoocytes and mammalian cell lines such as HEK293 cells. Overlapping pharmacological inhibitory and channel conductance and permeability profiles makes distinguishing between these channel types sometimes difficult. Selective pharmacology for Cx43 hemichannels (Gap19 peptide), probenecid or FD&C Blue #1 (Brilliant Blue FCF, BB FCF) for Panx1, and A740003, A438079, or oxidized ATP (oATP) for P2X7 channels may be the best way to distinguish between these three cell permeabilizing channel types. Endogenous connexin, pannexin, and P2X₇expression should be considered when performing exogenous cellular expression channel studies. Cell pair electrophysiological assays permit the relative assessment of the connexin hemichannel/gap junction channel ratio not often considered when performing isolated cell hemichannel studies.

Rotigaptide is proposed to exert its anti-arrhythmic effects by improving myocardial gap-junction communication. To directly investigate the mechanisms of rotigaptide action, we treated cultured neonatal murine ventricular cardiomyocytes with clinical pharmacological doses of rotigaptide and directly determined its effects on gap-junctional currents. Neonatal murine ventricular cardiomyocytes were enzymatically isolated and cultured for 1–4 days. Primary culture cell pairs were subjected to dual whole cell patch-clamp procedures to directly measure gap-junctional currents (I_j) and voltage (V_j). Rotigaptide (0–350 nM) was applied overnight or acutely perfused into 35 mm culture dishes. Rotigaptide (35–100 nM) acutely and chronically increased the resting gap-junction conductance (g_j), and normalized steady-state minimum g_j (G_min) by 5–20%. Higher concentrations produced a diminishing response, which mimics the observed therapeutic efficacy of the drug. The inactivation kinetics was similarly slowed in a therapeutic concentration-dependent manner without affecting the V_j dependence of inactivation or recovery. The effects of 0–100 nM rotigaptide on ventricular g_j during cardiac action potential propagation were accurately modelled by computer simulations which demonstrate that clinically effective concentrations of rotigaptide can partially reverse conduction slowing due to decreases in g_j and inactivation. These results demonstrate that therapeutic concentrations of rotigaptide increase the resting gap-junction conductance and reduce the magnitude and kinetics of steady-state inactivation in a concentration-dependent manner. Rotigaptide may be effective in treating re-entrant forms of cardiac arrhythmias by improving conduction and preventing the formation of re-entrant circuits in partially uncoupled myocardium.