Xianming Lin

Connexin40 and connexin43 determine gating properties of atrial gap junction channels

While ventricular gap junctions contain only Cx43, atrial gap junctions contain both Cx40 and Cx43; yet the functional consequences of this co-expression remain poorly understood. We quantitated the expression of Cx40 and Cx43 and their contributions to atrial gap junctional conductance (g(j)). Neonatal murine atrial myocytes showed similar abundances of Cx40 and Cx43 proteins, while ventricular myocytes contained at least 20 times more Cx43 than Cx40. Since Cx40 gap junction channels are blocked by 2 mM spermine while Cx43 channels are unaffected, we used spermine block as a functional dual whole cell patch clamp assay to determine Cx40 contributions to cardiac g(j). Slightly more than half of atrial g(j) and <or=20% of ventricular g(j) were inhibited. In myocytes from Cx40 null mice, the inhibition of ventricular g(j) was completely abolished, and the block of atrial g(j) was reduced to <20%. Compared to ventricular gap junctions, the transjunctional voltage (V(j))-dependent inactivation of atrial g(j) was reduced and kinetically slowed, while the V(j)-dependence of fast and slow inactivation was unchanged. We conclude that Cx40 and Cx43 are equally abundant in atrium and make similar contributions to atrial g(j). Co-expression of Cx40 accounts for most, but not all, of the differences in the V(j)-dependent gating properties between atrium and ventricle that may play a role in the genesis of slow myocardial conduction and arrhythmias.

N-terminal residues in Cx43 and Cx40 determine physiological properties of gap junction channels, but do not influence heteromeric assembly with each other or with Cx26

The cytoplasmic N-terminal domain in the connexins (Cx) has been implicated in determining several properties including connexin hetero-oligomerization, channel gating and regulation by polyamines. To elucidate the roles of potentially crucial amino acids, we produced site-directed mutants of connexins Cx40 and Cx43 (Cx40E12S,E13G and Cx43D12S,K13G) in which the charged amino acids at positions 12 and 13 were replaced with serine and glycine as found in Cx32. HeLa, N2a and HEK293 cells were transfected and studied by immunochemistry and double whole-cell patch clamping. Immunoblotting confirmed production of the mutant proteins, and immuno-fluorescence localized them to punctuate distributions along appositional membranes. Cx40E12S,E13G and Cx43D12S,K13G formed homotypic gap junction channels that allowed intercellular passage of Lucifer Yellow and electrical current, but these channels exhibited negligible voltage-dependent gating properties. Unlike wild-type Cx40, Cx40E12S,E13G channels were insensitive to block by 2 mM spermine. Affinity purification of material solubilized by Triton X-100 from cells co-expressing mutant Cx43 or mutant Cx40 with wild-type Cx40, Cx43 or Cx26 showed that introducing the mutations did not affect the compatibility or incompatibility of these proteins for heteromeric mixing. Co-expression of Cx40E12S,E13G with wild-type Cx40 or Cx43 dramatically reduced voltage-dependent gating. Thus, whereas the charged amino acids at positions 12 and 13 of Cx40 or Cx43 are not required for gap junction assembly or the compatibility of oligomerization with each other or with Cx26, they strongly influence several physiological properties including those of heteromeric channels.

Molecular interaction and functional regulation of connexin50 gap junctions by calmodulin.

Cx50 (connexin50), a member of the α-family of gap junction proteins expressed in the lens of the eye, has been shown to be essential for normal lens development. In the present study, we identified a CaMBD [CaM (calmodulin)-binding domain] (residues 141-166) in the intracellular loop of Cx50. Elevations in intracellular Ca2+ concentration effected a 95% decline in gj (junctional conductance) of Cx50 in N2a cells that is likely to be mediated by CaM, because inclusion of the CaM inhibitor calmidazolium prevented this Ca2+-dependent decrease in gj. The direct involvement of the Cx50 CaMBD in this Ca2+/CaM-dependent regulation was demonstrated further by the inclusion of a synthetic peptide encompassing the CaMBD in both whole-cell patch pipettes, which effectively prevented the intracellular Ca2+-dependent decline in gj. Biophysical studies using NMR and fluorescence spectroscopy reveal further that the peptide stoichiometrically binds to Ca2+/CaM with an affinity of ~5 nM. The binding of the peptide expanded the Ca2+-sensing range of CaM by increasing the Ca2+ affinity of the C-lobe of CaM, while decreasing the Ca2+ affinity of the N-lobe of CaM. Overall, these results demonstrate that the binding of Ca2+/CaM to the intracellular loop of Cx50 is critical for mediating the Ca2+-dependent inhibition of Cx50 gap junctions in the lens of the eye.

Regulation of Connexin43 Gap Junctional Conductance by Ventricular Action Potentials

Abstract— Transjunctional voltage regulates cardiac gap junctional conductance, but the kinetics of inactivation were considered too slow to affect cardiac action potential propagation. Connexin43 (Cx43) is abundantly expressed in the atrial and ventricular myocardium and the rapid ventricular conduction tissues (ie, His-Purkinje system) of the mammalian heart and is important to conduction through these cardiac tissues. The kinetics of Cx43 voltage gating were examined at peak action potential voltages using simulated ventricular myocardial action potential waveforms or pulse protocols exceeding 100-mV transjunctional potentials. Junctional current responses approximate the action potential morphology but conductance calculations reveal a 50% to 60% decline from peak to near constant plateau values. Junctional conductance recovers during phase 3 repolarization and early diastole to initial values. The bases for these transient changes in junctional conductance are the rapid decay kinetics in tens of milliseconds at peak transjunctional voltages (Vj) of 130 mV and the gradual increase in junctional conductance as Vj returns toward 0 mV. The decay time constants change e-fold per 22.1 mV above the half-inactivation voltage for Cx43 gap junctions of ±58 mV. A realistic dynamic model for changes in junctional resistance between excitable and nonexcitable cells during cardiac action potential propagation was developed based on these findings. This dynamic model of cardiac gap junctions will further our understanding of the role gap junctions play in the genesis and propagation of cardiac arrhythmias. The full text of this article is available online at http://www.circresaha.org.

Enhancement of ventricular gap-junction coupling by rotigaptide

AIMS 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. METHODS AND RESULTS 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. CONCLUSION 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.

Cx30.2 can form heteromeric gap junction channels with other cardiac connexins.

Since most cells in the heart co-express multiple connexins, we studied the possible heteromeric interactions between connexin30.2 and connexin40, connexin43 or connexin45 in transfected cells. Double-label immunofluorescence microscopy showed that connexin30.2 extensively co-localized with each co-expressed connexin at appositional membranes. When Triton X-100 solubilized connexons were affinity purified from co-expressing cells, connexin30.2 was isolated together with connexin40, connexin43, or connexin45. Co-expression of connexin30.2 with connexin40, connexin43, or connexin45 did not significantly reduce total junctional conductance. Gap junction channels in cells co-expressing connexin30.2 with connexin43 or connexin45 exhibited voltage-dependent gating intermediate between that of either connexin alone. In contrast, connexin30.2 dominated the voltage-dependence when co-expressed with connexin40. Our data suggest that connexin30.2 can form heteromers with the other cardiac connexins and that mixed channel formation will influence the gating properties of gap junctions in cardiac regions that co-express these connexins.

An amino‐terminal lysine residue of rat connexin40 that is required for spermine block

Spermine blocks connexin40 (Cx40) gap junctions, and two cytoplasmic amino‐terminal domain glutamate residues are essential for this inhibitory activity. To further examine the molecular basis for block, we mutated a portion of a basic amino acid (HKH) motif on the Cx40 amino‐terminal domain. Replacement of the Cx40 H15 + K16 residues with the Q15 + A16 sequence native to spermine‐insensitive connexin43 (Cx43) gap junctions increased the equilibrium dissociation constant (Kd) and reduced the maximum inhibition by spermine. The corresponding electrical distance (δ) approximation was decreased by about 50%. The transjunctional voltage (Vj)‐dependent gating of homotypic Cx40 H15Q + K16A mutant gap junctions was also significantly reduced. The minimum normalized steady‐state junctional conductance (Gmin) increased from 0.17 to 0.72, with an increase in the half‐inactivation voltage from 48 to 60 mV. However, the unitary junctional conductance (γj; 160 pS) was only slightly altered, and the relative cation/anion conductance and permeability ratios were unchanged from wild‐type Cx40 gap junction channels. The relative K+/Cl− permeability (PK/PCl) ratio increased from six to ten when [KCl] was reduced to 25% of normal. These data suggest that the HKH motif at positions 15–17 is important to the conformational structure of the putative voltage sensor and spermine receptor of Cx40, without causing significant alteration of the electrostatic surface charge potentials that contribute to the ion selectivity of this gap junction channel.

Atrial fibrillation-associated Connexin40 mutants make hemichannels and synergistically form gap junction channels with novel properties

Mutations of Cx40 (GJA5) have been identified in people with lone chronic atrial fibrillation including G38D and M163V which were found in the same patient. We used dual whole cell patch clamp procedures to examine the transjunctional voltage (V j) gating and channel conductance properties of these two rare mutants. Each mutant exhibited slight alterations of V j gating properties and increased the gap junction channel conductance (γ j) by 20–30 pS. While co‐expression of the two mutations had similar effects on V j gating, it synergistically increased γ j by 50%. Unlike WTCx40 or M163V, G38D induced activity of a dominant 271 pS hemichannel.

Functional formation of heterotypic gap junction channels by connexins-40 and -43.

Connexin40 (Cx40) and connexin43 (Cx43) are co-expressed in the cardiovascular system, yet their ability to form functional heterotypic Cx43/Cx40 gap junctions remains controversial. We paired Cx43 or Cx40 stably-transfected N2a cells to examine the formation and biophysical properties of heterotypic Cx43/Cx40 gap junction channels. Dual whole cell patch clamp recordings demonstrated that Cx43 and Cx40 form functional heterotypic gap junctions with asymmetric transjunctional voltage (Vj) dependent gating properties. The heterotypic Cx43/Cx40 gap junctions exhibited less Vj gating when the Cx40 cell was positive and pronounced gating when negative. Endogenous N2a cell connexin expression levels were 1,000-fold lower than exogenously expressed Cx40 and Cx43 levels, measured by real-time PCR and Western blotting methods, suggestive of heterotypic gap junction formation by exogenous Cx40 and Cx43. Imposing a [KCl] gradient across the heterotypic gap junction modestly diminished the asymmetry of the macroscopic normalized junctional conductance – voltage (Gj-Vj) curve when [KCl] was reduced by 50% on the Cx43 side and greatly exacerbated the Vj gating asymmetries when lowered on the Cx40 side. Pairing wild-type (wt) Cx43 with the Cx40 E9,13K mutant protein produced a nearly symmetrical heterotypic Gj-Vj curve. These studies conclusively demonstrate the ability of Cx40 and Cx43 to form rectifying heterotypic gap junctions, owing primarily to alternate amino-terminal (NT) domain acidic and basic amino acid differences that may play a significant role in the physiology and/or pathology of the cardiovascular tissues including cardiac conduction properties and myoendothelial intercellular communication.

Specificity of the connexin W3/4 locus for functional gap junction formation

ABSTRACT The N-terminal (NT) domain of the connexins forms an essential transjunctional voltage (Vj) sensor and pore-forming domain that when truncated, tagged, or mutated often leads to formation of a nonfunctional channel. The NT domain is relatively conserved among the connexins though the α- and δ-group connexins possess a G2 residue not found in the β- and γ-group connexins. Deletion of the connexin40 G2 residue (Cx40G2Δ) affected the Vj gating, increased the single channel conductance (γj), and decreased the relative K+/Cl− permeability (PK/PCl) ratio of the Cx40 gap junction channel. The conserved α/β-group connexin D2/3 and W3/4 loci are postulated to anchor the NT domain within the pore via hydrophilic and hydrophobic interactions with adjacent connexin T5 and M34 residues. Cx40D3N and D3R mutations produced limited function with progressive reductions in Vj gating and noisy low γj gap junction channels that reduced the γj of wild-type Cx40 channels from 150 pS to < 50 pS when coexpressed. Surprisingly, hydrophobic Cx40 W4F and W4Y substitution mutations were not compatible with function despite their ability to form gap junction plaques. These data are consistent with minor and major contributions of the G2 and D3 residues to the Cx40 channel pore structure, but not with the postulated hydrophobic W4 intermolecular interactions. Our results indicate an absolute requirement for an amphipathic W3/4 residue that is conserved among all α/β/δ/γ-group connexins. We alternatively hypothesize that the connexin D2/3-W3/4 locus interacts with the highly conserved FIFR M1 motif to stabilize the NT domain within the pore.