Abstract-Intercellular communication through gap junctions coordinates vascular tone by the conduction of vasomotorresponses along the vessel wall. Gap junctions in arterioles are composed of different connexins (Cxs) (Cx40, Cx37, Cx45, Cx43), but it is unknown whether Cxs are interchangeable. We used mice with a targeted replacement of Cx40 by Cx45 (Cx40KI45) to explore whether Cx45 can functionally replace Cx40 in arterioles. Arterioles were locally stimulated using acetylcholine, bradykinin, adenosine, and K ϩ in the cremaster of Cx40KI45, Cx40-deficient (Cx40ko), and wild-type mice, and diameter changes were assessed by intravital microscopy. Additionally, arterial pressure was measured by telemetry and Cx expression verified by immunofluorescence. Acetylcholine initiated a local dilation of a similar amplitude in all genotypes (Ϸ50%), which was rapidly conducted to upstream sites (1200 m distance) without attenuation in wild type. In marked contrast, the remote dilation was significantly reduced in Cx40ko (25Ϯ3%) and Cx40KI45 (24Ϯ2%). Likewise, dilations initiated by bradykinin application were conducted without attenuation up to 1200 m in wild type but not in Cx40ko and Cx40KI45. Adenosine-induced dilations and K ϩ -induced constrictions were conducted similarly with decaying amplitude in all genotypes. Arterial pressure was strongly elevated in Cx40ko (161Ϯ1 versus 116Ϯ2 mm Hg) but only moderately in Cx40KI45 (133Ϯ8 mm Hg). This demonstrates that Cx40 function is critical for the conduction of acetylcholine and bradykinin dilations and cannot be substituted by Cx45. Therefore, unique properties of Cx40 are required for endothelial signal conduction, whereas nonspecific restoration of communication maintains additional functions related to blood pressure control.

Abstract-N-terminal mutations in the KCNQ1 channel are frequently linked to fatal arrhythmias in newborn children and adolescents but the cellular mechanisms involved in this dramatic issue remain, however, to be discovered. Here, we analyzed the trafficking of a series of N-terminal truncation mutants and identified a critical trafficking motif of KCNQ1. This determinant is located in the juxtamembranous region preceding the first transmembrane domain of the protein. Three mutations (Y111C, L114P and P117L) implicated in inherited Romano-Ward LQT1 syndrome, are embedded within this domain. Reexpression studies in both COS-7 cells and cardiomyocytes showed that the mutant proteins fail to exit the endoplasmic reticulum. KCNQ1 subunits harboring Y111C or L114P exert a dominant negative effect on the wild-type KCNQ1 subunit by preventing plasma membrane trafficking of heteromultimeric channels. The P117L mutation had a less pronounced effect on the trafficking of heteromultimeric channels but altered the kinetics of the current. Furthermore, we showed that the trafficking determinant in KCNQ1 is structurally and functionally conserved in other KCNQ channels and constitutes a critical trafficking determinant of the KCNQ channel family.Computed structural predictions correlated the potential structural changes introduced by the mutations with impaired protein trafficking. In conclusion, our studies unveiled a new role of the N-terminus of KCNQ channels in their trafficking and its implication in severe forms of LQT1 syndrome. Key Words: channels Ⅲ KCNQ Ⅲ LQT Ⅲ membrane Ⅲ trafficking T he long QT syndrome (LQTS) is a cause of sudden cardiac death and is characterized by an increased QT interval on patients' ECG. The prolongation of the QT interval predisposes patients to cardiac arrhythmias known as torsades de pointes, eventually leading to ventricular fibrillation. Over the last decade, inherited mutations in genes encoding cardiac ionic channels or associated partners have been identified in 4 different congenital LQT syndromes (LQT1-4). For example, mutations in KCNQ1 have been associated with either autosomal dominant Romano-Ward (RW) or recessive Jervell and Lange-Nielsen (JLN) LQT1 syndromes. [1][2][3] KCNQ1 encodes the pore forming ␣-subunit of a voltage gated K ϩ channel, which associates with an accessory subunit, KCNE1, in the heart to form channels responsible for the slow component of the delayed repolarizing K ϩ current (I Ks ). 4,5 KCNQ1 mutations associated with LQT1 alter channel function through different mechanisms. Carboxy terminal mutations often disrupt channel assembly, alter regulatory subunit association and cause mistrafficking. 6 -9 Mutations in the pore region and transmembrane domains (TMDs), on the other hand, produce dominant negative effects on potassium permeation and KCNQ1 channel gating functions. 10 -12 Although more than a hundred different LQT1-causing mutations have been reported in KCNQ1, only a few are located in its cytoplasmic N-terminus. 2,3 In general, these mutations...

Abstract-The electrical activity in heart is generated in the sinoatrial node and then propagates to the atrial and ventricular tissues. The gap junction channels that couple the myocytes are responsible for this propagation process. The gap junction channels are dodecamers of transmembrane proteins of the connexin (Cx) family. Three members of this family have been demonstrated to be synthesized in the cardiomyocytes: Cx40, Cx43, and Cx45. In addition, each of them has been shown to form channels with unique and specific electrophysiological properties. Understanding the conduction phenomenon requires detailed knowledge of the spatiotemporal expression pattern of these Cxs in heart. The expression patterns of Cx40 and Cx43 have been previously described in the adult heart and during its development. Here we report the expression of Cx45 gene products in mouse heart from the stage of the first contractions (8.5 days postcoitum [dpc]) to the adult stage. The Cx45 gene transcript was demonstrated by reverse transcriptase-polymerase chain reaction experiments to be present in heart at all stages investigated. Between 8.5 and 10.5 dpc it was shown by in situ hybridization to be expressed in low amounts in all cardiac compartments (including the inflow and outflow tracts and the atrioventricular canal) and then to be downregulated from 11 to 12 dpc onward. At subsequent fetal stages, the transcript was weakly detected in the ventricles, with the most distinct expression in the outflow tract. Cx45 protein was demonstrated by immunofluorescence microscopy to be expressed in the myocytes of young embryonic hearts (8.5 to 9.5 dpc). However, beyond 10.5 dpc the protein was no longer detected with this technique in the embryonic, fetal, or neonatal working myocardium, although it could be shown by immunoblotting that the protein was still synthesized in neonatal heart. In the major part of adult heart, Cx45 was undetectable. It was, however, clearly seen in the anterior regions of the interventricular septum and in trace amounts in some small foci dispersed in the ventricular free walls. Cx45 gene is the first Cx gene so far demonstrated to be activated in heart at the stage of the first contractions. The coordination of myocytes during the slow peristaltic contractions that occur at this stage would thus appear to be controlled by the Cx45 channels. (Circ Res. 1999;84:1365-1379.)Key Words: connexin 45 Ⅲ heart Ⅲ development T he gap junctions are clusters of transmembrane channels that mediate direct communication between the cytoplasmic compartments of adjacent cells. These channels, permeable to ions and small molecules (Ͻ900 Da), including second messengers, are the structural components responsible for intercellular electrical and metabolic coupling. The proteins that form these channels are encoded by a multigene family, the connexin (Cx) family. Each Cx forms channels that have unique properties of conductance and permeability. The structure and the oligomerization of Cxs into gap junction channels, and the propertie...