Regulation of Cx45 hemichannels mediated by extracellular and intracellular calcium.

Connexin45 (Cx45) hemichannels (HCs) open in the absence of Ca(2+) and close in its presence. To elucidate the underlying mechanisms, we examined the role of extra- and intracellular Ca(2+) on the electrical properties of HCs. Experiments were performed on HeLa cells expressing Cx45 using electrical (voltage clamp) and optical (Ca(2+) imaging) methods.

Influence of v5/6-His tag on the properties of gap junction channels composed of connexin43, connexin40 or connexin45.

HeLa cells expressing wild-type connexin43, connexin40 or connexin45 and connexins fused with a V5/6-His tag to the carboxyl terminus (CT) domain (Cx43-tag, Cx40-tag, Cx45-tag) were used to study connexin expression and the electrical properties of gap junction channels. Immunoblots and immunolabeling indicated that tagged connexins are synthesized and targeted to gap junctions in a similar manner to their wild-type counterparts. Voltage-clamp experiments on cell pairs revealed that tagged connexins form functional channels.

Functional differences between human Cx37 polymorphic hemichannels.

A polymorphism in the human Cx37 gene (C1019T), resulting in a non-conservative amino acid change in the regulatory C-terminus of the Cx37 protein (P319S), has been proposed as a prognostic marker for atherosclerosis. We have recently demonstrated that Cx37 hemichannels control the initiation of atherosclerotic plaque development by regulating ATP-dependent monocyte adhesion in atherosclerosis-susceptible apolipoprotein E-deficient mice. In this study, we have measured the electrical properties of Cx37 hemichannels (HCs) and gap junction channels (GJCs) with voltage-clamp methods.

Gap junction channels and cardiac impulse propagation.

The role of gap junction channels on cardiac impulse propagation is complex. This review focuses on the differential expression of connexins in the heart and the biophysical properties of gap junction channels under normal and disease conditions. Structural determinants of impulse propagation have been gained from biochemical and immunocytochemical studies performed on tissue extracts and intact cardiac tissue.

Subconductance states of Cx30 gap junction channels: data from transfected HeLa cells versus data from a mathematical model.

Human HeLa cells expressing mouse connexin30 were used to study the electrical properties of gap junction channel substates. Experiments were performed on cell pairs using a dual voltage-clamp method. Single-channel currents revealed discrete levels attributable to a main state, a residual state, and five substates interposed, suggesting the operation of six subgates provided by the six connexins of a gap junction hemichannel.

Pitfalls when examining gap junction hemichannels: interference from volume-regulated anion channels.

Human HeLa cells transfected with mouse connexin45 were used to explore the experimental conditions suitable to measure currents carried by gap junction hemichannels. Experiments were performed with a voltage-clamp technique and whole-cell recording. Lowering [Ca(2+)](o) from 2 mM to 20 nM evoked an extra current, I (m), putatively carried by Cx45 hemichannels.

Electrical propagation in synthetic ventricular myocyte strands from germline connexin43 knockout mice.

To characterize the role of connexin43 (Cx43) as a determinant of cardiac propagation, we synthesized strands and pairs of ventricular myocytes from germline Cx43-/- mice. The amount of Cx43, Cx45, and Cx40 in gap junctions was analyzed by immunohistochemistry and confocal microscopy. Intercellular electrical conductance, gj, was measured by the dual-voltage clamp technique (DVC), and electrical propagation was assessed by multisite optical mapping of transmembrane potential using a voltage-sensitive dye.

Cardiac connexins Cx43 and Cx45: formation of diverse gap junction channels with diverse electrical properties.

HeLa cells expressing rat connexin43 (Cx43) and/or mouse Cx45 were studied with the dual voltage-clamp technique. Different types of cell pairs were established and their gap junction properties determined, i.e.

The electrical behaviour of rat connexin46 gap junction channels expressed in transfected HeLa cells.

Pairs of human HeLa cells expressing rat connexin46 were used to study the electrical properties of gap junction channels with the dual voltage-clamp method. The steady-state conductance ( g(j,ss)) had a bell-shaped dependence on transjunctional voltage ( V(j)). The parameters of the Boltzmann fit were: V(j,0)=42 mV, g(j,min)=0.

The electrophysiology of gap junctions and gap junction channels and their mathematical modelling.

In most tissues of vertebrates, gap junctions control the exchange of ions and small molecules between adjacent cells, thus co-ordinating the cellular activities. The application of the dual voltage-clamp method to cell pair preparations enables one to elucidate the electrical properties of gap junctions and gap junction channels. The conductive and kinetic data obtained at the multichannel and single channel level led to a generalised concept for the operation of gap junction channels.

Expression and regulation of connexins in cultured ventricular myocytes isolated from adult rat hearts.

Gap junctions were assayed during re-differentiation of adult rat cardiomyocytes in long-term culture to gain insight into the processes of remodeling. Double immunostaining allowed the localization of connexins Cx40, Cx43, and Cx45 between myocytes and demonstrated co-expression and co-localization in individual cells and gap junction plaques, respectively. Immunoblots showed differential time-dependent changes in connexin expression and phosphorylation.