Richard D. Veenstra

Molecular cloning and functional expression of human connexin37, an endothelial cell gap junction protein.

Gap junctions allow direct intercellular coupling between many cells including those in the blood vessel wall. They are formed by a group of related proteins called connexins, containing conserved transmembrane and extracellular domains, but unique cytoplasmic regions that may confer connexin-specific physiological properties. We used polymerase chain reaction amplification and cDNA library screening to clone DNA encoding a human gap junction protein, connexin37 (Cx37). The derived human Cx37 polypeptide contains 333 amino acids, with a predicted molecular mass of 37,238 D. RNA blots demonstrate that Cx37 is expressed in multiple organs and tissues (including heart, uterus, ovary, and blood vessel endothelium) and in primary cultures of vascular endothelial cells. Cx37 mRNA is coexpressed with connexin43 at similar levels in some endothelial cells, but at much lower levels in others. To demonstrate that Cx37 could form functional channels, we stably transfected communication-deficient Neuro2A cells with the Cx37 cDNA. The induced intercellular channels were studied by the double whole cell patch clamp technique. These channels were reversibly inhibited by the uncoupling agent, heptanol (2 mM). The expressed Cx37 channels exhibited multiple conductance levels and showed a pronounced voltage dependence. These electrophysiological characteristics are similar to, but distinct from, those of previously characterized connexins.

Multiple connexins confer distinct regulatory and conductance properties of gap junctions in developing heart.

Multiple gap junction proteins (connexins) and channels have been identified in developing and adult heart. Functional expression of the three connexins found in chick heart (connexin42, connexin43, and connexin45) by stable transfection of communication-deficient neuro2A (N2A) cells revealed that all three connexin cDNAs are capable of forming physiologically distinct gap junctions that differ in their transjunctional voltage dependence and unitary channel conductances. The transjunctional voltage dependences of connexin45 and connexin42 closely resembled those of 4-day and 18-day embryonic chick heart gap junctions, respectively. The multiple channel conductances between 80 and 240 pS, including the predominant 160 pS channel, observed in embryonic chick heart were also common to connexin42. The expression of multiple gap junction channels with distinct conductance and regulatory properties within a given tissue may account for developmental changes in intercellular communication.

Purkinje and ventricular activation sequences of canine papillary muscle. Effects of quinidine and calcium on the Purkinje-ventricular conduction delay.

We have studied in vitro preparations of canine right and left papillary muscles to determine the excitation sequences of the Purkinje and ventricular cells, using both monopolar surface electrodes and intracellular microelectrodes. Our results show, for right papillary muscles, that the Purkinje layer covers the basal part of the muscle, and that activation from the right bundle branch propagates over all of the Purkinje layer, but directly activates the underlying ventricular layer only at specific junctional sites. Left papillary muscles have attachments to both apical and basal Purkinje strands and the Purkinje layer covers the entire muscle, but, as for right papillary muscles, activation from the Purkinje layer to the ventricular layer occurs only at basal junctional sites. Antidromic conduction in papillary muscles (propagation from the ventricular layer to the Purkinje layer) can occur at regions other than the specific sites through which the Purkinje layer activates the ventricular layer. At the identified junctional sites, the Purkinje cell action potential duration is significantly shorter than in the free-running strand, but it remains longer than that of the ventricular cells. The time delay at the junctional sites is increased by quinidine, increased calcium concentration, and increased pacing frequency.

Propagation through electrically coupled cells. Effects of a resistive barrier

Action potential propagation through cardiac tissue occurs in a spatially inhomogeneous three-dimensional electrical syncytium composed of discrete cells with regional variations in membrane properties and intercellular resistance. In comparison with axons, cardiac tissue presents some differences in the application of core conductor cable theory. We have used analytical and numerical techniques to contrast the propagation of action potentials along nerve axons and along cardiac strands, including an explicit inclusion of cellular anatomical factors (the surface-to-volume ratio), the strand radius, and the regional distribution of longitudinal resistance. A localized decrease in the number of gap junctions will produce a functional resistive barrier, which can lead to unidirectional block of propagation if the tissue on two sides of the barrier in either excitability or passive electrical load. However, in some circumstances, a resistive barrier separating regions of different electrical load can actually facilitate propagation into the region of larger electrical load.

Propagation through electrically coupled cells. Effects of regional changes in membrane properties.

The normal process of excitation of the heart involves propagation of action potentials through cardiac regions of different anatomy and different intrinsic membrane properties. Although our understanding of these properties is still incomplete, it is well accepted that the parameters measured from a single cell penetration in an electrical syncytium (e.g., action potential duration, rate of rise, and velocity) reflect not only the properties of that cell but also the electrotonic interactions with other cells to which the recorded cell is electrically coupled. We have used simulation techniques to predict the spatial distribution of action potential parameters resulting from discretely localized alterations in the intrinsic membrane properties of some of the cells of an electrical syncytium. We have shown that the resulting spatial distribution is markedly different for alterations in plateau and pacemaker currents vs. rising phase currents, and that other factors, such as the site of stimulation and the underlying spatial pattern of cell-cell coupling resistance, also modify the spatial distribution of action potential properties resulting from a discrete regional change in intrinsic membrane properties.

Amino terminal glutamate residues confer spermine sensitivity and affect voltage gating and channel conductance of rat connexin40 gap junctions

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 (Vj) 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 (Gmin) and Vj 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 Vj 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 Vj‐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.

Gating of mammalian cardiac gap junction channels by transjunctional voltage

Numerous two-cell voltage-clamp studies have concluded that the electrical conductance of mammalian cardiac gap junctions is not modulated by the transjunctional voltage (Vj) profile, although gap junction channels between low conductance pairs of neonatal rat ventricular myocytes are reported to exhibit Vj-dependent behavior. In this study, the dependence of macroscopic gap junctional conductance (gj) on transjunctional voltage was quantitatively examined in paired 3-d neonatal hamster ventricular myocytes using the double whole-cell patch-clamp technique. Immunolocalization with a site-specific antiserum directed against amino acids 252-271 of rat connexin43, a 43-kD gap junction protein as predicted from its cDNA sequence, specifically stained zones of contact between cultured myocytes. Instantaneous current-voltage (Ij-Vj) relationships of neonatal hamster myocyte pairs were linear over the entire voltage range examined (0 less than or equal to Vj less than or equal to +/- 100 mV). However, the steady-state Ij-Vj relationship was nonlinear for Vj greater than +/- 50 mV. Both inactivation and recovery processes followed single exponential time courses (tau inactivation = 100-1,000 ms, tau recovery approximately equal to 300 ms). However, Ij recovered rapidly upon polarity reversal. The normalized steady-state junctional conductance-voltage relationship (Gss-Vj) was a bell-shaped curve that could be adequately described by a two-state Boltzmann equation with a minimum Gj of 0.32-0.34, a half-inactivation voltage of -69 and +61 mV and an effective valence of 2.4-2.8. Recordings of gap junction channel currents (ij) yielded linear ij-Vj relationships with slope conductances of approximately 20-30 and 45-50 pS. A kinetic model, based on the Boltzmann relationship and the polarity reversal data, suggests that the opening (alpha) and closing (beta) rate constants have nearly identical voltage sensitivities with a Vo of +/- 62 mV. The data presented in this study are not consistent with the contingent gating scheme (for two identical gates in series) proposed for other more Vj-dependent gap junctions and alternatively suggest that each gate responds to the applied Vj independently of the state (open or closed) of the other gate.

Voltage-Dependent Blockade of Connexin40 Gap Junctions by Spermine

The effects of spermine and spermidine, endogenous polyamines that block many forms of ion channels, were investigated in homotypic connexin (Cx)-40 gap junctions expressed in N2A cells. Spermine blocked up to 95% of I(j) through homotypic Cx40 gap junctions in a concentration- and transjunctional voltage (V(j))-dependent manner. V(j) was varied from 5 to 50 mV in 5-mV steps and the dissociation constants (K(m)) were determined from spermine concentrations ranging from 10 micro M to 2 mM. The K(m) values ranged from 4.9 mM to 107 micro M for 8.6 < or = V(j) < or = 37.7 mV, within the physiological range of intracellular spermine for V(j) > or = 20 mV. The K(m) values for spermidine were > or = 5 mM. Estimates of the electrical distance (delta) for spermine (z = +4) and spermidine (z = +3) were 0.96 and 0.76 respectively. Cx40 single channel conductance was 129 pS in the presence of 2-mM spermine and channel open probability was significantly reduced in a V(j)-dependent manner. Similar concentrations of spermine did not block I(j) through homotypic Cx43 gap junctions, indicating that spermine selectively blocks Cx40 gap junctions. This is contrary to our previous findings that large tetraalkylammonium ions, also known to block several forms of ion channels, block junctional currents (I(j)) through homotypic connexin Cx40 and Cx43 gap junctions.

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.