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.
Keywords: Connexin40; Cx37, connexin37; Cx40, connexin40; Cx43, connexin43; Cx45, connexin45; E1, first extracellular loop domain; EDTA, Ethylenediaminetetraacetic acid; FITC, fluorescein isothiocyante; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; Gj, normalized junctional conductance; Gj,max, maximum normalized gj; Gj,min, mimimum normalized gj; I1 and I2, whole cell currents for cell 1 and cell 2; Ij, junctional current; Kon, inactivation on-rate; N2a, mouse Neuro2a; NT, N-terminal domain; Popen, open probability; RT-PCR, real-time PCR; Rel1 and Rel2, whole cell patch electrode resistance values for cell 1 and cell 2; Rin, renal insulinoma; TBS, Tris buffered saline; TRITC, tetramethylrhodamine isothiocyanate; V1 and V2, command voltage clamp potentials for cell 1 and cell 2; V1/2, half-inactivation voltage; Vj, transjunctional voltage; connexin43; gap junctions; gj, junctional conductance; heterotypic; ij, single gap junction channel current; mCx30.2/hCx31.9, mouse connexin30.2/human connexin31.9; pS, picoSiemen; spermine; transjunctional voltage gating; wt, wild-type; ΔI2, change in I2 in response to an applied Vj gradient produced by changing V1; γj, single gap junction channel conductance; τdecay, exponential decay time constant.