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

Abstract

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.

Figure 1.

Bright-field (A) and epifluorescent (B) illumination of a heterotypic Cx40/Cx43 N2a cell pair after membrane labeling with and 24 hours in culture. (C) Western blots for Cx40, Cx43, and Cx45 protein expression in parental N2a (left lane), stable rat Cx40-N2a clone (left center lane), stable rat Cx43-N2a clone (right center lane), or transiently expressed murine Cx45 in parental N2a cells (right lane) relative to GAPDH. No endogenous Cx40, Cx43, or Cx45 expression was detected with picomole sensitive detection kit. (D) Endogenous Cx40, Cx43, and Cx45 was detectable with femtomolar detection. E, Real-time PCR results for endogenous murine Cx45 or exogenous rat Cx40 or Cx43 mRNA expression in parental or stable Cx N2a cell clones relative to GAPDH. Exogenously expressed Cx43 or Cx40 mRNA levels were 0.6–1.0 times the GAPDH level in their respective stable cell clones whereas endogenous Cx mRNA levels were <0.001 in all parental and stable N2a cell clones.

Figure 2.

Normalized steady-state junctional current (A) and conductance (B) – transjunctional voltage (I_j – V_j and G_j – V_j) curves for 7 heterotypic Cx43/Cx40 cell pairs. V_j was defined relative to the N2a-Cx40 cell pair. Rectification is produced by the enhanced V_j-dependent gating on the Cx40 negative (Cx43 positive) side of the gap junction and reduced gating when the V_j polarities are reversed. Normalized steady-state I_j – V_j (C) and G_j – V_j (D) curves produced under conditions of unilateral 50% reductions in [KCl] exhibited increased rectification when low [KCl] was present on the Cx40-side and became more symmetrical when [KCl] was lowered on the Cx43-side of the gap junction (N = 6 each). Normalized steady-state I_j – V_j (E) and G_j – V_j (F) curves produced by pairing the mutant Cx40E9,13K double mutant with Cx43 dramatically reduced the rectification by reducing both polarities of V_j-dependent gating.

Figure 3.

Kinetics of inactivation for heterotypic Cx43/Cx40 gap junctions. (A) Junctional current (I_j) decay time constants (τ) for a Cx43/Cx40 heterotypic cell pair when V_j = +120 mV relative to the Cx43 or Cx40 expressing cell. The inactivation rates were dramatically faster and the steady-state open probabilities were significantly (P_open) lower when V_j was positive on the Cx43-side of the gap junction. (B) The V_j-dependent inactivation (K_on) rates as determined by the equation K_on = (1-P_open)/τ when V_j was positive on the Cx43-side of the junction. (C) The same K_on calculation when V_j was positive on the Cx40 side of the heterotypic gap junction. (D) The first-order inactivation rates for the Cx43 and Cx40 V_j positive heterotypic gap junction are plotted relative to each other. The numbers of experiments (n) are indicated next to the symbol.

Figure 4.

(A) Single gap junction channel currents (i_j) recorded from one heterotypic Cx43/Cx40 cell pair depicting a 120 pS channel at V_j = +50 mV and 95 pS channel at −50 mV relative to the Cx40 cell. (B) A different Cx43/Cx40 heterotypic cell pair exhibited mostly 50 or 70 pS channel conductance (γ_j) states at ±40 mV relative to the Cx40-expressing cell. (C) Summarized γ_j data from 8 cell pairs in a frequency histogram and multi-gaussian curve fits suggested 2 peaks at approximately 51 pS and 90 pS (N = 237 events). (D) The average i_j – V_j relationship for 4 Cx43/Cx40 cell pairs and linear curve fits for all channel events above or below 65 pS in amplitude. These pooled channel events averaged 52 or 94 pS, indicative of Cx43 γ_j values.

Figure 5.

(A) Average I_j – V_j curves for heterotypic Cx43/Cx40 gap junctions obtained with symmetrical internal patch pipette solutions (black symbols and lines) or in the presence of 2 mM spermine (gray symbols and lines) added to the Cx40-side of the heterotypic gap junction. The lines indicate the steady-state curve obtained with a continuous 200 ms/mV V_j ramp and the symbols indicate the steady-state I_j value obtained at the end of a 20 sec V_j pulse to the indicated V_j value. (B) The same as in panel A except the 2 mM spermine was added to the Cx43-side of the heteroypic Cx43/Cx40 gap junction. (C) Actual current traces from 2 different experiments illustrating the effect of 2 mM spermine added to the Cx43-side of a Cx43/Cx40 heterotypic gap junction during the −/+/−50 mV V_j pulse sequence of the cationic block V_j clamp protocol. (D) The V_j-dependent spermine inhibition curves for homotypic Cx40 (•) or Cx43 (▪) gap junctions and the heterotypic Cx43/Cx40 gap junction with spermine added to either the Cx40-positive or Cx43-positive side of the gap junction. The fraction of I_j block for the heterotypic gap junction was calculated relative to the heterotypic gap junction currents in the absence of spermine for each side of the junction.