Monovalent cation permeation through the connexin40 gap junction channel. Cs, Rb, K, Na, Li, TEA, TMA, TBA, and effects of anions Br, Cl, F, acetate, aspartate, glutamate, and NO3

Abstract

The unitary conductances and permeability sequences of the rat connexin40 (rCx40) gap junction channels to seven monovalent cations and anions were studied in rCx40-transfected neuroblastoma 2A (N2A) cell pairs using the dual whole cell recording technique. Chloride salt cation substitutions (115 mM principal salt) resulted in the following junctional maximal single channel current-voltage relationship slope conductances (gamma 1 in pS): CsC1 (153), RbC1 (148), KC1 (142), NaC1 (115), LiC1 (86), TMAC1 (71), TEAC1 (63). Reversible block of the rCx40 channel was observed with TBA. Potassium anion salt gamma j are: Kglutamate (160), Kacetate (160), Kaspartate (158), KNO3 (157), KF (148), KC1 (142), and KBr (132). Ion selectivity was verified by measuring reversal potentials for current in rCx40 gap junction channels with asymmetric salt solutions in the two electrodes and using the Goldman-Hodgkin-Katz equation to calculate relative permeabilities. The permeabilities relative to Li+ are: Cs+ (1.38), Rb+ (1.32), K+ (1.31), Na+ (1.16), TMA+ (0.53), TEA+ (0.45), TBA+ (0.03), Cl- (0.19), glutamate+ (0.04), and NO(3)- (0.14), assuming that the monovalent anions permeate the channel by forming ion pairs with permeant monovalent cations within the pore thereby causing proportionate decreases in the channel conductance. This hypothesis can account for why the predicted increasing conductances with increasing ion mobilities in an essentially aqueous channel were not observed for anions in the rCx40 channel. The rCx40 effective channel radius is estimated to be 6.6 A from a theoretical fit of the relationship of relative permeability and cation radius.

Figure 1

Rat connexin40 single channel activity with IPS KCl (Table I). (A) Whole cell currents from a rat Cx40 transfected N2A cell pair during a −40-mV step applied to cell 1 from a common holding potential of 0 mV, resulting in a transjunctional voltage (V_j) of −40 mV. Junctional currents appear as opposite polarity signals. A 10-s interval of a 120-s recording is shown to illustrate the closed (ground, I_j = 0) state and open states of the four channels observed during the pulse. Both current traces were low pass filtered at 40 Hz and digitized for illustration at 1 kHz. (B) All points histogram (solid line) compiled from the cell 2 current trace. The first 10 s of the recording were omitted due to nonstationary channel activity. The parameters for the probability density function (pdf, dashed line) include four channels each with a single channel current of 6.1 pA (152.5 pS), 0.32 open probability, and closed and open state current variances of 0.5 pA. (C) Whole cell currents from the same cell pair as A during a +40-mV step applied to cell 1. This current trace also shows four open channels, each with a single channel current of 5 pA (125 pS). (D) All points histogram of the cell 2 current trace shown in C. Each channel had an open probability of 0.27 and current variance of 0.4 pA. The closed state current variance was 0.55 pA. (E) Single channel current-voltage relationship for the same cell pair illustrated in A and C. Each dot represents the junctional current amplitude of an observed channel for an applied V_j pulse. The single channel slope conductance (γ_j) of 139 pS was determined from the linear regression fit (solid line) of the data (r   > 0.99).

Figure 2

Single channel junctional current-voltage relationships using substituted cation chloride salt IPSs (Table I). Each symbol refers to a different cell pair. The composite slope conductance (γ_j) obtained by linear regression fit (solid line) of the single channel current amplitudes from all of the cell pairs (N) indicated. (A) IPS CsCl, γ_j = 152.5 pS, 5 cell pairs. (B) IPS RbCl, γ_j = 148.4 pS, 3 cell pairs. (C) IPS KCl, γ_j = 141.5 pS, 6 cell pairs. (D) IPS NaCl, γ_j = 115.1 pS, 4 cell pairs. (E) IPS LiCl, γ_j = 86.1 pS, 7 cell pairs. (F) IPS TMACl, γ_j = 71.0 pS, 6 cell pairs. (G) IPS TEACl, γ_j = 63.1 pS, 4 cell pairs. The slope conductances listed here are not statistically different from the mean calculated from the individual single channel junctional current-voltage relationships for each cell pair (see results). Correlation coefficients were >0.99 for all graphs.

Figure 3

Experiments with IPS TBACl. (A) Whole cell currents during an experiment in which one electrode (cell 1) was filled with IPS KCl and the other (cell 2) was filled with IPS KCl in which TBACl was dissolved to yield a final concentration of 5 mM TBACl. A significantly greater number of open channel current transitions is shown during positive relative to negative polarity V_j pulses applied to cell 1. (B) Expanded view of A illustrating the disappearance of discrete channel opening from +35 to −40 mV. (C) Expanded view of A showing the recovery of discrete channel opening from −40 to +25 mV. (D) The i_j-V_j relationship for this experiment. The γ_j of 141 pS is equal to the mean conductance obtained with symmetric IPS KCl which is consistent with an insignificant TBA current.

Figure 4

Single channel junctional current-voltage relationships using substituted anion potassium salt IPSs (Table I). Each symbol refers to a different cell pair. The composite slope conductance (γ_j) obtained by linear regression fit (solid line) of the single channel current amplitudes from all of the cell pairs (N) indicated. (A) IPS Kglutamate, γ_j = 160.4 pS, 4 cell pairs. (B) IPS Kacetate, γ_j = 159.8 pS, 4 cell pairs. (C) IPS Kaspartate, γ_j = 158.0 pS, 2 cell pairs. (D) IPS KNO₃, γ_j = 157.2 pS, 3 cell pairs. (E) IPS KF, γ_j = 148.0 pS, 5 cell pairs. (F) IPS KCl, γ_j = 141.5 pS, 6 cell pairs. (G) IPS KBr, γ_j = 131.5 pS, 5 cell pairs. The slope conductances listed here are not statistically different from the mean calculated from the individual single channel junctional current-voltage relationships for each cell pair (see results). Correlation coefficients were >0.99 for all graphs.

Figure 5

Comparison of theoretical aqueous and junctional rCx40 single channel conductances. The theoretical linear relationships (dashed lines) of the conductance with mobility were calculated from the Goldman-Hodgkin-Katz current equation assuming ion permeabilities directly proportional to their diffusion coefficients. (A) Junctional (open circles) and theoretical (filled circles) aqueous relative single channel conductance are plotted versus cation mobility. The relationship of junctional conductance with cation mobility was fit by linear regression (solid line, r = 0.96). The conductance sequence Cs⁺ > Rb⁺ > K⁺ > Na⁺ > Li⁺ > TMA⁺ > TEA⁺ resulted from the cation-Cl substitutions. (B) Junctional (open circles) and theoretical (filled circles) aqueous relative single channel conductance are plotted versus anion mobility. The relationship of junctional conductance ratios with anion mobility decreased only slightly up to a mobility of 7.4 × 10⁻⁴ cm²/V·s but declined sharply for greater mobilities. This was in marked contrast to the linearly increasing conductance ratios predicted for an aqueous channel (dashed line).

Figure 6

(A) Single channel current-voltage relationships obtained for the purpose of determining reversal potentials to confirm the relative conductances measured with the symmetric salt solutions. I_j-V_j relationships from four cell pairs using asymmetrical 115 mM KCl/LiCl IPSs. The linear regression fit (solid line, r = 0.99) of all channel current amplitudes indicates a reversal potential (I_j = 0) of 9.9 mV. (B) Single channel junctional current-voltage relationships obtained for the purpose of determining the rCx40 relative permeabilities of Li⁺ and Cl⁻. The i_j-V_j relationships (n = 4) were determined when one cell of a rCx40 transfected N2A cell pair was dialyzed with IPS LiCl (115 mM LiCl) and the other cell of the pair with a lower LiCl concentration (30 mM) and mannitol (145 mM) for osmotic balancing. The linear regression fit (solid line, r = 0.99) of all channel current amplitudes indicates a reversal potential (I_j = 0) of 15.8 mV. (C) I_j-V_j relationships from four cell pairs using asymmetrical 115 mM Kglutamate/KCl IPSs. The linear regression fit (solid line, r = 0.99) of all channel current amplitudes indicates a reversal potential (I_j = 0) of 9.9 mV.

Figure 7

Relationship between rCx40 gap junction channel relative ion permeability (P_X/P_Li) as listed in Table V and hydrated radius (Nightingale, 1959) for the monovalent cations studied. The theoretical fit of the hydrodynamic equation suggests a pore radius of 6.6 ± 0.7 Å.