INTRODUCTION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION CONCLUSIONS APPENDIX REFERENCES

Gap junction channels are unique among ion channels because they connect two intracellular compartments while traversing the extracellular environment. Hexamers of homomeric or heteromeric connexin subunits form a plasmalemmal transmembrane hemichannel that interacts with another hemichannel in a homotypic or heterotypic manner to form the intact gap junction channel. Gap junctions functionally integrate the coupled cells via electrical and chemical diffusion of ions and second messengers essential to tissue homeostasis and primary physiological function (e.g., excitation, secretion, contraction, transport). The ionic permeabilities of rat connexin-40, -43, -46, and Xenopus connexin38 (rCx40, rCx43, rCx46, and Cx38) homogeneous connexin hemichannel or gap junction channels, reveal quantitatively similar K⁺/Cl permeabilities of 10/1 (Trexler et al., 1996; Beblo and Veenstra, 1997; Wang and Veenstra, 1997; Zhang et al., 1998). Furthermore, the relative permeability sequence for the group IA monovalent cations exhibited only minor deviations from their relative aqueous mobility sequence. These single salt ionic permeabilities and unitary channel conductances are the only information currently available about the ion-permeation pathway through connexin channels. Furthermore, although some three-dimension structure of hemichannels and gap junction channels are emerging, there are no direct structural correlates that define which protein domains line the transmembrane pore (Perkins et al., 1997, Unger et al., 1999).

Ionic affinities and interactions have always been integral to the development of mechanistic models for ion permeation through ion-selective channels. Structural correlates were obtained by combining mutational analysis with electrophysiological analysis of ionic conductance, permeability, or block of putative pore-forming sequences (French and Shoukimas, 1985; Yellen, 1987; Choi et al., 1993; Hille, 1992). For example, ionic blockade by tetraethylammonium (TEA⁺) ions was instrumental in the identification of the signature pore segment of several voltage-dependent potassium channels (Armstrong and Binstock, 1965; MacKinnon and Yellen, 1990). Furthermore, TEA⁺ and larger tetraalkylammonium cations, such as tetrabutylammonium (TBA⁺), tetrapentylammonium (TPeA⁺), and tetrahexylammonium (THxA⁺) ions, are known to block ion permeation through the ryanodine receptor, nicotinic acetylcholine receptor, neuronal chloride, and anthrax toxin channels (Sanchez and Blatz, 1995; Sanchez et al., 1986; Tinker et al., 1992; Blaustein and Finkelstein, 1990a,b; Blaustein et al., 1990). Previously, we observed lower single-channel conductance (_j) and ionic permeability ratios (_TAA/K or P_TAA/K) in the rCx40 and rCx43 channels with 115 mM tetramethylammonium (TMA⁺) and TEA⁺ ions (in Cl salts; Beblo and Veenstra, 1997; Wang and Veenstra, 1997). The _TAA/K or P_TAA/K ratios also closely followed their relative monovalent cation aqueous mobilities. However, unilateral addition of 115 mM TBA⁺ abolished rCx40 junctional currents (I_j) when the transjunctional potential (V_j) was positive on the tetraalkyammonium (TAA⁺) side even though I_j was observed when V_j was reversed (KCl side positive, Beblo and Veenstra, 1997). Prolonged exposure to 115 mM TBA⁺ proved toxic to the cells. We have investigated the actions of larger TAA⁺ ions on the macroscopic junctional conductance (g_j) of rCx40 gap junctions by unilateral addition of millimole amounts of TBA⁺, TPeA⁺, and THxA⁺ ions. We demonstrate, for the first time, ionic blockade of a homotypic connexin gap junction channel by large TAA⁺ ions in a concentration- and voltage-dependent manner. Furthermore, we have modeled the voltage-dependent dissociation constants K_m(V_j) for TPeA⁺ and THxA⁺ block of the rCx40 channel. The one- and two-site models both predict that TPeA⁺ and THxA⁺ permeate through the rCx40 pore. Bilateral addition of TPeA⁺ reduced ionic blockade in exact proportion to the unilateral K_m(V_j) for the opposite side [TPeA⁺], consistent with single-site interactions between two TPeA⁺ ions acting with identical binding affinities. Finally, we demonstrate that the addition of an octyl side chain increases the affinity and kinetics for block.

	MATERIALS AND METHODS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION CONCLUSIONS APPENDIX REFERENCES

Electrophysiological recording

Stable rCx40 transfected neuro2A (N2A) cell cultures were prepared and maintained as previously described (Beblo et al., 1995). N2A cell cultures were washed 3-5 times with HEPES-buffered saline immediately prior to use and placed on the stage of an inverted phase contrast microscope (Olympus IMT-2, Lake Success, NY). The bath saline contained (in mM): 142 NaCl, 1.3 KCl, 0.8 MgSO₄, 0.9 NaH₂PO₄, 1.8 CaCl₂, 4.0 CsCl, 2.0 TEACl, 5.5 dextrose, 10 HEPES, pH 7.4 (titrated with 1 N NaOH), 310 mosm. All junctional current recordings were performed using conventional double whole cell recording techniques using two Biologic RK-300 (Claix, France) or Axopatch 1D (Axon Instruments, Foster City, CA) patch clamp amplifiers (Veenstra and Brink, 1992; Beblo et al., 1995). Patch electrodes (PG52151-4, WPI, Inc., Sarasota, FL) had tip resistances of 4-6 M prior to G seal formation and patch break when filled with 140 mM KCl internal pipette solution (IPS). The standard KCl IPS contained (in mM): 140 KCl, 4.0 CsCl, 2.0 TEACl, 3.0 CaCl₂, 5.0 K₄BAPTA, 1.0 MgCl₂, 25 HEPES, pH 7.4 (titrated with 1 N KOH), 310 mosm. MgATP was added daily to achieve a final concentration of 3.0 mM. TAACl salts were added unilaterally as indicated for each experiment. TBACl, TPeACl, THxACl, and Tetraheptylammonium chloride (THepACl) were purchased from Aldrich Chemical (Milwaukee, WI) and stored as a 1-M stock solution in 18 M-cm water or 70% ethanol and diluted as required with KCl IPS. The final osmolarity of the TAACl+KCl IPS was not adjusted because the maximum dose of 10 mM TPeACl altered the final IPS volume by 1% and the total osmolarity by 6%. For most [TAA⁺], the IPS osmolarity was altered by 3%. All experiments were performed at room temperature (20-22°C). Off-line current and voltage data recordings were stored on VCR tape using a Neurocorder DR-484 2/4 channel digitizer (Cygnus Technology, Delaware Water Gap, PA) at 10 kHz direct from the patch clamp amplifier. All analyzed currents were digitized at 2 kHz and low-pass filtered at 100 Hz (WPI LPF-30) unless otherwise indicated. Analysis was performed using the DOSTAT analysis program (Manivannan et al., 1992). Final graphs and curve-fitting were performed using Kaleidagraph software (Synergy Software, Reading, PA).

To determine the magnitude of TAA⁺ block, a voltage protocol was written that sequentially altered the holding potential (V₁) of the TAACl + KCl-containing cell (cell 1) from negative to positive and back to negative potentials relative to the KCl-containing cell (cell 2) in 30-s intervals. The common holding potential (V₁ and V₂) was 40 mV for both cells. Cell 1 was stepped to this common potential for 1 s at the end of each 30-s voltage pulse and for 10 s between different /+/ command voltages to assess any change in the nonjunctional voltage clamp circuit. V_j = (V₁ + V₁)  V₂ and V₁ was altered in 5-mV increments from 10 to 50 mV and I_j = I₂. One example of the I_j recordings obtained from an experiment with 10 mM TPeACl at V_j = /+/40 mV is shown in Fig. 1. There were no time-dependent changes in I_j observed during the control V_j pulse to 40 mV. Some time-dependent decay and recovery of I_j was observed during the first 5 s of the subsequent block and recovery ±40-mV V_j pulses in all TAA⁺ experiments. I_j was averaged over the 29-s V_j pulse and junctional I-V relationships were plotted for every experiment as shown in Fig. 2. This plot demonstrates the reversibility of the TAA⁺-dependent block using this voltage protocol. The maximum junctional conductance (g_j,max) was calculated from the slope of the linear regression fit of the 10 to 30-mV I_j-V_j curve for each experiment.

View larger version (21K): [in this window] [in a new window]   FIGURE 1   Whole cell currents from the postjunctional cell (cell 2) of a rat Cx40-transfected N2A (rCx40) cell pair demonstrating voltage-dependent block of junctional currents (Ij) in the presence of 10 mM tetrapentylammonium chloride. Shown is an excerpt from a single experiment displaying initial and steady-state currents elicited by a transjunctional voltage step (Vj) of ±40 mV. This is one sequence of an entire protocol in which Vj is varied in 5-mV increments from ±10 to ±50 mV. Voltage polarity is in reference to the prejunctional cell (cell 1). TPeA+ ions were added only to the prejunctional cell (cell 1). For each Vj, cell 1 is stepped to a negative holding potential (negative Vj polarity, control) for 30 s, followed by a voltage step of equal amplitude and opposite polarity (positive Vj, block) for 30 s, and then returning to the control voltage for 30 s (recovery). Each 30-s step terminates with a 1-s interval where Vj = 0 mV. The common holding potential for both cells when Vj = 0 mV was 40 mV. Each +//+ voltage sequence is followed by a 10-s rest period (Vj = 0 mV). There is a significant reduction in Ij when Vj pulses of positive polarity are applied to cell 1 relative to steady-state currents obtained by the preceding and subsequent Vj pulses of negative polarity.

View larger version (29K): [in this window] [in a new window]   FIGURE 2   Whole cell junctional current-voltage (I-V) relationship from the same 10 mM TPeA+ experiment shown in Fig. 1. The junctional I-V curve illustrates a reversible Vj-dependent and time-dependent block of rCx40 Ij at positive values that drive positively charged TPeA+ ions into the pore.

Synthesis of octyl-tributylammonium

Solutions of triethylammonium and tributylammonium were refluxed with equimolar amounts of 1-bromoocatane for 48 h in acetonitrile under an inert atmosphere (Halpern et al., 1982). The reaction mixtures were concentrated in vacuo, dissolved in water, extracted with ether, decolorized with activated charcoal, and lyophilized. All alkyl reagents were purchased from Aldrich Chemical. The chemical structures were confirmed by NMR spectroscopy. Stock solutions of 0.5 M N-octyl-triethylammonium (TEOA) and N-octyl-tributylammonium (TBOA) bromide were kept at 20°C until the day of use.

	RESULTS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION CONCLUSIONS APPENDIX REFERENCES

Concentration-dependence of TAA block

We first attempted to determine the magnitude and concentration-dependence of TBACl block of the rCx40 channel because the lack of I_j was first observed when this compound was added unilaterally at 115 mM to rCx40 N2A cells (Beblo and Veenstra, 1997). Stable I_j recordings were limited to 20 mM TBA⁺ added unilaterally because the input resistance of the TAA⁺-containing cell diminished over time. V_j-dependent gating was observed to commence at ±40 mV, consistent with the previous observations of Beblo et al. (1995). Because the analysis of voltage-dependent ionic blockade of an ion channel is best performed under conditions of constant (equal) open probability (P_o), all quantitative analysis of ionic blockade was limited to the linear portion of the junctional I-V relationship for each experiment (i.e., N × P_o = constant). Reversible ionic blockade of I_j by unilateral TBA⁺ at V_j = +35 or +40 mV was 10% of control I_j at 1 or 2 mM TBA⁺ and achieved a maximum of 40% with 10 or 20 mM TBA⁺ (Fig. 3 A). The reduction in I_j was even more pronounced at higher voltages where V_j-dependent gating is also more prevalent. The average g_j was 1.36 ± 0.56 nS (n = 3) for 1 mM TBACl, 5.02 ± 2.69 nS (n = 3) for 2 mM TBACl, 4.61 nS (n = 1) for 5 mM TBACl, 2.05 ± 1.58 nS (n = 5) for 10 mM TBACl, and 3.35 ± 2.24 nS (n = 4) for 20 mM TBACl. The average g_j was 2.96 ± 2.13 nS (n = 16) for all TBACl experiments, which was not statistically different from previous observations of g_j with this clone of rCx40-transfected N2A cells (Beblo et al., 1995). Due to the limited V_j and TBA⁺ concentration ranges where partial block of rCx40 g_j were observed in Fig. 3, no further analysis with TBACl was attempted.

View larger version (26K): [in this window] [in a new window]   FIGURE 3   (A) Normalized steady-state junctional I-V curves for 1 mM TBA+ (n = 3) and 10 mM TBA+ (n = 5) added unilaterally to cell 1. Transjunctional voltage (Vj)-dependent closure is evident at ±35 mV and additional block of Ij at +Vj is due to the presence of 10 mM TBA+. The solid line is the junctional I-V predicted for rCx40 gap junctions using Eq. 4 to predict the Vj-dependence of gj as previously determined (Beblo et al.,1995, see text for details). Each experiment was normalized to the slope of the junctional I-V plot in the 10 to 30 mV range (linear range of Vj, see Beblo et al., 1995) and the normalized junctional I-Vs were pooled for each [TBA+]. Each data point represents the mean ± SD for each [TBA+]. (B) Normalized steady-state junctional I-V curves for 100 µM (n = 3), 1 mM (n = 3), and 10 mM TPeA+ (n = 4) added unilaterally to cell 1. The solid line again illustrates the Vj-dependent gj for rCx40 according to Eq. 4, whereas the three different dashed lines indicated in the legend illustrate the additional effect of Vj-dependent block by 0.1, 1.0, and 10 mM TPeA+ on rCx40 Ij according to Eq. 3. The Km(Vj) values for TPeA+ are given in Table 1. All data for each [TPeA+] were normalized as described for TBA+. Each data point represents the mean ± SD for each [TPeA+]. (C) Normalized steady-state junctional I-V curves for 10 µM (n = 3), 200 µM (n = 3), 1.0 mM (n = 7), and 3.5 mM THxA+ (n = 4) added unilaterally to cell 1. The indicated lines again predict the junctional I-V according to Eq. 3 (various dashed lines) or Eq. 4 (solid line) using the Km(Vj) values for THxA+ provided in Table 2. All data for each [THxA+] were normalized as described for TBA+. Each data point represents the mean ± SD for each [THxA+].

The next largest TAA⁺ ion in the series was TPeA⁺, and the same experimental protocols were applied to the concentration-dependent block of rCx40 g_j. The upper limit that could be tolerated in KCl IPS with this compound was 10 mM TPeACl. The mean junctional I-V relationships for several TPeA⁺ concentrations are presented in Fig. 3 B. The mean g_j was 4.00 ± 1.29 nS for 100 µM TPeACl (n = 3), 5.34 ± 3.04 nS for 200 µM TPeACl (n = 4), 4.00 ± 0.40 nS for 350 µM TPeACl (n = 3), 4.40 ± 2.27 nS for 500 µM TPeACl (n = 3), 9.56 ± 0.34 nS for 1 mM TPeACl (n = 3), 1.36 ± 0.31 nS for 2 mM TPeACl (n = 2), 3.61 ± 3.02 nS for 3.5 mM TPeACl (n = 5), 2.68 ± 1.91 nS for 5 mM TPeACl (n = 4), and 1.37 ± 0.18 nS for 10 mM TPeACl (n = 4). The average g_j was 4.01 ± 2.85 nS (n = 31) for all TPeCl experiments. I_j was reversibly reduced in a V_j- and concentration-dependent manner at all positive V_j values, as indicated by the linear junctional I-V at negative V_j (average of control and recovery I_j values, see Fig. 2). The junctional I-V relationships for each TPeA⁺ concentration were pooled after normalizing each experiment to its linear slope g_j. V_j-dependent gating was observed at ±45 mV in all experiments. I_j declined only when the holding potential was more positive in the TPeA⁺-containing cell, indicative of ionic block of the KCl permeation pathway (Tinker et al., 1992). The magnitude of the apparent TPeA⁺ block was significantly greater than with identical concentrations of TBA⁺ and achieved a maximum percent block of 64% at +40 mV with 10 mM TPeA⁺.

THxA⁺ was a more potent blocker than TPeA⁺ as evidenced by the junctional I-V relationships illustrated in Fig. 3 C. The increasing hydrophobicity was also more evident because THxACl had to be dissolved in 70% ethanol to achieve a 1 M stock solution and was not tolerated internally at concentrations 5 mM in KCl IPS. Experimentally, the maximum degree of block actually observed with THxA⁺ was the same as with TPeA⁺ but it was achieved with only 3.5 mM THxACl in the KCl IPS. The mean g_j was 10.28 ± 3.13 nS for 10 µM THxACl (n = 3), 6.31 ± 2.25 nS for 35 µM THxACl (n = 2), 5.49 ± 1.88 nS for 50 µM THxACl (n = 3), 3.61 ± 2.16 nS for 100 µM THxACl (n = 4), 6.13 ± 0.44 nS for 200 µM THxACl (n = 3), 12.16 ± 6.80 nS for 350 µM THxACl (n = 5), 6.96 ± 6.19 nS for 500 µM THxACl (n = 4), 2.72 ± 1.87 nS for 1 mM THxACl (n = 7), 2.14 ± 0.84 nS for 2 mM THxACl (n = 6), and 1.47 ± 0.94 nS for 3.5 mM THxACl (n = 4). The mean g_j (5.34 ± 4.67 nS, n = 41) for all THxA⁺ experiments was similar to the TPeACl g_j values. This demonstrates that the increasing amount of I_j blockade is a result of the addition of THxA⁺ in place of TPeA⁺ ions and not to differences in the I_j recordings. Furthermore, higher g_j values would lead to less observable I_j block because the series resistance error of the transjunctional voltage clamp increases with increasing g_j. The fraction of the applied V_j that actually drops across the junctional resistance (R_j) is proportional to the sum of the patch pipette resistances divided by the sum of both pipette resistances and R_j (= 1/g_j). This is true even when the whole cell patch electrode resistances are constant and 1% of the cellular input resistance. Hence, the data are consistent with the increasing effectiveness of progressively larger TAA⁺ ions in producing I_j blockade.

Time-dependence of TAA block

The junctional I-V relationships displayed in Fig. 3, A-C were the mean steady-state junctional I-V relations for each [TAA⁺] of the TBACl, TPeACl, and THxACl experiments performed. In some experiments, a similar voltage protocol was used to determine the magnitude of the time-dependent or time-independent (instantaneous) TAA⁺ block and relief from block (unblock) with these organic cations. In these experiments, the 1-s rest interval at the common holding potential of 40 mV was omitted from the /+/ V_j sequence. The pulse duration was 30 s per pulse and the V_j increment was 5 mV. The net amount of instantaneous block and unblock observed upon  and ± V_j polarity reversal results from the relative values of k_off(V_j)/k_on[TAA]. Analysis of the instantaneous and final I_j values of all block and recovery V_j pulses from 17 TPeA⁺ (three 350-µM, one 500-µM, two 2-mM, four 3.5-mM, three 5.0-mM, and four 10-mM) and 27 THxA⁺ (seven 350-µM, ten 500-µM, five 1-mM, three 2-mM, and two 3.5-mM) experiments did not reveal any instantaneous block of I_j by either compound at any [TAA⁺] or V_j. Conversely, there was some instantaneous unblock that varied inversely with [TAA⁺] and only slightly with V_j. Below 500 µM TPeA⁺, the recovery from steady-state block was instantaneous at all V_j values. Figure 4 illustrates the amount of steady-state block of I_j that recovered instantaneously upon polarity reversal of V_j for 2 and 10 mM TPeA⁺. The complete I_j traces from one 2-mM and one 10-mM TPeA⁺ experiment at /+/ 40 mV are shown in panels A and C. The transitions from the steady-state block to the instantaneous recovery for these two experiments are displayed in panels B and D. The percentage of instantaneous unblock appeared to be concentration-dependent, decreasing from 51.3 ± 3.1% at 2 mM TPeA⁺ to 22.2 ± 4.6% at 10 mM TPeA⁺ for all V_j values examined (20-40 mV in 5-mV increments, panel E). Approximately 30% instantaneous unblock was observed for 3.5 and 5.0 mM TPeA⁺. THxA⁺ also exhibited a concentration-dependent instantaneous recovery from steady-state block. Instantaneous unblock at 350 µM THxA⁺ appeared to be V_j-dependent, decreasing from 100% at 20 mV to 45% at 40 mV. This apparent V_j-dependent instantaneous relief of block was not evident at any of the higher [THxA⁺] where <50% instantaneous unblock was observed for all V_j values (data not shown).

View larger version (36K): [in this window] [in a new window]   FIGURE 4   Instantaneous relief of block (unblock) upon /+ Vj step to the indicated value for 2 mM and 10 mM TPeA+. Instantaneous block during the +/ Vj step was not observed for any [TPeA+] as shown in panels A and C for 2 and 10 mM TPeA+. The instantaneous relief of block by TPeA+ decreased with increasing [TPeA+] as observed in panels B and D from these same two experiments. The percentage (mean ± SD) of instantaneous block or unblock was calculated by dividing the change in instantaneous Ij by the difference in steady-state Ij for the control/block and block/recovery Vj steps (% instantaneous unblock = [(inst. Ij,recovery  |ss Ij,block|)/(ss Ij,recovery  |ss Ij,block|)] × 100) for all 2 (n = 2) and 10 mM (n = 4) TPeA+ experiments.

Because the entire steady-state block by both TPeA⁺ and THxA⁺ developed during the +V_j pulse, the time-dependence of the decay phase of the I_j traces was fit with single- and double-exponential functions to determine the time constant(s) for steady-state block at each V_j. The difficulty with this analysis was that the magnitude of the steady-state block was typically only 2-4 times greater than the magnitude of the steady-state current fluctuations. This condition also applied to the (lower amplitude) time-dependent rising phase of the subsequent recovery (unblock) I_j traces. Therefore, the mean time constants and the experimental variability of the exponential fits were in excess of 100 ms for all [TAA⁺] and V_j values. Individual fluctuations of I_j during a blocking or unblocking V_j pulse had time constants of <100 msec.

Voltage-dependent dissociation constants for TAA block

To determine the equilibrium constants for the blocking reaction, the fractional current (I_j,K+TAA/I_j,K) was plotted as a function of V_j for all concentrations of TPeACl and THxACl and fitted with the equation

(1)

Alternatively, the data were fit with the following expression, which assumes that complete blockade is never achieved

(2)

The minimum unblocked fractional I_j (I_j,min) could result from partial blockade of the open rCx40 channel currents (i.e., a TAA⁺-induced subconductance state), the inability to block a naturally occurring rCx40 subconductance state (i.e., a TAA⁺-insensitive V_j-dependent subconductance state; G_j,min = 0.30, Beblo et al., 1995), or a nonspecific background current that is insensitive to TAA⁺-dependent block (i.e., a significant series resistance error in the junctional voltage clamp). The calculated K_m(V_j) ± SD values for [TPeA⁺] and [THxA⁺] are summarized in Table 1. These estimated K_m(V_j) values were used to fit the steady state junctional I-V relationships in Fig. 3, B and C, according to the expression

(3)

when G_j,max = 1 (normalized slope conductance for each experiment) and G_j,min is the estimated TAA-insensitive portion of G_j,max. The maximum junctional conductance (g_j,max) was determined from the slope of the linear regression fit of all I-V points between 10 and 30 mV for each experiment and G_j = g_j/g_j,max. The g_j,max for all experiments averaged between 1 and 12 nS (see Results, Concentration-dependence of TAA block), and G_j,max was defined to be 1. Normalized I_j equals G_j · V_j and the actual I-V curve is readily obtained by multiplying the normalized I_j values by g_j,max for each experiment. Figure 3, A-C, is representative pooled normalized I_j-V_j curves for the selected concentrations of TBA⁺, TPeA⁺, and THxA⁺. The same procedures were applied to the bilateral TPeA⁺ I-V curves (see Fig. 11), except that g_j was determined from the linear slope conductance between 10 and +10 mV of each experimental I-V curve because TAA⁺ block is minimal at low V_j values. No K_m(V_j) values were calculated for TBACl and the steady-state junctional I-V relationships in Fig. 3 A were fitted with the expression

(4)

that describes the predicted V_j-dependence of rCx40 from previous observations (Beblo et al., 1995). In Eqs. 3 and 4, G_j,max = 1 because all the experiments were normalized to their instantaneous slope conductances, 50 mV was the half-inactivation voltage (50 mV for V_j values), and 0.12 (+0.12 for V_j values) was the slope factor of the Boltzmann curve that best described the V_j-dependent gating of g_j for rCx40. These data indicate that a concentration-dependent and V_j-dependent blocking mechanism can describe the nonlinear steady-state junctional I-V relationships observed in Fig. 3.

Complete block of I_j was not achieved within the experimental voltage range of ±50 mV, and the calculated fit of the experimental data with Eq. 2 provided a more accurate fit of the data because Eq. 1 requires that I_j  0. Examples of a few voltage-dependent dose-response curves for TPeA⁺ and THxA⁺ are shown in Fig. 5.

View larger version (34K): [in this window] [in a new window]   FIGURE 5   Dose-response curves for unilateral TPeA+ and THxA+ reduction of rCx40 Ij. The steady-state unblocked fraction of Ij was determined for each [TAA+] and Vj by dividing the steady Ij at +Vj by steady-state Ij at Vj (control and recovery pulses averaged together) for every independent experiment. Each data point is the mean ± SD for n experiments at each [TAA+]. (A) Dose-response curves for TPeA+ at the indicated Vj values. The curved lines are theoretical fits of the data using Eq. 2 (see text for details). The parameters of the fit are listed in Table 1. (B) Dose-response curves for THxA+ at the indicated Vj values. The curved lines are theoretical fits of the data using Eq. 2 (see text for details). The parameters of the fit are listed in Table 1.

Barrier models for TAA block of the rCx40 channel

Since Woodhull (1973) derived a barrier model for voltage-dependent ionic block, voltage-dependent K_m have determined the relative energy profile of ion channels at the site of block by determining the fraction () of the applied transmembrane voltage field sensed by the ions. That original derivation was for an asymmetrical inside-outside membrane channel. In the case of a homotypic gap junction channel, we have a symmetrical inside-inside double-membrane channel where the permeant ions remain electrically isolated from the extracellular potential. Given that the applied V_j will be across two identical halves of the rCx40 gap junction channel, two possibilities exist. There could be a single ionic binding site formed by both homotypic rCx40 hemichannels that could sense the entire applied V_j or that each hemichannel contains its own ion binding site which could sense a maximum of V_j/2 of the applied V_j. To determine which one of these models best describes the TAA⁺-dependent block of the rCx40 gap junctions, we derived similar expressions to the original Woodhull derivation for a symmetrical single-site and symmetrical two-site permeation pathway with or without a central compartment into which an ion could dissociate. The energy profiles for these one-site and two-site models are illustrated in Fig. 6, A-C. The mathematical derivations are provided in the Appendix. The K_m(V_j) values listed in Table 1 that lie within the range of experimental [TAA⁺] for TPeA⁺ and THxA⁺ were fitted with each of the expressions derived in the Appendix that correspond to the original Woodhull model for a permeant or nonpermeant blocking ion, Eqs. A6 and A7, respectively, and Eq. A16, for a permeant blocking ion traversing a two-site, four-barrier model. Only the two-site, four-barrier model yields a different expression for block by TAA⁺ ions under unilateral conditions (see Appendix). The fraction of V_j sensed by these monovalent cations at the site(s) of block were determined from these theoretical fits to the experimental K_m(V_j) data presented in Fig. 7. The  and relative kinetic rate constants (e.g., b₁/b₁) for the fitted curves are listed in Table 2. If the blocking TAA⁺ is impermeant, only Eq. A7 applies to all three models. Only the K_m(V_j) values 20  V_j  40 mV were used because the estimated K_m(V_j) values for 10 and 15 mV were highly variable and near the upper experimental [TAA⁺] limit. V_j-dependent gating of g_j also became increasingly prominent when V_j  ±40 mV. The original Woodhull derivation for an impermeant blocking ion (Eq. A7) predicts an electrical distance () of 1. This implies that the ion traverses the entire V_j field across the homotypic rCx40 gap junction channel. When we use the more appropriate Woodhull expression for a slightly permeant blocking ion (Eqs. A6), we obtain   1.7. This implies that, as soon as we permit TAA⁺ ions to be on both sides of the blocking site, more than one ion (TAA⁺ or K⁺) occupies the pore. If two sites were included within the pore, one associated with each rCx40 hemichannel, a   2 is obtained. This again implies one permeant ion per site. Due to the high variability in the experimental K_m values and the predicted small differences between the one- and two-site models, the available experimental data unfortunately cannot distinguish between the one-site and two-site models. One test of whether each side of the channel has identical K_m(V_j) values for the larger TAA⁺ and how they might interact requires bilateral addition of TAA⁺ ions. The data from two sets of bilateral TPeA⁺ experiments are presented in the Results section.

View larger version (23K): [in this window] [in a new window]   FIGURE 6   Model energy barrier diagrams for a symmetrical one-site (A) and two-site three- (B) or four-barrier (C) ion channel. Derivations for the voltage-dependent equilibrium dissociation constant (Km(Vj)) appear in the Appendix according to the association and dissociation rate constants (e.g. k1 or 1) indicated in the diagram.

View larger version (33K): [in this window] [in a new window]   FIGURE 7   Theoretical fits of the Vj-dependent Km values for TPeA+ and THxA+. The Km(Vj) values provided in Tables 1 and 2 over the +20- to +40-mV range were fitted with equations A6, A7, and A16 (see Appendix). Eqs. A6 and A7 represent the Woodhull (1973) derivation for a permeant and impermeant blocking ion with the exception that the channel is assumed to be symmetrical. Eq. A16 predicts the Km(Vj) values for a symmetrical channel with two ion binding sites. TPeA+ and THxA+ are also assumed to be permeant for Eq. A16. The estimated electrical distance () and relative kinetic rate parameters from the fitted lines are listed in Table 2.

View this table: [in this window] [in a new window]   TABLE 2   Electrical distance () estimates for TPeA+ and THxA+

Single-channel analysis of TAA block of the rCx40 channel

All of the experiments presented in Figs. 2-5 and 7 were obtained from macroscopic conductance data from rCx40 N2A cell pairs. On occasion, single-channel currents were observed due to a lower g_j of <1 nS and different V_j protocols were performed to obtain unitary channel conductance (_j) and open probability (P_o) data from these multichannel records. One experiment each with 2 mM TPeA⁺ and THxA⁺ are shown in Figs. 8 and 9. Both examples demonstrate a reversible reduction in P_o when V_j is positive with respect to the TAA⁺-containing cell. These were the only two experiments that produced complete single-channel current-voltage relationships without the use of pharmacological uncoupling agents. Uncoupling agents such as octanol must be avoided because they also reduce P_o (Veenstra and DeHaan, 1988; Takens-Kwak et al., 1992). The junctional I-V relationships were also plotted for the current amplitudes observed in the all points histograms (panels B, D, and F) from both observed multichannel experiments (Fig. 10). The main state conductance of the rCx40 channel in the presence of TPeA⁺ or THxA⁺ was 150 ± 5.6 pS (n = 2). There were numerous rapid channel events observed in Figs. 8 and 9 that appear to be of reduced amplitude when filtered at 100 Hz and digitized at 2 kHz. These recording conditions have a temporal resolution of 6 ms for a full-amplitude event. Some of this data was resampled at 1 kHz low-pass bandwidth and digitized at 10 kHz to resolve these brief duration events with 1-ms resolution for full-amplitude events. There were no new peaks in the amplitude histograms to suggest the presence of a subconductance state of the rCx40 channel although the open channel noise did increase due to the wider recording bandwidth. Hence, the brief events observed in Figs. 8 C and 9 C during the TPeA⁺ and THxA⁺ blocking pulses achieve full amplitude openings and closings within the temporal resolution of the recording system.

View larger version (48K): [in this window] [in a new window]   FIGURE 8   Blockade of rCx40 unitary channel currents by unilateral addition of 2 mM TPeA+. Panels A-F are representative of a /+/45-mV Vj sequence obtained from a rCx40 cell pair with low gj (1 nS). Panels A, C, and E illustrate all of the multichannel levels observed during each 30-s Vj pulse with IPS KCl in both pipettes and 2 mM TPeA+ added only to cell 1. Panels B, D, and F are the all-points junctional current amplitude histogram for the entire 30-s Vj pulse. Six unitary channels are observed in the control (A and B) and recovery (E and F) 45-mV Vj pulses with N = 1 or 2 or 3 open channels being the predominant peaks accounting for 79% of the cumulative open time (cumulative open probability (Po) = 0.79). Closed probability (Pc) was 0.06 excluding the 1-s Vj = 0 baseline interval during each 45-mV Vj pulse. During the +45-mV blocking pulse (C and D), Pc increased to 0.65 and the N = 2 or 3 open-channel peaks were nearly nonexistent (Po  0.05). Only the N = 1 open-channel peak is clearly evident in the histogram (Po = 0.27), indicating that TPeA+ blocked all six rCx40 channels 94% of the time as determined from a probability density function fit of the closed and N = 1 current peaks with N = 6 total channels. The unitary junctional current-voltage relationship is shown in Fig. 10.

View larger version (45K): [in this window] [in a new window]   FIGURE 9   Blockade of rCx40 unitary channel currents by unilateral addition of 2 mM THxA+. Panels A, C, and E are again representative current recordings that illustrate all of the open channel levels observed during each /+/40-mV Vj pulse. There was a maximum of N = 2 channels observed in this experiment. Each Vj pulse was 2 min in duration for this experiment and the +Vj pulses were obtained by hyperpolarizing cell 2 by 40 mV in this example from a common holding potential of 40 mV for both cells. Panels B, D, and F are the junctional current amplitude histograms for each 2-min recording. The open probability for the N = 1 open-channel state was 0.15 for the control (panel B) and 0.23 for the recovery (panel F) pulses and <0.01 during the blocking Vj pulse (panel D). A second channel appeared only briefly during the entire 6-min recording (panels E and F, Po = 0.01 for N = 2 state). The channel openings illustrated in panel C were the only observed channel events during the +40-mV Vj pulse. The unitary junctional current-voltage relationship is shown in Fig. 10.

View larger version (34K): [in this window] [in a new window]   FIGURE 10   Single-channel junctional current-voltage relationships in the presence of either 2 mM TPeA+ or THxA+. The unitary amplitudes of the rCx40 channels present in the current amplitude histograms of Figs. 8 and 9 (panels B, D, and F) were plotted as a function of Vj, and a linear regression fit was performed on all of the data points for each experiment. The single-channel slope conductances (j) were 146 and 154 pS for the open rCx40 channels in the presence of 2 mM TPeA+ and THxA+, respectively. These j values are similar to the j of 142 pS reported previously for the rCx40 in 115 mM KCl + 20 mM X+ (12 Na+, 8 Cs+, and 3 TEA+; Beblo and Veenstra, 1997).

Bilateral TAA as a test of the symmetry of block

If TAA⁺ is added bilaterally, the steady-state junctional I-V should be predicted by using the K_m(V_j) values already determined from unilateral addition of TAA⁺, provided that TAA⁺ entering the pore from either side binds and blocks independently of the trans side [TAA⁺]. No interaction is expected to occur if two independent blocking sites exist or if a single blocking site is vacated prior to TAA⁺ arriving from the opposite side upon V_j polarity reversal. The TPeA⁺ and THxA⁺ block and unblock data indicate that unblock is more rapid than block. Hence, bilateral addition of TAA⁺ will not provide an accurate test of the one-site or two-site models unless interactions are observed between bilateral [TAA⁺] as reported for voltage-gated potassium channels (Newland et al., 1992). To test this hypothesis, 500 µM TPeA⁺ or 2 mM TPeA⁺ was added to the trans side relative to 2 mM TPeACl. TPeA⁺ was chosen because it was the more hydrophilic of the two blocking ions and 2 mM  the experimentally observed K_m(V_j) values (see Table 1). No interaction between trans/cis TPeA⁺ ions is predicted by the 0.5/2 mM I-V curve (long dashed line) in Fig. 11 A. This result would indicate no apparent change in the K_m(V_j) for either trans ([TAA⁺]₂) 500 µM or cis ([TAA⁺]₁) 2 mM TPeA⁺, i.e., independent binding. Contrary to the unilateral block data, 500 µM trans TPeA⁺ produced no block, and 2 mM cis TPeA⁺ block was reduced from the unilateral condition (trans [TPeA⁺] = 0). This phenomenon was observed before and after normalization of the three experimental I-V curves.

View larger version (31K): [in this window] [in a new window]   FIGURE 11   (A) Normalized steady-state junctional I-V curve for bilateral 500 µM/2 mM TPeA+. The solid line is the junctional I-V predicted for rCx40 gap junctions using Eq. 4 (see text for details). The open circles represent the mean ± SD of three experiments on rCx40 gap junctions where 2 mM TPeA+ was present in cell 1 (cis) with 500 µM TPeA+ present in cell 2 (trans). Each experiment was normalized to the linear slope conductance of the I-V between 10 and +10 mV. The long dashed line (0.5/2 mM) illustrates the amount of block expected for unilateral addition of TPeA+ at the respective concentrations. The short dashed line (0.5/2 model) was determined using Eq. 5 (see text for details) which predicts a reduction in the 2-mM cis TPeA+ block in exact proportion to the expected block by 500-µM trans TPeA+. (B) Normalized steady-state junctional I-V curve for bilateral 2/2 mM TPeA+. The open circles represent the mean ± SD of two experiments on rCx40 gap junctions where 2 mM TPeA+ was present in both cells 1 (cis) and 2 (trans). The long dashed line (2/2 mM) illustrates the amount of block expected for unilateral addition of TPeA+ at the respective concentrations. No block is observed in either direction, consistent with Eq. 5 when equal concentrations of the blocking ion are present on both sides of the channel. Only the intrinsic Vj-gating of rCx40 is observed (Eq. 4) under these conditions.

To model the observed shift in the I-V curve, we added the amount of I_j block expected from unilateral trans 500 µM TPeA⁺ to the 2 mM cis TPeA⁺ I-V curve. This theoretical result (Eq. 5 and Fig. 11 A, short dashed line) closely matched the experimental 500 µM/2 mM I-V curve (Fig. 11 A, open circles ± SD, N = 3). This result is expected if 500 µM TPeA⁺ acting from the opposite side blocks I_j in exact proportion to the K_m(V_j) values determined from the unilateral 500 µM TPeA⁺ experiments (Figs. 3 B, 5 A, and Table 1). However, we can only estimate the amount of block by plotting I_j,K+[TAA]1/I_j,K+[TAA]2. For the unilateral case, [TAA]₂ = 0 (Eqs. 1 and 2). This model predicts that anytime [TAA⁺]₁ = [TAA⁺]₂, no net TAA⁺ block will be observed because TAA⁺ blocks I_j in identical proportion from either side of the channel. Furthermore, the intrinsic V_j-gating of rCx40 g_j is unaltered, thus indicating that TPeA⁺ occupancy from one or both sides does not shift the V_j dependence of rCx40. Eq. 5 was obtained from Eq. 3 by adding the difference in g_j expected from the block produced by [TAA⁺]₂ based on the unilateral K_m values.

(5)

which reduces to Eq. 3 whenever [TAA⁺]₂ = 0. This equation only applies when [TAA⁺]₁  [TAA⁺]₂ because the general expression
defines the proportion of G_j,max blocked by [TAA⁺], the general expression
defines the proportion of G_j,max not blocked by [TAA⁺], and
Therefore, whenever [TAA⁺]₁ = [TAA⁺]₂, this expression reduces to Eq. 4.

This prediction was tested by two bilateral 2-mM TPeA⁺ experiments. The results are illustrated in Fig. 11 B, where no TPeA⁺ block of rCx40 g_j was observed in either direction, and the experimental data agree closely with the expected V_j-dependence of rCx40. These results are consistent with TPeA⁺ binding with equal affinity from opposite sides of the channel and reducing the expected 2 mM cis TPeA⁺ block by the precise amount expected from 500 µM or 2 mM trans TPeA⁺ binding at an identical site at all V_j values. We do not know from macroscopic junctional I-V curves if g_j is already reduced proportionately by the respective cis and trans [TPeA⁺] because we cannot independently determine g_j in the absence of net cationic K⁺/TPeA⁺ flux depending upon the polarity of V_j as was performed under unilateral conditions. For the bilateral TPeA⁺ experiments, g_j was normalized to the slope of the junctional I-V curve between 10 mV < V_j < +10 mV where TPeA⁺ block is minimal. The slope conductances were 0.80, 1.27, and 7.06 nS for the three 500 µM/2 mM TPeA⁺ experiments and 4.48 and 5.31 nS for the two 2/2-mM TPeA⁺ experiments.

These results are best explained by alternating occupancy by cis or trans TPeA⁺ binding at one or more sites with a constant K_m(V_j). The simplest explanation for multiple sites is the presence of two identical sites, although this model cannot definitively distinguish between two blocking ions interacting at one or two sites. A further test of the two-site hypothesis requires single-channel block data under bilateral conditions. Either rapid block occurs due to the alternating occupancy by cis/trans TPeA⁺ or the channel open and closed dwell times are increased and reduced by trans TPeA⁺ relative to the unilateral 2-mM TPeA⁺ condition. Unfortunately, the latter case cannot be readily tested because gap-junction channel kinetics are too slow to acquire a reliable number of channel events to calculate the open and closed time distributions under unilateral and bilateral TPeA⁺ conditions. The observation of only full-channel amplitude events, as occurred in the unilateral experiments, or the observation of temporally unresolved flicker block of the rCx40 channel would favor the independent one-site model. The observation of full-amplitude channel events upon polarity reversal are indicative of the site(s) becoming unoccupied prior to block from the opposite side, a scenario most likely to occur with a single site. Rapid flicker or partial channel block (an apparent subconductance state) can result from simultaneous occupancy of one or two identical sites. Figure 12 demonstrates the continuous presence of flicker block under bilateral TPeA⁺ conditions, consistent with cis/trans interactions at a single site.

View larger version (46K): [in this window] [in a new window]   FIGURE 12   Blockade of rCx40 unitary channel currents by bilateral addition of 2-mM and 500-µM TPeA+. Panels A-F are representative of a /+/35-mV Vj sequence obtained from a rCx40 cell pair with low gj ( 1 nS). Panels A, C, and E illustrate all of the multichannel levels observed during each 30-s Vj pulse with IPS KCl in both pipettes to which 2 mM TPeA+ was added to cell 1 (cis) and 500 µM TPeA+ was added to cell 2 (trans). Panels B, D, and F are the all-points junctional current amplitude histogram for the entire 30-s Vj pulse. Approximately four unitary channels are observed in the control (A and B) and recovery (E and F) 35-mV Vj pulses with N = 1 or 2 or 3 open channels being the predominant peaks. The open channels accounting for 100% of the cumulative recording time (cumulative open probability (Po) = 1.0), excluding the 0.5-s Vj = 0 baseline interval during each 35-mV Vj pulse. During the +35-mV blocking pulse (C and D), the closed probability did not increase, and the N = 1 or 2 open-channel peaks were most prevalent (Po = 1.0). All open and closed probability measurements were based on cumulative time distributions and no probability density function fits of the open-channel histograms with N = 4 total channels were attempted due to inability to resolve unitary channel current amplitudes.

Hydrophobic interactions at the site of block

Because the hydrophobicity of the TAA⁺ ions increased with molecular size, we synthesized TEOA⁺ and TBOA⁺ ions to examine the effect of hydrophobicity on the block of Cx40 gap junctions. TBOA⁺ possesses the same molecular mass as TPeA⁺, however, the highest [TBOA⁺] that was tolerated for the duration of the 15-min V_j protocol was 1 mM. Figure 13 illustrates that 1 mM TBOA⁺ blocked only the V_j-sensitive component of I_j, but did so with faster kinetics than TPeA⁺ (see Fig. 4). The amount of block is approximately equal to the block observed with TBA⁺. The V_j pulse illustrated in panel A demonstrates the V_j-dependent gating of Cx40 during the 40-mV control and recovery pulses and the apparent lack of this V_j-gating during the +40-mV test pulse. Higher temporal resolution of the transition from the control to block V_j pulse reveals the rapid block by TBOA⁺ to steady-state I_j values within 100 ms (panel B). A similar time course was revealed for the unblock of TBOA⁺ from the steady-state I_j value to the full-peak value during the transition from the block to recovery V_j pulse (panel C). The normalized I-V curve from four 1-mM TBOA⁺ experiments illustrates the rapid block of I_j by TBOA to control steady-state values.

View larger version (42K): [in this window] [in a new window]   FIGURE 13   Increased kinetics of block by 1 mM TBOA+. The complete 40-mV Vj protocol from one of four 1-mM TBOA+ experiments illustrates the rapid block to steady-state levels of Ij (panel A). The control/block and block/recovery transitions are shown at higher temporal resolution in panels B and C as indicated. The normalized instantaneous () and steady-state () I-V curves for all four 1-mM TBOA+ experiments are plotted in panel D. The mean value of Ij from 10 to 20 ms after the switch in Vj polarity was taken as the value of instantaneous Ij. The curved line depicts the Vj-dependence of Cx40 according to Eq. 4.

TEOA⁺ also produced V_j-dependent reductions in I_j at a concentration of 1 mM. In one macroscopic g_j experiment, the kinetics of block was slow, similar to that shown for TPeA⁺ (data not shown). Two multichannel experiments further revealed a reduction in the open probability of the main Cx40 channel state with no reduction in channel current amplitude when V_j  +30 mV (data not shown). These results indicate that the mechanism of block is the same as that observed for TPeA⁺ and THxA⁺.

	DISCUSSION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION CONCLUSIONS APPENDIX REFERENCES

Ionic block of a gap junction

Ionic blockade has not been described for any gap-junction channel. The observation that TBA⁺ did not carry current through rCx40 gap-junction channels (Beblo and Veenstra, 1997) led us to further examine the block of rCx40 gap junctions by larger TAA⁺ ions. The results presented in this manuscript demonstrate that TBA⁺, TPeA⁺, and THxA⁺ directionally reduce I_j in a manner that is fully reversible by changing the V_j polarity (Figs. 1 and 2). The direction of block was always in the direction of cationic current flow from the TAA⁺-containing cell and was concentration-dependent (Fig. 3). Ionic blockade is also typically voltage-dependent (Woodhull, 1973). Our results demonstrate a decreasing K_m with increasing positive V_j (Fig. 5) for both TPeA⁺ and THxA⁺ in the rCx40 channel, consistent with this hypothesis. Because we are studying a double-membrane channel, we derived alternative expressions to the classical Woodhull equation for symmetrical (homomeric homotypic) and asymmetric (homomeric heterotypic) gap junctions, with V_j defined relative to the TAA⁺-containing cell (Fig. 6 and Appendix). We obtained equivalent electrical distance () values of 1 ion per site for both TPeA⁺ and THxA⁺ irrespective of the mathematical expression used to fit the data (Fig. 7). Mathematical descriptions reveal only minor differences between the one-site and two-site models that depend entirely on the V_j-dependent distribution of internal ions to produce any difference in the observed K_m(V_j) values (Appendix). Multichannel recordings from low g_j rCx40 cell pairs revealed a V_j-dependent reduction in open probability without a reduction in the single-channel current amplitude (Figs. 8-10). Macroscopic g_j experiments revealed symmetrical block by TPeA⁺ with identical K_m(V_j) values to those determined from the unilateral experiments, indicative of a common site of interaction (Fig. 11). However, single-channel current recordings under bilateral TPeA⁺ conditions revealed only "noisy" open-channel currents at all V_j values that made determination of _j impossible (Fig. 12). The unilateral application of 1 mM TBOA⁺ and TEOA⁺ also produced directional block resembling that of TPeA⁺, thus revealing a role for a hydrophobic interaction in increasing the affinity for block (Fig. 13). The kinetics of TBOA⁺ block was significantly faster than TPeA⁺, despite their possessing identical chemical formula weights, suggestive of a further role of tethering the ion on the kinetics of block (Figs. 4 and 13).

Prior to these observations, all pharmacological blockade of g_j was produced by the external addition of amphipathic hydrocarbons (Johnston et al., 1980; Burt, 1989; Burt and Spray, 1989; Davidson and Baumgarten, 1988, Wu et al., 1993; Guan et al., 1997). All of these amphipathic compounds are thought to act via partitioning into the biological phospholipid membrane, a mechanism consistent with the general anesthetic properties of the long chain alkyl alcohols, fatty acids, and halothane (Spray and Burt, 1990; Takens-Kwak et al., 1992; Bastiannse et al., 1993). Two of these uncoupling agents, octanol and heptanol, are known to act by reducing channel open probability (Veenstra and DeHaan, 1988; Takens-Kwak et al., 1992). This occurs in a voltage-independent manner and may involve deformations in the lipid bilayer-protein interface, resulting in altered channel conformations (Lundbæk and Andersen, 1999). The dependency of block on cationic current direction is not consistent with block by a lipophilic pathway, although hydrophobic TAA⁺ interactions within a channel pore are known to occur (Armstrong,