An amino-terminal lysine residue of rat connexin40 that is required for spermine block

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1 J Physiol (2006) pp An amino-terminal lysine residue of rat connexin40 that is required for spermine block Xianming Lin, Edward Fenn and Richard D. Veenstra Department of Pharmacology, SUNY Upstate Medical University, 750 East Adams Street, Syracuse, NY 13210, USA Spermine blocks connexin40 (Cx40) gap junctions, and two cytoplasmic amino-terminal domain glutamate residues are essential for this inhibitory activity. To further examine the molecular basis for block, we mutated a portion of a basic amino acid (HKH) motif on the Cx40 amino-terminal domain. Replacement of the Cx40 H15 + K16 residues with the Q15 + A16 sequence native to spermine-insensitive connexin43 (Cx43) gap junctions increased the equilibrium dissociation constant (K d ) and reduced the maximum inhibition by spermine. The corresponding electrical distance (δ) approximation was decreased by about 50%. The transjunctional voltage (V j )-dependent gating of homotypic Cx40 H15Q + K16A mutant gap junctions was also significantly reduced. The minimum normalized steady-state junctional conductance (G min ) increased from 0.17 to 0.72, with an increase in the half-inactivation voltage from 48 to 60 mv. However, the unitary junctional conductance (γ j ; 160 ps) was only slightly altered, and the relative cation/anion conductance and permeability ratios were unchanged from wild-type Cx40 gap junction channels. The relative K + /Cl permeability (P K /P Cl )ratio increased from six to ten when [KCl] was reduced to 25% of normal. These data suggest that the HKH motif at positions is important to the conformational structure of the putative voltage sensor and spermine receptor of Cx40, without causing significant alteration of the electrostatic surface charge potentials that contribute to the ion selectivity of this gap junction channel. (Resubmitted 28 August 2005; accepted after revision 7 November 2005; first published online 10 November 2005) Corresponding author R. D. Veenstra: Department of Pharmacology, SUNY Upstate Medical University, 750 East Adams Street, Syracuse, NY 13210, USA. veenstrr@upstate.edu Gap junctions are modulated by transjunctional voltage (V j ), intracellular Ca 2+ ion and proton concentrations, and receptor signalling cascades involving protein kinases (Harris, 2001; Lampe & Lau, 2004). Recently it was demonstrated that another polyvalent cation, spermine, blocks homomeric connexin40 (Cx40) gap junctions in a concentration- and V j -dependent manner (Musa & Veenstra, 2003). The spermine block of Cx40 gap junctions was subsequently demonstrated to be dependent on the presence of two glutamic acid residues located at positions 9 and 13 on the cytoplasmic amino-terminal (NT) domain of Cx40 (Musa et al. 2004). However, replacement of the naturally occurring lysine residues of connexin43 (Cx43) with these two glutamic acid residues from Cx40 did not confer spermine sensitivity to Cx43. This observation led to speculation that other connexin NT residues or cytoplasmic domains are required for the overall structure of the putative Cx40 polyamine receptor. Mutations incorporating the Cx40 E13K mutation also exhibited the loss of V j -dependent gating and long-duration unitary gap junction channel events. The multiplicity of the Cx40 E13K mutational effects complicate the mechanistic analysis of spermine block, but also imply a commonality of function. The NT domain of the connexins is postulated to control the voltage polarity of V j gating (Verselis et al. 1994). The connexin NT domain is thought to form an α helix that points inward towards the connexin channel pore due to the formation of a helix loop helix motif that positions the first half of the NT domain within the V j field (Purnick et al. 2000a). Polar or charged NT amino acid mutations alter the V j -gating properties of several connexins, and also affect the corresponding hemichannel and gap junction channel conductances (Verselis et al. 1994; Oh et al. 1999; Musa et al. 2004; Tong et al. 2004). It is generally observed that the V j -gating polarity is oppositely affected by the valence of the charge substitution on NT amino acids up to position 10 in the β-group of connexins (Purnick et al. 2000b). This raises the possibility that at least the first half of the connexin NT domain translocates towards the centre of the channel when the appropriate V j polarity is applied to that side of the gap junction, although the Cx32 R15Q Charcoat-Marie-Tooth disease (CMTX) mutation also slightly alters the V j gating of homotypic or DOI: /jphysiol

2 252 X. Lin and others J Physiol heterotypic Cx26 and Cx32 gap junctions (Abrams et al. 2001). Cx40 and Cx43 possess other alternative amino acid loci on their respective NT domains, including positions of this initial cytoplasmic connexin domain. We demonstrate that substituting the Q15 and A16 residues found in Cx43 for the naturally occurring H15 and K16 residues of Cx40 dramatically diminished the inhibition of Cx40 gap junction currents (I j )byintracellular spermine. Significant alterations in the V j -gating properties of Cx40 were also observed. However, in contrast to previous findings, the H15Q + K16A mutations did not significantly affect the unitary channel conductance of the homomeric homotypic and heterotypic or heteromeric mutant/wild-type (wt) Cx40 gap junction channels. These experiments demonstrate that polar and charged amino acid residues beyond the midpoint of the α type connexin NT domain are vital to the structural channel components required for V j -dependent gating and spermine block of gap junctions. Furthermore, the Cx40 H15Q + K16A mutations demonstrate a separation of function between the V j -dependent gating processes and the conductance and ionic selectivity properties of the Cx40 gap junction channel. Methods Cell culture conditions Stable murine neuro2a (N2a) neuroblastoma cell clones of rat connexin40 (Cx40) were grown in culture media containing 90% minimum essential medium (MEM), 10% heat-inactivated fetal bovine serum, 0.1 mm non-essential amino acids, 2.5 mm l-glutamine, 0.25 g l 1 G418 sulphate, and 10 U µg 1 ml 1 penicillin/streptomycin (Invitrogen, Carslbad, CA, USA). Confluent cell monolayers were passaged weekly and cells were plated in a 35 mm culture dish for electrophysiological examination the next day. Parental N2a cells were grown in MEM culture media minus the G418 sulphate. All T25 culture flasks were kept in a 5% CO 2 humidified incubator at 37 C. Expression of site-directed mutations The rat Cx40 cdna was mutated by PCR-based site-directed mutagenesis, and inserted into the ptracer-cmv2 vector (Invitrogen) by directional cloning using two restriction endonucleases. The mutant DNA sequence was confirmed by automated DNA sequence analysis by the Upstate Medical University DNA Core Facility. A plasmid preparation was prepared using the EndoFree plasmid maxi kit according to the manufacturer s instructions and stored at 20 C (Qiagen, Valencia, CA, USA). Transient transfections were performed on 80% confluent parental N2a cells grown in24-well culture plates on a daily basis. Each well was transfected for 4 h with approximately 1 µg DNA and 3 µl oflipofectamine 2000 in 100 µl ofopti-mem, according to manufacturer s instructions (Invitrogen). The cells were then split into two 35 mm culture dishes containing 3 ml of MEM culture medium and incubated overnight. Green-fluorescent-protein (GFP)-positive cell pairs were identified under epifluorescent illumination on the stage of an Olympus IMT-2 inverted phase-contrast microscope at 470 nm excitation and >500 nm emission wavelengths. Electrophysiological measurements All dual whole-cell voltage-clamp experiments were performed using two RK-400 patch clamp amplifiers (Molecular Kinetics, Inc., Pullman, WA, USA) at room temperature (20 22 C) on the stage of an Olympus IMT-2 microscope equipped with phase-contrast optics for observation of live cells. The 35 mm cell culture plates were rinsedandbathedwithserum-freesaline (mm:nacl 142, KCl 1.3, CsCl 4, TEACl 2, MgSO 4 0.8, NaH 2 PO 4 0.9, CaCl 2 1.8, dextrose 5.5, Hepes 10, ph 7.4). Patch pipettes were filled with a KCl internal pipette solution (IPS KCl; mm:kcl 140, CsCl 4, TEACl 2, MgCl 2 1, CaCl 2 3, BAPTA 5, Hepes 25, ph 7.4). IPS potasssium gluconate contained 140 mm potassium gluconate in place of KCl. The final osmolarity of all external and internal solutions was adjusted to 310 mosmol l 1. Raffinose (Sigma, St Louis, MO, USA) was added to the 25, 50 and 75% IPS KCl to maintain the osmolarity. The K + activities of each IPS were measured using ion-selective electrodes. Mg 2 ATP (3 mm) was added fresh daily to the IPS KCl. Astocksolution of 0.5 m spermine(hcl) 4 (Calbiochem, La Jolla, CA, USA) was diluted daily as required with IPS KCl. Patch electrodes had a measured bath resistance (R el ) of 3 6 M and dual whole-cell patch electrode series resistance was corrected for using previously described methods (Veenstra, 2001a). Whole-cell currents were digitally sampled at 1 or 4 khz using pclamp8.2 software (Axon Instruments) after low-pass filtering at 100 or 500 Hz with a four-pole Bessel filter (LPF-2, Warner Instruments). Statistical curve fitting of experimental data was performed using the least-squares method of iteration in the Clampfit8.2 application (Axon Instruments). Secondary connexin structure modelling The full-length rat Cx40, Cx43, connexin26 (Cx26) and connexin32 (Cx32) primary amino acid sequences were analysed using the PREDICTOR application (Combet et al. 2000). This program compares the output of eight different secondary-structure predictor programs (DPM, DSC, GOR4, HNNC, PHD, Predator, SIMPA96, and SOPM) to arrive at a consensus prediction.

3 J Physiol Functional consequences of Cx40 H15Q + K16A mutations 253 Results Effect of Cx40H15Q + K16A mutations on spermine block Previously it was shown that substitution of the Cx40 position 9 or 13 glutamic acid residues with the corresponding position 9 or 13 lysine residues from Cx43 reduced or eliminated the spermine block of homotypic mutant Cx40 gap junctions (Musa et al. 2004). However, the reciprocal K9E and K13E mutations into Cx43 did not confer spermine sensitivity to the homotypic mutant Cx43 gap junctions. This led us to consider the involvement of other disparate amino acid sequences between Cx40 and Cx43 as structural components of the Cx40 spermine-blocking site. The amino-terminal rat Cx40 and Cx43 primary amino acid sequences and their predicted consensus secondary structures are shown in Fig. 1 along with the same for Cx26 and Cx32. The previously mutated sites are italicized and, for this study, we replaced the Cx40 H15 and K16 residues with the Q15 and A16 residues from Cx43. These amino acid residues are predicted to reside on a cytoplasmic α helix formed by the NT domain of these two α group connexins (Combet et al. 2000). Spermine inhibition dose response curves were produced by adding spermine unilaterally to the cell receiving the voltage-clamp pulses, as previously described (Musa & Veenstra, 2003). The holding potential was 40 mv for both cells and cell 1 was sequentially stepped to negative (control), positive (block), and negative (recovery) V j values in 5 mv increments from 5 to 50 mv. Figure 2A demonstrates the increasing time- and V j -dependent inhibition of Cx40 gap junction current (I j )by2mm spermine during increasingly positive V j pulses from a single experiment. The amount of inhibition of steady-state I j increases with increasing spermine concentration and V j,asexpected (Fig. 2B). The data were pooled by dividing the steady-state junctional conductance (g j )bythe slope conductance (g j,max ) measured between 5 and 20 mv for each experiment, calculating the normalized I j ( = V j [g j /g j,max ]) for each experiment, and averaging the normalized I j for each test concentration of spermine. The dose response curves were calculated by dividing the steady state I j obtained at positive V j by the steady-state I j obtained at the equivalent negative V j for each experiment (Fig. 2C). Again, the average amount of inhibition was plotted at each V j for all spermine concentrations tested. The mean I j values at each V j were fitted with the equation: I j(kcl+spermine) /I j(kcl) = 1/(1 + [spermine]/k d ) The V j -dependent K d values for spermine inhibition of Cx40 I j are provided in Table 1. The effect of the Cx40 H15Q + K16A mutations on spermine block was examined using the same procedures as previously described. The inhibition of Cx40 H15Q + K16A I j from a single experiment is displayed in Fig. 2D. Some block of Cx40 H15Q + K16A gap junctions is still evident, but the magnitude of the block is obviously reduced. The spermine block was still concentration and V j dependent (Fig. 2E). Representative dose response curves are shown in Fig. 2F, and the V j -dependent K d values are provided in Table 1. Effective electrical distance estimates for wtcx40 and H15Q + K16A gap junctions V j -dependent K d values indicate that the blocking molecule senses a fraction of the voltage field at the site of occlusion. Woodhull (1973) ascribed this phenomenon to atwo-barrier binding-site model where the inner well (binding site) lies an electrical distance (δ)from the inside of the channel. The V j -dependent K d values from Table 1 were fit with the expression: ( ) ( ) b 1 zδfvj K d = exp RT b 1 derived for a homotypic gap junction channel where Z = valence, F = Faraday s constant, R = molar gas constant, and T = absolute temperature ( k) (Musa et al. 2001). The results are displayed graphically in Fig. 3, and the corresponding relative dissociation/association rate constants (b 1 /b 1 ) and zδ values are provided in Table 2. Spermine has a valence of +4 atphysiological ph, so the equivalent δ values are 0.82 for the wtcx40 and 0.48 Figure 1. The primary amino acid sequences, predicted secondary structures, and predicted conserved primary connexin sequence for the cytoplasmic amino-terminal (NT) domains of rat Cx40, Cx43, Cx26 and Cx32 Previously mutated Cx40 and Cx43 amino acid residues are italicized (Musa et al. 2004). The amino acid motif targeted for mutagenesis and functional analysis in this study is indicated by the box, and the beginning of the first transmembrane (M1) domain is underlined. The consensus secondary structure prediction represents the average output from eight different secondary structure prediction program alogrithms (DPM, DSC, GOR4, HNNC, PHD, Predator, SIMPA96, and SOPM; Combet et al. 2000). Secondary structures: c, random loop or coil; h, α-helix; e, extended β-strand;?, unknown.

4 254 X. Lin and others J Physiol for the mutant Cx40 H15Q + K16A gap junctions. The H15Q + K16A mutation reduced the fraction of the V j field sensed by the spermine molecule at the blocking site to approximately 50% of the assumed maximum A (zδ = 4.0; Musa & Veenstra, 2003). From Table 1, it is also apparent that the H15Q + K16A mutation increased the V j -dependent minimum K d by an order of magnitude from 200 µm to 2 mm. D B E C F Figure 2. Functional current-voltage and dose-response curues for spermine Double whole-cell current (I 1 and I 2 )recordings obtained with 2 mm spermine added unilaterally to cell 1 and 140 mm KCl in both patch pipettes (A). Junctional current (I j = I 2 measured from baseline V j = 0mVsteps) was reduced in a time-dependent manner only when a positive transjunctional voltage (V j ) pulse (middle pulse) was applied to cell 1. Each current trace represents a ±10 mv increase in V j. B, the normalized steady-state I j V j curves for representative [spermine]. Each data point represents the mean value for between three and seven experiments. C, the V j -dependent dose response curves for inhibition of wild-type Cx40 I j by spermine calculated from the steady state I j V j relationships. Double whole-cell current recordings (D), normalized steady-state junctional current voltage relationships (E), and V j -dependent spermine dose response curves (F) for homotypic Cx40 H15Q + K16A gap junctions in 140 mm KCl. Each data point in E and F represents the mean value for between three and seven experiments. The K d values are listed in Table 1.

5 J Physiol Functional consequences of Cx40 H15Q + K16A mutations 255 Table 1. Voltage-dependent dissociation constants for spermine inhibition of Cx40 gap junction current (I j ) Wild-type rat Cx40 symmetrical 140 mm KCl V j (mv) K d (mm) S.E.M r Mutant rat Cx40 H15Q + K16A V j (mv) K d (mm) S.E.M r Kinetics of spermine blockade One of the predictions of the above Woodhull model analysis is that the relative association and dissociation rates should be, respectively, decreased and increased by the H15Q + K16A mutation. The kinetics of spermine association at positive V j values were quantified by fitting the decay phase of I j during the positive V j steps with a first-order exponential function at all V j and [spermine]. The spermine on-rates (k on )were calculated using the expression: k on = 1 P open τ decay [spermine] where τ decay was the decay constant under both experimental conditions. The corresponding off-rates (k off )were calculated from the expression: by the fitted curve: k on = ( ± ) exp[(v j 12.8 mv) /(20.6 ± 10.4 mv)] ± (r = 0.80) which is close to the 50% decrease in the observed zδ value for this mutant gap junction. The higher amplitude and on-rate at low V j is taken as an indication of increased accessibility of spermine with the Cx40 H15Q + K16A gap junction channel despite the lower K d values. This can occur if the opposing dissociation rates are increased even more than the association rates, as was apparent by the immediate and complete reversal of block with the negative V j steps in Fig. 2D. Figure 4B illustrates the exponential fit of the k off = P open τ decay or from the V j -dependent K d expression: k off = k on K d The rising phase of I j was fit with a first-order exponential function to determine the k off values at negative V j where k off = 1/τ rise.atpositive V j values, P open is the fraction of I j that remained on at the end of the V j pulse, and the steady-state P open = 1atallnegativeV j values. The on-rates were plotted as a function of V j for the wtcx40 and mutant Cx40 H15Q + K16A gap junctions in Fig. 4A and C. Theconcentration-dependent on-rates were exponentially distributed with respect to V j.the first-order equation for the wtcx40 concentration- and V j -dependent spermine association rates was: k on = ( ± ) exp[(v j 8.9 mv) /(14.4 ± 4.0 mv)] ± (r = 0.70) The V j sensitivity of the symmetric mutant Cx40 H15Q + K16A gap junctions was decreased by 42% relative to the control wtcx40 condition, as indicated Figure 3. V j -dependence of spermine K d values The V j dependence of the K d values for the wtcx40 and Cx40 H15Q + K16A gap junctions was assessed by fitting the K d distribution curves with the equation: K d = ( b 1 /b 1 ) exp ( zδf Vj / ) RT which is based on the one-site, two-barrier model from Woodhull (1973). The solution for each theoretical curve is provided in Table 2. The fraction of the V j field sensed by a single spermine molecule (z =+4) at the inhibitory site was decreased from 82 to 48% by the H15Q + K16A mutation in Cx40.

6 256 X. Lin and others J Physiol Table 2. Woodhull model values for Cx40 gap junctions + spermine wtcx40 Cx40 H15Q Parameter symmetric KCl + K16A b 1 /b S.E.M zδ S.E.M r Woodhull (1973). Spermine has a valence (z) of+4.0 at ph 7.4. r, correlation coefficient. The exponential fit of the k off values for the mutant Cx40 H15Q + K16A gap junctions: k off = ( ± ) exp[(v j 8.3 mv) /(4.9 ± 0.6 mv)] ± (r = 0.96) is illustrated in Fig. 4D. Theapparent saturation of the K d and the increasing k off values at these higher positive V j values are suggestive of maximal occupancy and subsequent spermine dissociation from the wtcx40 gap junction channel with increasing positive V j. V j -dependent spermine dissociation rates for wtcx40 gap junctions: k off = ( ± ) exp[(v j mv) /(12.8 ± 1.2 mv)] ± (r = 0.68) V j gating of wtcx40 and Cx40 H15Q + K16A gap junctions The steady state I j V j relationships for the wtcx40 and mutant Cx40 H15Q + K16A gap junctions suggest that the V j gating properties were altered by these experimental A B C D Figure 4. Spermine on-and off-rates for wild-type and mutant Cx40 gap junctions V j -dependent spermine association (A) and dissociation (B) rates for the wtcx40 gap junctions. The H15Q + K16A mutation dramatically reduced the V j sensitivity of the spermine association rates (C) and increased the off-rates for spermine dissociation (D) with the homotypic Cx40 gap junction.

7 J Physiol Functional consequences of Cx40 H15Q + K16A mutations 257 conditions. To examine this aspect, 200 ms mv 1 V j ramps from 0 to ±120 mv were applied to homotypic wtcx40 and Cx40 H15Q + K16A gap junctions (Fig. 5A D). The ensemble-averaged I j of five A V j ramps from each experiment were normalized to the ±5 to±20 mv slope conductance (g j,max ) and the data from between six and eight experiments were pooled to construct the steady-state G j V j curves. The G j V j B C D E F Figure 5. V j gating of homotypic and hetrotypic wild-type and mutant Cx40 gap junctions Representative junctional whole-cell current (I j = I 2 )responses to ±80 mv V j pulses applied to cell 1 of a pair of wild-type Cx40 (A), Cx40 H15Q + K16A (C), and heterotypic wtcx40/cx40 H15Q + K16A (E) gap junctions to illustrate the presence or near absence of time-dependent V j gating. The normalized steady-state junctional conductance voltage (G j V j )relationships for the wtcx40 (B) Cx40 H15Q + K16A (D), and heterotypic wtcx40/cx40 H15Q + K16A (F) gap junctions were fit with a Boltzmann equation to quantify their V j -dependent gating properties (see Table 3).

8 258 X. Lin and others J Physiol Table 3. Boltzmann gating properties of homotypic and heterotypic Cx40 gap junctions Wild-type Cx40 (n = 6) Cx40 H15Q + K16A (n = 7) Cx40 H15Q + K16A/wtCx40 (n = 10) V j +V j V j Parameter V j +V j V j G max 1.04 ± ± ± ± ± ± ± ± ± G min 0.16 ± ± ± ± ± ± ± ± ± V 1/2 (mv) 48.8 ± ± ± ± ± ± ± ± ± 0.3 z 3.31 ± ± ± ± ± ± ± ± ± 0.03 r Denotes data fitted with two independent Boltzmann functions. relationships were fit with the Boltzmann equation: [ ] Gmax [exp(a(v j V G j = 1 / 2 ))] + G min 1 + [exp(a(v j V 1 / 2 ))] where G max = 1, G j = g j /g j,max (g j,max is the ±5to±20 mv normalized slope conductance for each experiment), G min is the minimum value of g j /g j,max, A is the slope factor for the Boltzmann curve (= zf/rt at 20 C), and V1 / 2 is the half-inactivation voltage. The Boltzmann distribution for the wcx40 G j V j curve illustrated in Fig. 5 is provided in Table 3. The H15Q + K16A mutation exhibited biphasic gating characteristics, first decreasing and then increasing with increasing V j of either polarity. This biphasic G j V j relationship was fitted with two Boltzmann functions in series. The solutions to the biphasic Boltzmann curves are provided in Table 3. The V1 / 2 for the two gating phases of the Cx40 H15Q + K16A gap junction were near 40 and 80 mv, and the increase in G j at higher voltages averaged about 50% of the initial decline in G j.this complex V j gating behaviour has not been previously described for homotypic gap junctions. The heterotypic Cx40 H15Q + K16A/wtCx40 gap junction retained the complex biphasic V j gating characteristics at negative V j (positive relative to the Cx40 H15Q + K16A hemichannel). The G max was shifted towards negative V j with respect to the wtcx40 hemichannel, and the net gating charge was decreased at negative and positive V j values in relation to the homotypic Cx40 H15Q + K16A or wtcx40 gap junctions (Fig 5E and F, Table 3). These data indicate that the H15 and K16 residues are critical to the V j -dependent gating function of Cx40 gap junctions. No reversal of the Cx40 gating polarity was apparent with these mutations. Sidedness of spermine block It was assumed that spermine inhibits Cx40 I j from the positive V j side of the junction, consistent with the V j gating polarity of Cx40 gap junctions and the valence of spermine. As a direct test of this assumption, 2 mm spermine was applied unilaterally to the wtcx40 or the Cx40 H15Q + K16A side of heterotypic Cx40 H15Q + K6A/wtCx40 gap junctions. If the assumption is true, more spermine block will be observed when 2 mm spermine is added to the wtcx40 side of the junction and V j is positive. When V j is negative, the first-order kinetics predict that spermine will dissociate from the gap junction. Figure 6A illustrates the findings that the block produced by 2 mm spermine added to the wtcx40 side of the heterotypic gap junction closely resembled the wtcx40 I j V j relationship (compare to Fig. 2B). Conversely, when 2 mm spermine was added to the Cx40 H15Q + K16A side of the heterotypic gap junction, the I j V j relationship closely resembled that of the Cx40 H15Q + K16A gap junction (compare to Fig. 2E). Thus it is concluded that spermine, like V j -dependent gating, occludes the positive side of the Cx40 gap junction. It was also observed that the Cx40 H15Q + K16A essentially eliminated the block of Cx40 gap junctions by 5 mm tetrapentylammonium ions (Musa et al. 2001). Comparison of the two I j V j curves in Fig. 6B clearly indicates that the block by large tetraalkylammonium ions was almost completely abolished (<10% inhibition at all V j )bythe H15Q + K16A mutations. Coexpression of Cx40 H15Q + K16A gap junctions The effect of the mutant Cx40 H15Q + K16A protein on the V j -gating properties of wild-type Cx40 and Cx43 gap junctions was examined by transient coexpression of the Cx40 H15Q + K16A subunit in stable wtcx40 or wtcx43 transfected N2a cell clones. Coexpression of the Cx40 H15Q + K16A protein with its wild-type counterpart eliminated most of the V j -dependent gating of the heteromeric gap junctions (Fig. 7A and Table 4). The most notable difference between the homomeric Cx40 H15Q + K16A and heteromeric Cx40 H15Q + K16A plus wtcx40 gap junctions was the disappearance of the slight increase in G j at high V j values. The coexpressed wtcx40 + Cx40 H15Q + K16A G j V j relationship was fitted with a parallel double Boltzmann function of the form: G j = p 1 G wtcx430 Cx40 H15Q+K16A + (1 p 1 ) Gj where G Cx j equals the exact solution to the Boltzmann function for that particular homotypic connexin gap junction (i.e. Table 3). The thin continuous line in Fig. 7A

9 J Physiol Functional consequences of Cx40 H15Q + K16A mutations 259 depicts the solution to the parallel double Boltzmann function with p 1 equal to 0.13 or 0.17 for the homotypic wtcx40 gap junction at the respective negative or positive V j values. Other than the visual deviation of this model fit from the data at higher V j values, it was not possible to distinguish between parallel homomeric homotypic and heteromeric wtcx40 and Cx40 H15Q + K16A channel populations. This theoretical model suggests that the mutant V j gating phenotype was predominant (85% of G j ) under these conditions. Low g j recordings revealed the activity of 150 ps channels (Fig. 7B), consistent with previous observations of the unitary channel conductance (γ j )properties of wild type and now also the mutant H15Q + K16A Cx40 gap junctions (see Fig. 8). To determine if the Cx40 H15Q + K16A protein was also functionally dominant in terms of the sensitivity to spermine, it was coexpressed with wtcx40 in the presence of unilateral 2 mm spermine (Fig. 7C). The average normalized I j V j relationship demonstrated reduced spermine block relative to wtcx40 gap junctions (compare with Fig. 2B). Comparison of the fraction of unblocked I j in the presence of 2 mm spermine was identical to homotypic Cx40 H15Q + K16A gap junctions and distinctly less than that of wtcx40 gap junctions (Fig. 7D). Hence, the Cx40 H15Q + K16A protein was functionally dominant when coexpressed with wtcx40 in terms of its V j -dependent gating and block by intracellular spermine molecules. We also tested the ability of the Cx40 H15Q + K16A protein to alter the V j -gating and γ j properties of wtcx43 gap junctions. In contrast to the findings with wtcx40, the steady state G j V j curve for the Cx40 H15Q + K16A and wtcx43 coexpressed gap junctions retained a majority of the V j -dependent gating properties that could be ascribed to awtcx43 Boltzmann function (Fig. 7E, Table 4). The G j V j relationship for the wtcx43 + Cx40 H15Q + K16A coexpression experiments was also fitted with the parallel double connexin Boltzmann function: Low g j recordings confirmed the presence of 100 and 150 ps gap junction channels (Fig. 7F), indicative of the coexistence of parallel homomeric homotypic wtcx43 and mutant Cx40 H15Q + K16A gap junctions. Channel properties of wtcx40 and Cx40 H15Q + K16A gap junctions Previous results with the E9K and E13K mutations that altered the spermine blocking ability and V j gating properties of Cx40 gap junctions also altered G j = p 1 G wtcx43 j Cx40 H15Q+K16A + (1 p 1 ) Gj For wtcx43, the exact G Cx j solution was obtained from previous results on the same stable wtcx43 transfected N2a cells using the identical V j protocol (Lin et al. 2005). The average G min for both V j polarities was 0.23, the average V1 / 2 was ±69.2 mv, and the average gating charge valence was ±2.95 elementary charges (q) for wtcx43. The thin continuous line in Fig. 7E depicts the solution to the parallel double connexin Boltzmann function where p 1 was 0.72 or 0.69 for negative or positive V j,respectively. Hence, this function estimates that the relative ratio of homotypic homomeric wtcx43 to Cx40 H15Q + K16A gap junctions was 70% to 30% in favour of the wtcx43 phenotype. Again, the correlation coefficients for the two fitted curves were similar (r = 0.97 or 0.96, respectively). Figure 6. Sidedness of spermine block and mutant Cx40 TPcA block A, the unilateral application of 2 mm spermine to the wtcx40 (, n = 3) or mutant Cx40 H15Q + K16A (, n = 4) side of heterotypic wtcx40/cx40 H15Q + K16A gap junctions demonstrates that occlusion occurs on the positive V j side of the Cx40 gap junction channel. B, the normalized steady-state I j V j relationships in the presence of unilateral 5 mm tetrapentylammonium for wtcx40 (continuous line, modelled from Musa et al. 2001) and the Cx40 H15Q + K16A ( ) homotypic gap junctions illustrate the elimination of the block by quaternary ammonium ions.

10 260 X. Lin and others J Physiol A B C D E F Figure 7. Properties of wtcx40 and wtcx43 gap junctions cotransfected with Cx40 H15Q + K16A A, G j V j relationship for coexpressed wtcx40 + Cx40H15Q + K16A gap junctions. The mathematical fit of the standard Boltzmann G j V j curve (thick continuous line) is given in Table 4. The thin curved line depicts the predicted fit from parallel wtcx40 and Cx40 H15Q + K16A gap junctions with an average proportionality constant of 15% wtcx40 and 85% mutant Cx40. B, unitary gap junction channel activity obtained from one low g j wtcx40 + Cx40H15Q + K16A gap junction recording demonstrates the presence of a single population of 150 ps channels. C, the normalized steady-state I j V j curve for the cotransfected wtcx40 Cx40 H15QK16A gap junctions in the presence of 2 mm spermine. Each data point represents the average from eight experiments. D, the fraction of unblocked I j for the homotypic wtcx40 (filled square), Cx40 H15Q + K16A (open square), and the heteromeric wtcx40-cx40 H15QK16A (half-filled square) gap junctions in the presence of 2 mm spermine. E, the G j V j relationship for coexpressed wtcx43 + Cx40H15Q + K16A gap junctions demonstrates the presence of significant V j gating in this preparation. The mathematical fit of the standard G j V j Boltzmann curve (thick continuous line; Table 4) and parallel wtcx43 and Cx40 H15Q + K16A gap junctions (thin continuous line)

11 J Physiol Functional consequences of Cx40 H15Q + K16A mutations 261 Table 4. V j -dependent gating properties of Cx40 H15Q + K16A co-transfection experiments Wild-type Cx40 + Cx40 H15Q,K15A Wild-type Cx43 + Cx40 H15Q,K15A (n = 5) (n = 5) Parameter V j +V j V j +V j G max 1.01 ± ± ± ± G min 0.75 ± ± ± ± V1 / 2 (mv) 47.3 ± ± ± ± 0.4 z 2.92 ± ± ± ± 0.03 r their γ j properties (Musa et al. 2004). However, the coexpression experiments seem to indicate that the H15Q + K16A mutations had no effect on the γ j properties of Cx40 gap junction channels. The γ j of homotypic wtcx40 and Cx40 H15Q + K16A γ j are illustrated in Fig. 8. Representative examples of the unitary channel activity, the all-points amplitude histograms for a single-channel recording, and the composite single-channel current voltage (i j V j ) relationships are shown for both the wtcx40 and Cx40 H15Q + K16A homotypic gap junction channels. The slope conductances confirm that γ j is not reduced in the homotypic Cx40 H15Q + K16A channel and may actually be slightly (6%) elevated. The heterotypic Cx40 H15Q + K16A/wtCx40 channel did not exhibit any noticeable rectification (Fig. 9). Besides the γ j values of the homotypic wtcx40 and Cx40 H15Q + K16A gap junction channels, the reduction in γ j was similar for both channels when 140 mm KCl was replaced with140 mm potassium gluconate. Figure 10 illustrates the γ j values obtained in the presence of 140 mm potassium gluconate. The slope conductance of the wtcx40 gap junction channel was reduced to 146 ps, while the γ j of the Cx40 H15Q + K16A gap junction channel was similarly reduced to 149 ps. Despite the complete replacement of 140 mm Cl with gluconate, which is an organic anion with an aqueous mobility (25 C) of only 0.30 compared with 2.03 for Cl, the γ j of both channels was reduced by only 4 7%. This is indicative of a relatively cation-selective channel. A simple approximation of the relative cation/anion conductance of a gap junction channel can be obtained by scaling the anionic terms in the Goldman-Hodgkin-Katz (GHK) current equation by a relative permeability factor (R p ) to obtain the same KCl/potassium gluconate γ j ratio as the equimolar anion substitution experiments (Veenstra et al. 1995). c + I s = P s z 2 s F 2 [S] 1 [S] 2 e (z s FV j/rt) V j s 1 e (z s FV j/rt) a + R p P s z 2 s F 2 [S] 1 [S] 2 e (z s FV j/rt) V j s 1 e (z s FV j/rt) where P s is assumed to be the aqueous mobility of the solute s for all cations (c + ) and anions (a ). The R p values were 0.22 and 0.23 for the wtcx40 and Cx40 H15Q + K16A gap junction channels, respectively. These R p values are identical to the previously reported value of 0.22 for the wtcx40 channel and correspond to relative cation/anion conductance ratios of 4.5 and 4.3 for the wtcx40 and Cx40H15Q + K16A channels (Veenstra, 1996). To test whether the ionic permeability of the Cx40 channel was altered by the H15Q + K16A mutation, asymmetric 70/140 mm KCl experiments were performed on the homotypic wtcx40 and mutant Cx40 H15Q + K16A gap junction channels. The methods previously used to determine the relative cation-to-anion permeability of the Cx40 channel are demonstrated in Fig. 11 (Beblo & Veenstra, 1997; Veenstra, 2001b). Both pipette offsets were either nullified in the bath prior to G seal formation (Fig. 11A C) tocompensate for the aqueous Cl diffusion potential of the 50% KCl patch electrode or set equal to 0 mv (Fig. 11D F). The pipette offsets were 2.3 ± 1.3 mv and ± 0.7 mv for the 140 and 70 mm KCl patch electrodes relative to the bath Ag/AgCl 2 reference cell containing 140 mm KCl IPS (n = 7). This interelectrode offset of 16 mv is close to the expected 17 mv for the calculated Cl electrodiffusion potential. The i j V j curve in Fig. 11G indicates that the measured reversal potential (E rev )ofthe wtcx40 gap junction channel is not due to the nulling of the bath are illustrated. The double connexin fit predicts that an average of 70% wtcx43 and 30% Cx40 H15Q + K16A channels exist as homomeric homotypic gap junctions. F, single gap junction channel activity confirms the presence of both 100 ps (dashed line) and 150 ps (dotted lines) gap junction channels in the same low g j recordings.

12 262 X. Lin and others J Physiol pipette offsets prior to the establishment of the double whole-cell recording configuration. The corresponding K + /Cl relative permeability ratio (P K /P Cl ) for an E rev of 14.4 mv, adjusted to the measured K + activities, according to the GHK voltage equation was 6.2. The A previously reported value for P K /P Cl was approximately 8.0 (Beblo & Veenstra, 1997). The osmolarity was maintained at 310 mosmol l 1 by the addition of raffinose to the 50% [KCl] pipette solution. However, replacement of 70 mm KCl with an impermeant D B E C F n = 8 n = 3 Figure 8. Conductance properties of homotypic wild-type and mutant Cx40 channels Double whole-cell current recordings during ±V j pulses to the indicated value from a low g j pair of stable wtcx40 (A) or transiently Cx40 H15Q + K16A (D) transfected N2a cells demonstrating the presence of unitary channel current activity. The all-points histograms from the wtcx40 (B) and Cx40 H15Q + K16A (E) channel current recordings from the +V j pulse illustrate how channel current amplitudes were determined. The composite channel-current voltage (i j V j )relationship from eight wtcx40 (C) and three Cx40 H15Q + K16A (F) experiments were fit by linear regression to provide an estimate of the unitary channel conductance (γ j ) for these two homotypic gap junction channels. The H15Q + K16A mutation increased the Cx40 γ j by 6% at most.

13 J Physiol Functional consequences of Cx40 H15Q + K16A mutations 263 non-electrolyte also reduced the ionic strength of the pipette solution. To determine whether the equilibrium potential observed under asymmetric KCl conditions was due in part to the effect of altered ionic strength on the channel protein, the E rev was determined at three different asymmetric low KCl concentrations (Fig. 11H). The continuous line predicts the E rev for the wtcx40 channel with a constant P K /P Cl of 6.2 obtained from the 50/100% [KCl] experiments. The actual calculated P K /P Cl ratios were 5.9 and 10.5 for the 75/100% and 25/100% experiments. The P K /P Cl increase observed with decreased ionic strength is indicative of an increase in the electrostatic surface charge potential within the wtcx40 channel. The P K /P Cl of the homotypic mutant Cx40 H15Q + K16A gap junction channel was also determined under asymmetric KCl conditions. The channel currents and i j V j curve in Fig. 12 clearly indicate that the E rev and P K /P Cl values were indistinguishable from the wild type for this mutant Cx40 gap junction channel. Hence, despite the dramatic reductions in the ionic blocking ability of tetrapentylammonium and spermine molecules, the ionic conductance and permeability properties were not altered by the incorporation of the H15Q + K16A mutations. Discussion This study extends our previous findings about the molecular basis for spermine blockade of Cx40 gap junctions (Musa & Veenstra, 2003; Musa et al. 2004). The cytoplasmic polyamine receptor of the Cx40 protein is likely to require the two glutamic acid residues previously identified as being involved in this process (Musa et al. 2004). However, the apparently strict A C B D n = 4 Figure 9. Conductance properties of heterotypic mutant/wild-type Cx40 gap junction channel Whole cell 2 current (I 2 )recordings during ±30 V j pulses to the indicated value from a low g j heterotypic wtcx40/cx40 H15Q + K16A pair of N2a cells (A). V j was defined relative to the wtcx40 cell. The all-points histograms from the +30 mv (B) and 30 mv (C) channel-current recordings demonstrate a difference of <10% in i j. The heterotypic i j V j relationship (D) was linear, indicating a lack of rectification for this H15Q + K16A mutant/wt Cx40 gap junction channel.

14 264 X. Lin and others J Physiol requirement for the Cx40 E13 residue and the partial contribution from the E9 residue cannot explain the entire basis for the spermine-dependent inhibition of I j. These two acidic amino acid residues, when incorporated into the same primary locations of Cx43, did not confer spermine sensitivity to this homology-related protein (Sáez et al. 2003). In this study, we investigated the possible contribution of other alternative amino terminal sequence variations between rat Cx40 and Cx43. The HKH and QAY sequences of Cx40 and Cx43 at positions are predicted to lie one α helical turn away from the position 13 locus in the opposite direction from position 9 on the NT domains of these two connexins (Fig. 1). The tandem H15Q + K16A mutation in Cx40 diminishes the concentration- and V j -dependent inhibition of Cx40 I j by spermine (Fig. 2). The V j sensitivity of the spermine association rates and equilibrium dissociation constants were reduced by 50% and the spermine dissociation rates were essentially instantaneous upon V j polarity reversal (Figs 3 and 4, Table 2). The V j -dependent K d values did not decrease below 2 mm, and there was only partial ( 50%) I j inhibition at the minimum K d for the mutant Cx40 gap junction compared with 80% inhibition at the minimum K d of 200 µm for the wtcx40 gap junction. The Cx40 H15Q + K16A mutation also diminished the magnitude of the homotypic gap junction V j -dependent gating, although it is not readily apparent if any reduction in gating charge valence resulted from this homotypic charge substitution (Fig. 5, Table 3). The V j -dependent inhibition of Cx40 I j by 5 mm tetrapentylammonium (TPeA) ions was further reduced by the H15Q + Q16A mutation than with the E9K and E13K mutations (Fig. 6; Musa et al. 2001, 2004). The heterotypic Cx40 H15Q + K16A/wtCx40 gap junction clearly indicates that there is charge interaction between the A C B D Figure 10. Wild-type and mutant Cx40 gap junction channel conductances in Kgluconate Unitary channel current fluctuations (A) and slope conductance (B) for the wtcx40 gap junction channel in the presence of symmetrical 140 mm potassium gluconate. Similar unitary channel current (C) and slope conductance (D) values were obtained for the Cx40 H15Q + K16A gap junction channel under the same conditions. The 4 7% decrease in γ j for both channels with equimolar replacement of Cl with gluconate indicates that cations conduct the majority (80%) of ionic current through the Cx40 gap junction channel.

15 J Physiol Functional consequences of Cx40 H15Q + K16A mutations 265 mutant and wild-type homotypic hemichannels. The G max of this heterotypic gap junction was shifted 25 mv negative by the neutralization of the H15 and K16 amino acids of wtcx40, and the gating charge valence was reduced by approximately 50% at positive V j relative to the wtcx40 side of the gap junction. This heterotypic gap junction also retained the complex biphasic V j -dependent gating observed with the Cx40 H15Q + K16A gap junction, although any alteration in the gating charge valence was obscured by the limited 25% reduction in G j associated with the mutant hemichannel. A biphasic G j V j curve with respect to a single V j polarity was also observed in homotypic Cx45 Cx45 and heterotypic Cx45 Cx43EGFP gap junctions (Bukauskas et al. 2002). This phenomenon was attributed to a four-state contingent gating model consisting of two open-or-closed gates in series, one on each side of the channel. Our results with the Cx40 H15Q + K16A mutation are similar, but the magnitude of the increase in G j at high V j was greater than that observed for Cx45 despite a fourfold reduction in the overall magnitude of V j gating. This biphasic gating is consistent with a relaxation of V j -dependent closure as well as a contingent gating mechanism. Taken together, these results suggest that the spermine block, TPeA block, and intrinsic V j -dependent gating properties of the Cx40 gap junction share common structural determinants. This concept is further supported by the observation that the gating charge valence for both processes is approximately 3.2 equivalent charges (q) for the wtcx40 gap junction. Given that exogenously applied spermine serves as an inhibitory V j -dependent ligand, these observations are consistent with the NT domain of Cx40 serving as a receptor for an intrinsic or extrinsic gating particle that inactivates the channel. Because a homotypic gap junction is bilaterally symmetric, it cannot be definitively determined if the site of channel occlusion by spermine resides on the positive or negative V j side of the channel. It has been postulated that Cx40 gap junctions close with positive V j polarity (Valiunas et al. 2000). Since the mutant Cx40 H15Q + K16A gap junction exhibits only about 25% of the normal Cx40 V j gating and is an order of magnitude less sensitive to spermine, the polarity of Cx40 channel closure could be tested using the heterotypic mutant Cx40 H15Q + K16A/wtCx40 hemichannel combination. When added unilaterally, spermine will enter the gap junction channel when V j is positive on the same (cis) side. The electrical distance (δ) measurement of for the wtcx40 gap junction indicates that a single spermine molecule senses % of the V j field during channel occlusion, not which side of the gap junction is occluded (Fig. 3, Table 2). The inhibition of I j by unilateral 2 mm spermine resembled the block produced in wtcx40 or mutant Cx40 H15Q + K16A gap junctions depending on which cell contained spermine at positive V j polarity (Fig. 6). We therefore conclude that Cx40 gap junctions close with positive V j polarity whether it occurs by the endogenous fast V j gate or exogenously applied spermine. The V j -dependent gating properties of the coexpressed Cx40 H15Q + K16A subunit with the wtcx40 or wtcx43 connexins suggest that this mutant Cx40 subunit readily heteromerizes with wtcx40, but not wtcx43 gap junctions (Fig. 7, Table 4). The V j -dependent gating properties of the Cx40 H15Q + K16A wtcx40 gap junctions phenotypically resembled the mutant homotypic gap junction except for the loss of the complex biphasic increase in G j at higher V j values. The Cx40 H15Q + K16A wtcx43 gap junctions more closely resembled the V j -dependent gating behaviour of wtcx43 gap junctions. This parallel homomeric expression of Cx40 H15Q + K16A and wtcx43 gap junction channels was confirmed by the concomitant observation of distinct 100 and 150 ps channels (Fig. 7F). The Cx40 H15Q + K16A mutation is distinct from the previous E9K and E13K mutations in that the Cx40 gap junction channel properties are effectively unaltered. The unitary channel conductance (γ j ) of the Cx40 H15Q + K16A gap junction channel was at most 10 ps (6%) higher than the wtcx40 channel (Fig. 8). The heterotypic Cx40H15Q + K16A/wtCx40 gap junction channel possesses the same γ j profile on the positive side of the junction as the respective wtcx40 and mutant Cx40 H15Q + K16A gap junction channels (Fig. 9). Equimolar substitution of potassium gluconate for KCl also produces similar percentage reductions in γ j, indicative of similar cation-to-anion conductance ratios of 4.5:1 or 4.3:1 (Fig. 10). The selective permeability of the wtcx40 gap junction channel was previously postulated to be 8:1 in favour of cations (Beblo & Veenstra, 1997). This hypothesis was reexamined using the same experimental procedures this time with asymmetric KCl gradients across the homotypic wtcx40 gap junction (Veenstra, 2001b). The E rev measured 14.4 mv from the 70/140 mm KCl single channel i j V j relationship, which corresponds to a P K /P Cl of 6.2 when adjusted to the measured K + activities of 65 and 146 mm (Fig. 11). This P K /P Cl ratio is slightly lower than the estimate of 8.0 previously reported, but the ionic composition of the IPS was different and the P K /P Cl ratio was directly measured in these most recent experiments. The mutant Cx40 H15Q + K16A gap junction channel had an identical E rev measurement of 14.4 mv, and hence, the same selective ionic permeability as the wtcx40 channel (Fig. 12). The dependency of the P K /P Cl ratio on the ionic strength of the low KCl IPS was essentially identical for both homotypic gap junction channels. The increase in the P K /P Cl ratio from6to10whentrans KCl was reduced to 25% of control is indicative of electronegative surface charge effects on the low ionic strength side of the gap junction channel.

16 266 X. Lin and others J Physiol 570.2

17 J Physiol Functional consequences of Cx40 H15Q + K16A mutations 267 A C B D Figure 12. Ionic selectivity of H15Q + K16A Cx40 gap junction channel Asymmetric 70/140 mm KCl Cx40 H15Q + K16A gap junction channel recordings obtained after nulling the bath pipette offsets prior to dual whole-cell recording of I j at V j =±10 (A), ±30 (B), and ±40 mv (C). The summary i j V j relationship from four experiments yielded the same E rev of 14.4 mv, indicating that the Cx40 H15Q + K16A gap junction channel has the same ionic permeability as the wtcx40 channel. D, the P K /P Cl ratio increased from 6.2 at 75% and 50% trans [KCl] to 10.0 at 25% [KCl], nearly identical to the wtcx40 gap junction channel. A P K /P Cl ratio of 10:1 was reported for Cx46 hemichannels, while the endogenous Xenopus Cx38 hemichannels have a P K /P Cl ratio of about 4, indicative of modest cation selectivities for other connexin channels (Trexler et al. 1996; Zhang et al. 1998). We are presently re-examining the P K /P Cl ratio of the wtcx43 gap junction channel in the same context as the present Cx40 experiments. These observations with the wtcx40 and mutant Cx40 H15Q + K16A gap junction channels confirm our previous findings about this connexin and suggest that the ion permeation pathway is not significantly affected by the H15Q + K16A mutation, despite significant disruption of the V j -dependent gating and ionic blocking properties. The S26L mutation slightly altered the Cx32 V j -dependent gating without altering the ionic conductance (in CsCl) or selectivity of this Figure 11. Ionic selectivity measurements of wild-type Cx40 gap junction channel Asymmetric 70/140 mm KCl wtcx40 gap junction channel recordings obtained after nulling the bath pipette offsets prior to dual whole-cell recording of I j at V j =±10 (A), ±30 (B), and ±40 mv (C). The same asymmetric 70/140 mm KCl wtcx40 gap junction channel recordings were obtained without nulling the bath pipette offsets (bath V pipette = 0mVfor both cells) are shown in D F. The composite i j V j relationships from seven experiments with V pipette, either nulled in the bath or not, yield a channel current reversal potential (E rev )of 14.4 or 29.2 mv, respectively (G). This calculates to a K + :Cl permeability ratio (P K /P Cl )of The E rev of the wtcx40 gap junction channel was determined under three different asymmetric [KCl] conditions of 35 (25%), 70 (50%) and 105 mm (75%) relative to 140 mm (100%) KCl (H). The curved line drawn between the mean E rev values for the decreasing trans KCl concentrations is consistent with an P K /P Cl ratio according to Goldman-Hodgkin-Katz equilibrium permeability theory.