Abstract
Intracellular protons and calcium ions are two major chemical factors that regulate connexin43 (Cx43) gap junction channels and the synergism or antagonism between pH and Ca2+ has been questioned for decades. In this study, we assessed whether the calcium gating mechanism occurs independently of the pH gating mechanism by utilizing the Cx43-M257 (Cx43K258stop) mutant, a carboxyl-terminal (CT) truncated version of Cx43 lacking the pH gating domain. Dual whole cell patch clamp experiments were performed on Neuroblastoma-2a (N2a) cells or neonatal mouse ventricular myocytes (NMVMs) expressing either full length Cx43 or Cx43-M257 proteins. Addition of 1 μM ionomycin to normal calcium saline reduced Cx43 or Cx43-M257 macroscopic gap junction conductance (gj) to zero within 15 min of perfusion, while this response was prevented by omitting 1.8 mM CaCl2 from the external solution or adding 100 nM calmodulin (CaM) inhibitory peptide to the internal pipette solution. The ability of connexin calmodulin binding domain (Cx CaMBD) mimetic peptides and the Gap19 peptide to inhibit the Ca2+/CaM gating response of Cx43 gap junctions was also examined. Internal addition of a Cx50 cytoplasmic loop CaMBD peptide (200 nM) prevented the Ca2+/ionomycin-induced decrease in Cx43 gj, while 100 μM Gap19 peptide had no effect. Lastly, the transjunctional voltage (Vj) gating properties of NMVM Cx43-M257 gap junctions were investigated. We confirmed that the fast kinetic inactivation component was absent in Cx43-M257 gap junctions, but also observed that the previously reported facilitated recovery of gj from inactivating potentials was abolished by CT truncation of Cx43. We conclude that CT pH gating domain of Cx43 contributes to the Vj-dependent fast inactivation and facilitated recovery of Cx43 gap junctions, but the Ca2+/CaM-dependent gating mechanism remains intact. Sequence-specific Cx CaMBD mimetic peptides act by binding Ca2+/CaM non-specifically and the Cx43 mimetic Gap19 peptide has no effect on this chemical gating mechanism.
Introduction
Formed by a 20 member protein family, gap junctions are composed of a hexamer of connexin (Cx) subunits in each cell membrane that dock extracellularly to form an intercellular aqueous pore that facilitates the passage of ions, second messengers, fluorescent dyes, small nucleic acids, etc., virtually any solute under 1 kDa in molecular mass and 14 Å in width (1,2). Gap junctions are thought to be regulated by two general gating mechanisms, a fast transjunctional voltage (Vj) gating mechanism and a slow chemical gating mechanism (3). Chemical agents known to close gap junctions include intracellular calcium ions, intracellular pH, lipophiles, protein phosphorylation, and so on (4). Since the mid-1970s, intracellular calcium ions and pH have been known to uncouple gap junctions, though opinions varied about the relative importance of each to the uncoupling process first referred to as the “healing-over” in the heart (5-8). Though synergistic actions of cytosolic Ca2+ and H+ have been reported, opposing viewpoints on the operative role of Ca2+ ions or protons have persisted for decades without resolution (9-12).
Definitive evidence of a pH gating mechanism associated with connexin43 (Cx43), the connexin with the most widespread expression pattern, was provided when truncation of last 125 amino acids of the cytoplasmic carboxyl terminus (CT) abolished the pH sensitivity of Cx43 gap junction conductance (gj) (13). This pH gating mechanism for Cx43 gap junctions was further defined by the demonstration that the distal CT pH gating particle binds to a receptor domain located in region 119-144 of the Cx43 cytoplasmic loop (CL) in a pH-dependent manner (14). The calcium gating hypothesis evolved to the action of calmodulin (CaM) based on the inhibition of gap junction uncoupling by calmodulin (CaM) inhibitors and evidence that CaM binds to Cx32 and lens gap junctions (15-17). Since the identification of CaM binding domains (CaMBDs) on the cytoplasmic amino- and carboxyl termini of Cx32, additional connexin CaMBDs have been identified on the CL domain of Cx43, Cx50, Cx46 (sheep Cx44), and Cx45 (18-21). Previous studies in this laboratory indicated that Ca2+/CaM causes a gated closure of Cx43 and Cx50 gap junctions as evidenced by the reduced open probability of gap junction channels during perfusion of coupled cell pairs with the calcium ionophore ionomycin (20,22).
In this study, we tested the ability of the Ca2+/CaM to cause the gated closure of Cx43 gap junctions using the previously published CT-truncated version of Cx43, Cx43-M257 expressed in mouse neuro2a (N2a) cells. We also employed the Cx43+/K258stop mouse, which heterologously expresses the Cx43-M257 (K258stop) protein (23), to examine this chemical gating mechanism using an endogenous expression system, homozygous Cx43-M257 neonatal mouse ventricular cardiomyocytes (NMVMs). We found that superfusion of Cx43-M257 expressing cell pairs with 1.8 mM CaCl2 saline containing 1 μM ionomycin inhibited gap junction conductance (gj) by 100% in a calcium- and calmodulin-dependent manner. We further tested the ability of connexin CaMBD mimetic peptides to inhibit the Cx-Ca2+/CaM gating mechanism in a sequence-specific manner and found that 200 nM of the higher CaM affinity Cx50-3 peptide was sufficient to block the Ca2+/CaM-induced uncoupling of Cx43 gap despite the Cx43-3 CaMBD site possessing a distinct sequence from the Cx50 CaMBD. Conversely, the Gap19 peptide, which targets a CL sequence immediately adjacent to the known Cx43 CaMBD site (24), did not inhibit the Ca2+/CaM-induced uncoupling process. Finally, we also examined the Vj-gating properties of Cx43-M257 gap junctions in paired NMVMs and found that the fast kinetic component of the Vj-dependent gating mechanism was abolished as previously reported in Xenopus oocytes and N2a cells (25,26). The probability of the 50 pS subconductance state of Cx43 gap junction channels was similarly reduced in favor of the 100 pS main open state of the channel.
Additionally, the increased slope gj during the recovery from Vj-dependent inactivation previously observed only in primary NMVM gap junctions was also eliminated by the CT truncation of Cx43. Our findings indicate that the Ca2+/CaM-dependent gating mechanism of Cx43 gap junctions remains intact after deletion of the CT pH gating domain, that sequence-specific Cx CaMBD peptides function as non-specific CaMBDs to bind CaM, that the Gap19 peptide does not influence this gating mechanism, and the Cx43 distal CT domain is somehow involved in the “facilitated” recovery of gj after Vj-dependent inactivation.
Materials and Methods
Cell Cultures
Murine Neuro2a neuroblastoma (N2a) cells, grown to 70-90% confluency, were cultured in 12 well culture dishes containing MEM media supplemented with 10% fetal bovine serum and transiently transfected with 1 μg of plasmid cDNA containing full-length (WT) Cx43 or Cx43-M257 cDNA sequences using Lipofectamine2000 and OptiMEM according to manufacturer’s directions (ThermoFisher Scientific). Full length Cx43 pTracer-CMV2 and Cx43-M257 pIRES2-EGFP plasmids were purified using the EndoFree plasmid minikit (Qiagen). N2a cells were lightly trypsinized after 4 hrs and plated in 35 mm culture dishes overnight for patch clamp electrophysiology studies the next day.
All cardiomyocyte experiments were performed on enzymatically dissociated neonatal C57BL/6 murine ventricular myocytes cultured for 48–72 h according to published procedures (27). The newborn mice were euthanized under isoflurane anesthesia in accordance with procedures approved by the Institutional Animal Care and Use Committee (IACUC) conforming to the Guide for the Care and Use of Laboratory Animals published by the US National Institutes of Health (NIH Publication No. 85-23, revised 1996). Newborn litters from heterozygous Cx43-M257 (Cx43K258stop) mouse matings were collected and the ventricle and tail of each newborn pup was numbered for separate enzymatic dissociation and genotyping. Genomic DNA was obtained using the DNeasy Blood and Tissue kit (Qiagen) and subjected to polymerase chain reaction (PCR) analysis using Taq polymerase (ThermoFisher Scientific) and M257 forward (5’-CAA AAC ACC CCC CAA GGA ACC TAG) and reverse (5’ GCA TCC TCT TCA AGT CTG TCT TCG) primers as originally described (23).
Electrophysiology
Gap junctional current (Ij) recordings were acquired using dual whole cell patch clamp procedures (28). N2a or neonatal murine ventricular myocyte (NMVM) cell pairs were held at - 40 mV and a voltage step (ΔV) was applied to one cell while maintaining the holding potential (Vh = -40 mV) of the partner cell. The macroscopic junctional conductance (gj) was calculated by the equation: gj = -ΔI2/[(V1 + ΔV) - V2] where I1 and I2 are whole cell currents, V1 and V2 are command voltages, and (V1 + ΔV) - V2 = the transjunctional potential, Vj. For all experiments, gj was normalized by dividing the time-dependent gj by the initial gj measurement, or Gj = gj/gj,initial. To more accurately calculate gj, the whole cell patch electrode resistances, Rel1 and Rel2, were subtracted from the gj = -ΔI2/Vj calculation where the actual gj = apparent gj – (1/Rel1 + 1/Rel2). Rel = tcap/Cinput where τcap is the single cell capacitive current decay time constant and Cinput is the input resistance of each cell. The bath saline contained (in mM): 142 NaCl, 1.3 KCl, 4 CsCl, 2 tetraethylammonium chloride, 0.8 MgSO4, 0.9 NaH2PO4, 1.8 CaCl2, 5.5 dextrose, and 10 HEPES (titrated to pH 7.4 with 1 N NaOH). 1.8 mM CaCl2 was omitted from the Ca2+-free saline. Ionomycin (1 μM, I3909, Sigma-Aldrich) was added to the perfusion saline fresh daily. The internal pipette solution (IPS) contained (in mM): 140 KCl, 1.0 MgCl2, 0.3 KBAPTA, 25 HEPES (titrated to pH 7.40 with 1 N KOH).
The peptide inhibition studies of Ca2+/CaM-dependent uncoupling of Cx43 gap junctions was performed by adding the inhibitory peptides to the IPS of both cells on the day of use at concentrations dependent upon their measured Kd. Calmodulin (CaM) inhibition was achieved by adding 100 nM CaM inhibitory peptide corresponding to the CaMBD of CaM kinase II (Enzo Life Sciences, #BML-P200-0500) with a reported Kd of 52 nM. The Cx50 CaMBD peptide, corresponding to the Cx50 cytoplasmic loop sequence
141-SSKGTKKFRLEGTLLRTYVCHIIFKT-166 and its scrambled control (FKLYKCISFGGTEITTRSHVLTKKRL) were published previously (20). The CaMBD peptide was added to the IPS at a concentration of 200 nM. The Gap19 peptide (Sigma-Aldrich, #SML1426) was applied intracellularly at a concentration of 100 μM (24).
Immunocytochemistry
For immunocytochemical studies, NMVMs were cultured on poly-l-lysine coated coverslips for 48-72 hrs and immunolabeled for Cx43 protein using previously published procedures (27). Coverslips were rinsed in phosphate-buffered saline (PBS, pH 7.0), fixed with 4% paraformaldehyde in PBS, rinsed, permeabilized with 1% Triton X100 in PBS, and blocked with 2% goat serum in 1% Triton X100 PBS, all for 15 min at room temperature. Anti-Cx43 rabbit polyclonal amino-terminal (Abgent, #AP1541b) and mouse monoclonal carboxyl-terminal (ThermoFisher #35-5000) antibodies were diluted 1:200 in 2% goat serum, 1% triton X-100 PBS and added to the 12 mm diameter coverslip overnight at 4 °C. Each well was rinsed 3-5 times with PBS the next day and incubated with goat anti-rabbit Alexa Fluor 488 (ThermoFisher #11008) and anti-mouse Alexa Fluor 546 (ThermoFisher #11003) secondary antibodies, diluted 1:1500 in 10% goat serum PBS, for 30 min at room temperature. After rinsing, the cells were labeled with DAPI for 10 min, rinsed with PBS and finally with pure water, blotted dry, and mounted on a glass slide using ProLong antifade mounting reagent and cured overnight in the dark.
Sealed glass coverslips were viewed with an Olympus IX-70 microscope using a Sutter Instruments LS 175 W Xenon arc lamp epifluorescence illumination system and Lambda 10–2 filter wheel controller with 484/15 or 555/25 nm band pass excitation filters and FITC or FITC/Cy3/Cy5 dichroic mirror/emitter filter sets (Chroma Technology Corp, #41001 or #62005). DNA staining was observed with a 365/10 nm excitation filter and DAPI 460/50 nm dichroic mirror/emitter filter set (Chroma, 31000v2-dapi-hoechst-amca). Fluorescent micrographs were acquired with an Andor iXon 885 ECCD camera using Imaging Workbench 6.0 software (INDEC Systems, Santa Clara, CA). Exported TIF files were background subtracted (≈10%) and green/red/blue color processed using ImageJ software. Magnification was 600X using an Olympus PlanApo 1.40/0.17 aperture 60X oil immersion and 10X C-mount objectives.
Results
To examine the Ca2+/CaM gating of Cx43-M257 gap junctions, we employed the same ionomycin perfusion assay from our previous study on the full length wild-type (WT) Cx43 (22). When coupled WT or Cx43-M257 N2a cell pairs were superfused with normal saline containing 1 μM ionomycin and 1.8 mM CaCl2, junctional currents (Ij) in response to a -20 mV Vj pulse declined steadily from initial levels to zero during 8-12 min of perfusion at a rate of 1 ml/min (Fig. 1A, B). The average junctional conductance (gj) was 26.4 ± 6.2 nS for the WT Cx43 and 29.1 ± 7.7 nS for the Cx43-M257 cell pairs (mean ± SEM, n = 7, 6). The average normalized Gj (= gj(time)/gj,initial) declined to 0 nS within 8 min from the onset of perfusion for both WT Cx43 and Cx43-M257 gap junctions (Fig. 1D,E). Omission of the 1.8 mM CaCl2 from the perfusate or the addition of 100 nM CaM (CaMKII 290-309) inhibitory peptide to the whole cell patch pipette solutions prevented the rundown of Cx43-M257 Ij (Figs. 1C, E, F). The average gj was 18.6 ± 6.9 nS for the zero Ca2+ and 30.3 ± 6.9 nS for the CaM inhibitory peptide Cx43-M257 experiments (mean ± s.e.m., n = 6, 6). These results are consistent with previous findings using the full length Cx43 and are indicative of a calcium/calmodulin-dependent gating mechanism even with the absence of the Cx43 pH gating domain (13,22).
Calcium/calmodulin-dependence of Cx43 gap junctions expressed in N2a cells. A-C, Whole cell current traces from the partner cell 2 of an N2a cell pair during a -20 mV, 5 sec transjunctional (Vj) voltage pulse (inset, upper left) applied to cell 1 before (control) and during perfusion with 1 μM ionomycin, 1.8 mM CaCl2 saline. Current traces from representative experiments illustrate the decline in junctional current (Ij = -ΔI2) during ionomycin perfusion of an N2a cell pair expressing either full-length wild-type Cx43 (A) or carboxyl-tail truncated Cx43 (Cx43-M257, B) gap junctions, or the prevention of the calcium-induced decline in Ij by calmodulin (CaM) inhibition (C). D, The average (mean ± SEM, n = 7) decline in junctional conductance (Gj) during CaCl2/ionomycin perfusion of N2a-Cx43 cell pairs illustrating 100% Gj inhibition within 15 min of perfusion. E, Average Gj of N2a-Cx43-M257 cell pairs (n = 6) during perfusion with 1.8 mM CaCl2 saline + ionomycin (∎) illustrating complete uncoupling in the absence of the Cx43 pH gating domain, and the prevention of Gj inhibition by excluding CaCl2 from the 1 μM ionomycin saline perfusate (〇, n = 6). F, Inclusion of 100 nM CaM inhibitory peptide in the patch pipettes of both cells also prevents inhibition of N2a-Cx43-M257 Gj by calcium/ionomycin perfusion.
The above Cx43-M257 experiments were performed using the exogenous N2a cell expression system and a Cx43K258stop mouse was later developed that expresses the CT-truncated (K258stop = M257) form of Cx43 in a heterozygous manner (23). Thus, mouse ventricular cardiomyocytes were cultured from the hearts of newborn littermates from heterozygous Cx43+/K258stop mice matings. The genotype of each newborn pup was determined by PCR analysis of tail DNA samples and confirmed by immunocytochemical labeling of cultured cardiomyocytes using amino-terminal and carboxyl-terminal anti-Cx43 antibodies (Fig. 2A,B). The Cx43-NT antibody recognizing both forms of Cx43 was secondarily labeled with Alexa Fluor488 (green) and the Cx43-CT antibody, which would recognize only the full-length WT Cx43, was labeled with Alexa Fluor546 (red). Homozygous Cx43-M257 cardiomyocytes were devoid of Cx43-CT immunolabeling (Fig. 2A) whereas the WT Cx43 cardiomyocytes were immunolabeled by both anti-Cx43 antibodies (Fig. 2B). Perfusion of Cx43-M257 and WT Cx43 paired cardiomyocytes with 1.8 mM CaCl2 + 1 μM ionomycin saline resulted in complete uncoupling within 8 min of perfusion, similar to the results obtained in N2a cells. The average gj was 54.2 ± 6.2 and 39.3 ± 4.7 nS for the Cx43-M257 and WT-Cx43 cardiomyocyte pairs (n = 7, 7).
Calcium/CaM-dependent uncoupling of Cx43-M257 ventricular cardiomyocytes. A, Immunofluoresent labeling of Cx43-M257 cardiomyocyte gap junctions with anti-Cx43 aminoterminal (NT) and Alexa Fluor488 antibodies (green) and anti-Cx43 carboxyl-terminal (CT) and Alexa Fluor555 antibodies (red) illustrating the lack of the Cx43 CT domain in cardiomyocytes cultured from homozygous Cx43-M257 neonatal mice. B, Immunocytochemical labeling of wild-type Cx43 gap junctions illustrating the presence of the NT and CT domains in the littermate control cardiomyocytes. C, The average Gj of both wild-type and Cx43-M257 cardiomyocyte gap junctions was completely inhibited by calcium + ionomycin saline perfusion, confirming the results obtained by exogenous expression of Cx43 constructs in N2a cells.
We had previously shown that connexin mimetic peptides corresponding to the CaMBD of Cx43 and Cx50 were capable of preventing the uncoupling of their respective gap junctions during Ca2+-ionomycin perfusion (20,22). To test the specificity of these Cx CaMBD mimetic peptides, we tested different concentrations of the Cx50-3 peptide in WT Cx43 N2a cell pairs (Fig. 3A). We found that 200 nM Cx50-3 peptide was sufficient to completely prevent the decline in Gj during ionomycin perfusion of Cx43 gap junctions. The scrambled control Cx50-3 peptide, however, failed to prevent uncoupling during 10-12 min of Ca2+-ionomycin perfusion. These results suggest that any Cx mimetic peptide that is capable of binding CaM will interfere with the Ca2+/CaM-dependent gating mechanism in a connexin non-specific manner. The average gj was 14.9 ± 5.7 and 34.4 ± 8.1 nS for the Cx50-3 and scrambled control peptide experiments (n = 5, 3).
Effects of Connexin mimetic peptides on calcium/CaM-dependent uncoupling of Cx43 gap junctions. A, Connexin mimetic peptide of a confirmed Cx50 cytoplasmic loop (CL) calmodulin binding domain (CaMBDs) previously shown to inhibit Ca2+/CaM-dependent uncoupling was tested on Cx43 wild type gap junctions (20). Complete inhibition of Cx43 gap junction uncoupling was achieved with 200 nM Cx50-3 mimetic peptide added to both patch pipettes, but not the scrambled control Cx50 CaMBD peptide. B, A distinct Cx43 CL mimetic peptide reported to inhibit Cx43 hemichannel activity, but not Cx43 gap junction coupling, was examined for possible effects on Cx43 Ca2+/CaM-induced uncoupling (24). 100 μM Gap19 peptide added to both whole cell patch pipettes neither inhibited Cx43 Gj nor prevented the decline in Gj induced by 1.8 mM CaCl2/ionomycin saline perfusion.
The Cx43-3 CaMBD (136-158) is adjacent to the CL “L2” (119-144) region identified as the receptor for the CT pH gating domain of Cx43 (14). Recently, a Gap19 peptide targeting the central portion of the L2 region, 128-KQIEIKKFK-136, was developed and shown to inhibit Cx43 hemichannel function without inhibiting gj (24). Given the close proximity of the Gap19 region to the known Cx43 CaMBD, we wanted to test for possible effects of the Gap19 peptide on the Ca2+/CaM-dependent uncoupling of Cx43 gap junctions. In seven Ca2+-ionomycin perfusion experiments, inclusion of 100 μM Gap19 peptide in both patch pipettes did not prevent the uncoupling of WT Cx43 N2a cells pairs (Fig. 3B). The average gj was 31.8 ± 5.9 nS (n = 7). These results suggest that Gap19 does not affect the Ca2+/CaM-dependent gating mechanism of Cx43 gap junctions.
Truncation of the Cx43 CT was also reported to alter the Vj-gating of Cx43 gap junctions by eliminating the “fast” Vj-gating mechanism to the 50 pS subconductance state of Cx43 gap junction channels (25,26). We had previously observed a hysteresis in the steady state Gj–Vj curve only in NMVMs termed “facilitation” wherein a slow (200ms/mV) Vj ramp from ±120 mV back to 0 mV Vj resulted in a higher linear slope conductance at low Vj values than measured initially with increasing ±Vj values from 0 mV (27). The previous Vj-gating studies of Cx43-M257 gap junctions were performed in Xenopus oocytes and N2a cells. Thus, we examined the Vj-gating properties of myocardial Cx43-M257 gap junctions using cultured NMVMs. Application of the slow ±120 mV Vj ramp revealed the loss of facilitation in Cx43+/K258stop ventricular cardiomyocyte gap junctions during the return recovery phase of the Gj–Vj curve (Fig. 4A, B). The average gj of these Vj-gating experiments was 7.2 ± 1.4 nS since Vj-dependent inactivation can be detected only in low gj cell pairs.
Vj-gating and channel conductance properties of Cx43-M257 ventricular gap junctions. A, Average junctional current – transjunctional voltage relationship (Ij – Vj) from six Cx43-M257 ventricular cardiomyocyte cell pairs obtained during a continuous 200 msec per mV Vj ramp from 0 to ± 120 mV (inactivation) and back to 0 mV (recovery). Absent from these traces is the increased linear slope conductance during the recovery phase observed in wild type Cx43 ventricular cardiomyocyte gap junctions (27). B, Corresponding average Gj – Vj inactivation (—) and recovery (—) curves depicting the typical bell-shaped curve with Boltzmann equation fits of the data (see Table 1). Again, the lack of the “facilitation” of Gj is apparent, wherein facilitation is defined as a peak Gj during the recovery phase > 1 since Gj is normalized to the actual junctional conductance (gj) value for each cell pair at Vj = 0 mV prior to the application 200 msec/mV Vj inactivation and recovery ramps. C, The kinetics of Vj-dependent inactivation of wild type (WT) and Cx43-M257 ventricular cardiomyocytes were determined from 5-7 cell pairs in response to Vj pulses from -60 to -140 mV in 10 mV increments according to the methods of Lin et al. (27). Exponential fits of the slow inactivation rates (Kon,slow) from WT mice revealed an e-fold change in rate for every 17.2 ± 0.9 mV compared to 22.8 ± 1.7 mV for Cx43-M257 myocytes. The fast inactivation component was absent in Cx43-M257 myocytes, consistent with previous findings (25,26). D, Gj inactivation during a 1 Hz ventricular action potential waveform from WT (—) and Cx43-M257 (—) cardiomyocytes. Owing to the slower inactivation kinetics and reduced Vj-sensitivity of the Cx43-M257 gap junctions, Vj-dependent inactivation of Gj during the action potential was reduced to 10% compared to 50% for WT Cx43 cardiomyocyte gap junctions. E, Gap junction channel activity observed in a Cx43-M257 myocyte cell pair during one 7.5 sec, -60 mV Vj pulse. Larger unitary current events corresponded to single gap junction channel conductances (γj) of ≈100 pS were readily observed, but brief smaller unitary currents (*) with γj ≈ 60 pS were also observed. F, All points histogram of the entire I2 current trace shown in panel E illustrates the dominance of the 100 pS channel current events with smaller, less frequent events being lost in the noise of the dual whole cell current recording.
We also examined the kinetics of the Vj-dependent activation in NMVM cell pairs using -Vj pulses and the ventricular action potential as previously described. The inactivation kinetics during Vj pulses from -60 to -140 mV confirmed the loss of the fast inactivation time constant and the presence of a distinct slow inactivation time constant relative to WT Cx43 NMVM gap junctions (Fig. 4C). The elimination of the Cx43 fast inactivation time constant also eliminated nearly all of the Vj-dependent inactivation during the 1 Hz simulated ventricular action potential (Fig. 4D). The inactivation rates (Kon) were calculated using the expression Kon (ms-1) = 1/τinactrvation = A0•exp(Vj/Vk) + C where A0 is the rate constant amplitude in ms-1, C is the minimum rate (ms-1), and Vk is the voltage constant for the inactivation rate (27,29). Exponential fits of the WT slow inactivation rates and the M257 inactivation rates yielded rates of .0000244•exp(Vj/17.2) + 0.00236 for WT Cx43 NMVM gap junctions and 0.0000504•exp(Vj/22.8) - 0.000058 for Cx43-M257 NMVM gap junctions. These data reveal that deletion of the CT terminus of Cx43 not only eliminated the fast inactivation of Cx43 gap junctions, but also reduced the inactivation rate and Vj-sensitivity of the remaining slow inactivation component of Cx43 gap junctions. We also measured the unitary gap junction channel currents from three of these M257 NMVM experiments and observed an abundance of 100 pS channel events with only brief occurrences of apparent 60 pS channel events with open probabilities too low to be discernable in all points histograms of the gap junction channel current recordings (Fig. 4 E,F). Together, these data from Cx43-M257 NMVMs confirm the elimination of the fast inactivation component of Cx43 gap junctions and abundance of 100 pS gap junction channel conductance (γj) events, but also quantifies the reduction in the kinetics and Vj-sensitivity of the remaining slow inactivation component and demonstrates the removal of the of the facilitated recovery of gj from inactivating potentials of Cx43 gap junctions.
Discussion
The primary purpose of this study was to determine if the recently described Ca2+/CaM-dependent gating mechanism exists in the absence of the Cx43 CT pH gating domain which abolished the pH-sensitivity of Cx43 gj (13,18,22). Perfusion of Cx43-M257 N2a or NMVM cell pairs with 1 μM ionomycin saline containing normal 1.8 mM CaCl2 routinely induced 100% inhibition of Cx43 gj within 8-12 min from the onset of 1 ml/min perfusion (Figs. 1A,B,D,E and 2B). Consistent with previous findings using full length Cx43 expressed in N2a cells or wild-type NMVMs (22), omission of the 1.8 mM CaCl2 from the 1 μM ionomycin bath saline or addition of 100 nM CaMKII 290-309 CaM inhibitory peptide to both whole cell patch pipettes prevented the rundown of Cx43-M257 gj during 13 min of perfusion (Figs. 1C,E,F). Taken together, these data support the conclusion that the Ca2+- and CaM-dependent chemical gating mechanism of Cx43 gap junctions does not require the presence of the Cx43 distal CT pH gating particle.
The existence of Cx CaMBDs has been demonstrated by in vitro CaM binding assays with sequence-specific Cx mimetic peptides comprising the entire15-26 amino acid CaMBDs of Cx32, Cx43, sheep Cx44 (Cx46), Cx45, and Cx50 (17-21). In past studies, these Cx CaMBD mimetic peptides were used as inhibitory peptides to validate the functional role of the Cx-specific sequence in the Ca2+/CaM gating of the parent connexin (20,22). However, like the CaMKII 290-309 inhibitory peptide which corresponds to the high affinity CaMBD of CaMKII, these peptides likely function purely on the basis of binding CaM and acting as a “CaM sponge” when added in excess to an intracellular pipette solution. To test this hypothesis, we applied increasing concentrations of the Cx50 CaMBD peptide to WT Cx43 gap junctions and found that 200 nM of the Cx50-3 peptide was sufficient to prevent the Ca2+/CaM-dependent inhibition of Cx43 gj (Fig. 3A). This observation indicates that inhibition of the Ca2+/CaM gating process by these Cx-sequence specific CaMBD mimetic peptides does not necessarily prove the functionality of the corresponding domain, it also does not necessarily disprove the functional relevance of these identified Cx CaMBD domains. Additional experiments and novel approaches will be required to test the functionality of known Cx CaMBDs in a sequence-specific manner.
Another Cx43 mimetic peptide, the Gap19 nonapeptide, targeting CL residues 128-136 in the middle of the L2 pH receptor domain (residues 119-144) was shown to inhibit Cx43 hemichannel activity with an intracellular IC50 of 6.5 μM without affecting gj at substantially higher concentrations (400 μM) presumably by interfering with the CL-CT interaction (14,24). Since the Cx43 CaMBD peptide corresponds to CL residues 136-158, it is possible that Gap19 might interfere in the Ca2+/CaM gating mechanism (18). To test this hypothesis, we performed the Ca2+-ionomycin perfusion experiments on N2a-Cx43 cell pairs with 100 μM Gap19 added to both whole cell patch pipettes (Fig. 3B). 100% inhibition of Cx43 Gj was still achieved within 10-12 min of perfusion and no inhibition of gj was evident. Our results confirm that Gap19 does not affect Cx43 gj nor the Ca2+/CaM-dependent uncoupling mechanism.
The Vj-gating and γj properties of Cx43-M257 gap junctions were previously studied in exogenous Xenopus oocyte and N2a cell expression systems (25,26). Both studies reported a loss of the fast kinetic component of the Vj-gating mechanism and a lower Gmin for the steady state Gj – Vj curve. Additionally, gap junction channel recordings from Cx43-M257 N2a cell pairs revealed a loss of the low γj subconductance state, an increased open time for the remaining ≥ 100 pS main conductance state with primarily slow transitions between the open and closed states of the channel (26). Previously, we had observed a hysteresis in the steady state Gj -Vj curves obtained during the application of slow 24 sec, 0 to ±120 mV Vj ramps found only in primary NMVM cell pairs, not N2a-Cx43 cell pairs (27). The observed increase in the linear slope conductance at low Vj potentials, corresponding to the Gmax of the steady state Gj – Vj curves, occurred during the recovery (from inactivation) phase of the ±120 mV to 0 mV Vj ramp in NMVMs and was termed facilitation. This facilitated recovery of Gj was not affected by non-specific serine/threonine protein kinase inhbitors (e.g. 12 μM H7, data not shown) nor 100 nM rotigaptide, though the inactivation phase was reduced by these treatments (29).
Thus, we examined the effect of the Cx43 CT truncation on the Vj-gating properties on homozygous Cx43-M257 NMVMs. Our results confirmed the loss of the fast inactivation component of Cx43 gap junctions and predominance of the 100 pS main γj state of Cx43 gap junction channels seen in exogenous expression systems, but also the abolition of the facilitated recovery of Gj during decreasing Vj values after achieving steady state inactivation to Gmin (Fig. 4A-F). We did not, however, observe a reduction in Gmin compared to control WT NMVM gap junctions (27). The reduced rate and Vj-sensitivity of the remaining slow kinetic inactivation component of Cx43-M257 gap junctions implies that the Cx43 CT domain contributes some of the charge to the slow Vj gating mechanism of Cx43 gap junctions (25). Furthermore, the deletion of the facilitated recovery of Gj from inactivating potentials was only previously attained by non-selective histone deacetylase inhibition (pan-HDACI) by 100 nM trichostatin A or 1 μM vorinostat implies that protein acetylation directly (e.g. Cx43 CT domain) or indirectly (e.g. tubulin?) affects the Vj gating properties of Cx43 gap junctions (30).
In summary, we conclude that the calcium/calmodulin-dependent chemical gating mechanism of Cx43 gap junctions does not require the distal CT pH gating particle of Cx43. The Gap19 peptide targeting a portion of the Cx43 CL pH receptor domain also does not affect the Ca2+/CaM gating mechanism of Cx43, but the sequence-specific Cx-CaMBD mimetic peptides function as a non-specific CaM binding domain and do not necessarily infer the modulatory function of these domains in their respective connexin-specific gap junctions. Lastly, we confirmed in Cx43-M257 cardiomyocyte gap junctions that deletion of the Cx43 CT domain eliminates the fast inactivation component and enhances the probability of the 100 pS main γj state of Cx43 gap junctions, but also eliminates the facilitated recovery of gj from inactivation.
Acknowledgements
We thank Dr. Steven Taffet for the gift of the Cx43-M257 pIRES2-EGFP plasmid.
We thank Dr. Karen Maass for providing us with the Cx43+/K258stop mice and the methodology for genotyping these mice.
R.D.V. was supported by grants from the NIH HL042220, AHA 17GRNT33710031, Hendricks Fund, and Joseph C Georg Fund from the CNY Community Foundation.