Objective: Na+ current derived from expression of the principal cardiac Na+ channel, Nav1.5, is increased by activation of protein kinase A (PKA). This effect is blocked by inhibitors of cell membrane recycling, or removal of a cytoplasmic endoplasmic reticulum (ER) retention motif, suggesting that PKA stimulation increases trafficking of cardiac Na+ channels to the plasma membrane.
Methods: To test this hypothesis, green fluorescent protein (GFP) was fused to Nav1.5 (Nav1.5–GFP), and the effects of PKA activation were investigated in intact, living cells that stably expressed the fusion protein. Using confocal microscopy, the spatial relationship of GFP-tagged channels relative to the plasma membrane was quantitated using a measurement that could control for variables present during live-cell imaging, and permit an unbiased analysis for all cells in a given field.
Results: In the absence of kinase stimulation, intracellular fluorescence representing Nav1.5–GFP channels was greatest in the perinuclear area, with additional concentration of channels beneath the cell surface. Activation of PKA promoted trafficking of Na+ channels from both regions to the plasma membrane. Experimental results using a chemiluminescence-based assay further confirmed that PKA stimulation increased expression of Nav1.5 channels at the cell membrane.
Conclusions: Our results provide direct evidence for PKA-mediated trafficking of cardiac Na+ channels into the plasma membrane in living, mammalian cells, and they support the existence of multiple intracellular storage pools of channel protein that can be mobilized following a physiologic stimulus.
Voltage-gated Na+ channels are transmembrane proteins responsible for the action potential upstroke that initiates excitation–contraction coupling in the heart. In humans, a reduction in cardiac Na+ current (INa) can lead to serious arrhythmias. This is evidenced by the occurrence of sudden cardiac death in the Brugada Syndrome and bradyarrhythmias/heart block in cardiac conduction disease [1,2], in which sarcolemmal Na+ current is reduced . Thus, the number of Na+ channels that reside in the cardiac sarcolemma is a critical component for normal cellular excitability. For many membrane proteins, activity at the plasma membrane is tightly controlled by cellular responses to various hormonal stimuli . However, for Na+ channels, as well as cardiac ion channels in general, the basic mechanisms whereby cells regulate trafficking of channels into and out of the plasma membrane have not been fully elucidated. Given that disease-causing mutations in ion channel genes often disrupt normal protein trafficking [5,6], a greater understanding of this regulated process can have potential therapeutic import.
We have previously shown that stimulation of PKA causes an increase in Nav1.5 current that is prevented by inhibitors of cell membrane recycling (e.g., chloroquine), implying that kinase activation promotes trafficking of channels to the plasma membrane . In support of this hypothesis, PKA-mediated potentiation of INa in myocytes isolated from normal and peri-infarct regions of canine heart is suppressed by preincubation of cells in chloroquine . We have also shown that the PKA response is abrogated by mutation of an endoplasmic reticulum (ER) retention signal and nearby phosphorylation sites in the cytoplasmic I–II interdomain linker of Nav1.5 , suggesting release of channels from an intracellular storage compartment to mediate the effect. However to date, direct evidence for kinase-regulated trafficking of Nav1.5 channels by PKA has been lacking.
Although PKA activation consistently increases Nav1.5 current when the channel is expressed in Xenopus oocytes [7,10,11], results obtained using whole-cell recordings in mammalian cells have been controversial, with both an increase  and decrease  reported. The basis for this discrepancy is not clear. However, a likely contributing factor is loss of critical proteins or cofactors during whole-cell recordings in mammalian cells, which is less likely to occur during two-microelectrode recordings in oocytes. Green fluorescent protein (GFP) is widely used in heterologous expression systems as a reporter molecule to monitor protein localization. To test the hypothesis that PKA alters Na+ channel trafficking, we have constructed fusion proteins of Nav1.5 and GFP. After verifying in oocytes that the addition of GFP did not disrupt the PKA response, the effects of kinase activation were investigated using confocal microscopy and electrophysiology in intact, mammalian cells to preserve critical intracellular components of cell signaling. To further confirm our results, an independent method was employed to determine the effect of PKA stimulation on Na+ channel expression at the plasma membrane.
Reagent grade chemicals, as well as 8-chlorophenylthio cAMP (8-cpt-cAMP), 3-isobutyl-1-methylxanthine (IBMX), and forskolin, were obtained from Sigma (St. Louis, MO). Enzymes and buffers were purchased from Roche (Indianapolis, IN) and Promega (Madison, WI).
2.2. Plasmid construction
The Nav1.5 DNA construct (SCN5A; GenBank™ accession number M77235) was previously subcloned into a modified pSP64T vector for expression in Xenopus laevis oocytes . The DNA sequence for EGFP was amplified by PCR and cloned in frame to the 3′-terminal end of the Nav1.5 sequence immediately prior to the stop codon after nucleotide 6198. In the resulting fusion protein, the C-terminus of Nav1.5 was fused to the N-terminus of GFP (Nav1.5–GFP[C]). To generate a fusion protein with GFP fused to the N-terminus of Nav1.5 (Nav1.5–GFP[N]), EGFP was inserted in frame with Nav1.5 just before the start codon. For experiments using a chemiluminescence assay to quantitate surface expression of Na+ channels, three copies of the hemagglutinin (HA) epitope were inserted into the initial, extracellular portion of the Domain I S5–S6 loop of Nav1.5 (Nav1.5–HA) in pSP64T using recombinant PCR techniques as described previously [9,14]. For expression in HEK 293 cells, wild-type and mutant Nav1.5 DNA constructs were subcloned into the pCR3 vector. For all variant constructs, final products were sequenced to verify that the desired sequence was present.
2.3. Na+ channel expression
To generate HEK 293 cell lines that stably expressed Nav1.5–GFP fusion proteins, 4 μg of each pCR3/Nav1.5–GFP construct was added with Lipofectamine (Gibco; Carlsbad, CA) to a 60 mm dish containing 40% confluent cells grown in Dulbecco's modified Eagle's medium (DMEM, Sigma) supplemented with 10% serum. Transfection efficiency was ∼50–60% as assessed by the presence of green fluorescent cells after 24 h. After 48 h, 1–2.2 mg/ml of G418 (Sigma) was added to the medium, which was changed every 2 days. After 19 days, colonies of green cells were transferred to 96-well plates and maintained in 2.2 mg/ml of G418. Colonies surviving after 72 h were transferred to wells on a new dish. Following an additional 72 h, cells were transferred to 60 mm dishes, with an additional transfer after 5 days. Cells from 12 original colonies were amplified and stored.
Coexpression of the human β1 subunit (SCN1B) with mutant Nav1.5 constructs in Xenopus oocytes was performed as described previously for the wild-type channel . In mammalian cells, hβ1 was coexpressed using bicistronic plasmids that encoded additional markers (either CD8 for Nav1.5–GFP, or GFP for Nav1.5–HA) with an internal ribosomal entry site (IRES) to allow verification of hβ1 expression as previously reported [15,16]. For transient expression of Nav1.5 and Nav1.5–HA, HEK 293 cells were transfected with 2 μg of DNA and 1 μg of pGFP-IRES-hβ1 using Lipofectamine. The medium was changed after 6 h, with cells harvested for study after 48 h.
2.4. Western blot analysis
Proteins were separated by SDS-PAGE (6% gel) and transferred to a polyvinylidene difluoride membrane using wet transfer. Membranes were incubated with 5% nonfat dry milk in TBST (in mM: Tris-base 10, NaCl 150, Tween-20 0.1%, pH 7.6) overnight at 4 °C and probed with an anti-Na+ channel antibody (Upstate, Lake Placid, NY) at a dilution of 1:250 overnight at 4 °C. After removal of the primary antibody, horseradish peroxidase-conjugated secondary antibody (goat anti-rabbit, Jackson ImmunoResearch, West Grove, PA) was added at a dilution of 1:15,000 for 90 min at room temperature with gentle shaking. An enhanced chemiluminescence kit (ECL, Amersham, Arlington Heights, IL) was used for detection of protein bands.
2.5. Electrophysiologic recordings and data analysis
In Xenopus oocytes, Na+ current recordings were obtained using the two-microelectrode voltage-clamp technique as previously described [7,9]. For wild-type Nav1.5, α-subunit RNA was diluted with RNase-free water to achieve Na+ currents within 24 h that could be successfully voltage clamped (≤6 μA). For mutant constructs, measurable Na+ currents (≥1 μA) were expressed only after at least 48 h (2–3 days and 5–7 days for the C- and N-terminal GFP constructs, respectively), despite injection of undiluted α-subunit RNA. Pipettes were filled with 3 M KCl, and a standard extracellular bath solution was utilized (in mM: NaCl 96, KCl 2, CaCl2 1.8, MgCl2 1, HEPES 5; pH 7.5). For HEK cells, single cells were isolated for experimentation by exposure of near-confluent (∼80–90%) dishes to trypsin. To assay Na+ channel expression, whole-cell Na+ currents (INa) were obtained using an Axopatch 200 amplifier (Axon Instruments, Union City, CA) and were acquired using pCLAMP 8.2 (Axon Instruments) . Pipette resistance was 1–1.5 MΩ when filled with a pipette solution containing (in mM): NaF 10, CsF 110, CsCl 20, EGTA 10, HEPES 10 (pH 7.35 with CsOH). The bath solution contained (in mM): NaCl 145, KCl 4.5, CaCl2 1.5, MgCl2 1, HEPES 10 (pH 7.35 with CsOH). Data were collected 10–15 min after the whole-cell configuration was established. To investigate effects of PKA activation, perforated patch recording was used with amphotericin B as previously described . The pipette solution contained (in mM): Cs2SO4 75, NaCl 35, CsCl 20, MgCl2 8, HEPES 10 (pH 7.4). In both whole-cell and perforated patch recordings, ∼80–90% of the series resistance was compensated, yielding a maximum voltage error of ≤3 mV. Potent activation of PKA was achieved in Xenopus oocytes by bath superfusion of 8-cpt-cAMP 200 μM, IBMX 1 mM, and forskolin 10 μM ; in HEK cells, respective concentrations were 20 μM, 100 μM, and 20 μM. All experiments were conducted at room temperature (22±2 °C).
Analysis of data was performed using either pCLAMP 8.2 or custom programs designed to read and analyze pClamp data files as described previously [7,17]. Results are presented as mean±SEM.
2.6. Confocal laser scanning microscopy
Near-confluent (∼70%) HEK 293 cells expressing Nav1.5–GFP fusion proteins were imaged using a confocal microscope (LSM510) with a 63X/1.4 plan apochromat objective (Carl Zeiss, Inc.). Cells were plated onto a Matek dish (Matek Corporation, Ashland, MA); after 48 h, they were exposed to 1 μM Di-8-ANEPPS (Molecular Probes, Eugene, OR) for 3 min (followed by several washes) to identify the plasma membrane, with images obtained within 5–10 min. Both GFP and Di-8-ANEPPS were excited at 488 nm with the LSM510 Ar laser and detected with a 505–530 nm band pass or 585 nm long pass filter, respectively. The confocal aperture was adjusted to restrict detection to optical sections of 1 μm, and transmitted differential interference contrast (DIC) images were obtained sequentially for additional definition of the cell boundaries. To control for cross-talk between detection of GFP and Di-8-ANEPPS, single-labeled control cells (i.e., nontransfected HEK cells exposed to Di-8-ANEPPS, or cells expressing Nav1.5–GFP in the absence of Di-8-ANEPPS) were imaged under identical conditions as those used for dual-labeled probes to confirm proper signal isolation of each channel. Initial data analysis was performed using the LSM510 software.
2.7. Quantitation of Nav1.5 trafficking
The time-dependent spatial relationship of the GFP-tagged Nav1.5 channels with the Di-8-ANEPPS-labeled plasma membrane was measured using Metamorph image analysis software (Universal Imaging Corporation, Downington, PA). The dynamic range for both the green (GFP) and red (Di-8-ANEPPS) channels was maximized to achieve the highest precision and contrast in the image. The minimum threshold signal was set to the value of the background signal + 5%, while the maximal threshold was set to the maximum-1 (or 254 AU [arbitrary units]), so that the signal measured was both above background and on scale. For an entire field of cells, the pixel coordinates that contained both red and green signal were identified. Because staining by Di-8-ANEPPS was restricted to the plasma membrane, the green/red (G/R) ratio (defined as the percentage of red-containing pixels that also contained green signal) reflected colocalization of Nav1.5–GFP channels with the plasma membrane (within the resolution space, approximately 0.1 μm3). Statistical analyses utilizing repeated-measures ANOVA were performed using SPSS Version 11.5 (SPSS Inc., Chicago, IL), with P<0.05 considered statistically significant.
2.8. Chemiluminescence assay for surface expression of Na+ channels
HEK 293 cells were plated onto a 96-well dish using a dilute suspension of cells, and in a manner designed to optimize equal loading of cells and minimize inter-well variability. After 24 h, cells were transfected with Nav1.5–HA DNA plus SCN1B (using the pGFP-IRES-hβ1 construct that also expressed GFP), with a transfection efficiency of ∼40–50%. After 48 h when confluency was achieved, plasma membrane expression of Nav1.5 was assayed as previously described for mammalian cells . All steps were performed at room temperature. After chemiluminescence measurements, dishes were examined using phase contrast microscopy to identify wells in which significant cell loss occurred. For both stimulated and nonstimulated conditions, data from 16 wells in each experiment were pooled for 3 separate experiments to obtain the results reported.
3.1. Expression of Nav1.5–GFP channels
To investigate trafficking of cardiac Na+ channels in living cells, GFP was fused to either the C- or N-terminus of Nav1.5 (Nav1.5–GFP[C] and Nav1.5–GFP[N], respectively). Because PKA potentiation of Nav1.5 current is reproducibly observed in Xenopus oocytes, electrophysiology experiments were performed using this heterologous system to verify that the addition of GFP did not disrupt channel function or the PKA response. Expression of Nav1.5–GFP(C) resulted in Na+ currents that resembled wild-type Nav1.5 currents (Fig. 1A), except for changes in the voltage dependence of channel gating (activation curve midpoint, or V½, was −28±1 mV, compared to −33±2 mV for Nav1.5; n=5, 10 each, P<0.01; for channel availability or inactivation, V½ was −69±1 mV, and −64±1 mV for Nav1.5; n=5, 8 each, P<0.01). As for wild-type Nav1.5 , bath superfusion of 8-cpt-cAMP, IBMX, and forskolin to stimulate PKA increased Na+ currents derived from the GFP-tagged channel (Fig. 1B, +36±9% at −20 mV [from 2.4±0.9 to 3.2±0.9 μA]; P<0.01). Similar findings were obtained for Nav1.5–GFP(N) currents (INa increased +58±1% with PKA activation; n=7). However, in both oocytes and HEK cells, expression for the N-terminal GFP construct was markedly reduced, and subsequent experiments were conducted using Nav1.5 with GFP attached to the C-terminus (hereafter referred to as Nav1.5–GFP).
In mammalian cells, expression of Nav1.5–GFP channels was assayed using multiple approaches. Using Di-8-ANEPPS to visualize the plasma membrane, HEK 293 cells that stably expressed Nav1.5–GFP were imaged using confocal microscopy, with evidence of robust Na+ channel expression at the plasma membrane (Fig. 2A). This was further confirmed during whole-cell recordings of expressed Na+ currents (Fig. 2B; INa was 6.9±0.3 nA [1431±319 pA/pF], n=10) and Western blotting (Fig. 2C).
The effects of PKA activation on Nav1.5–GFP currents were examined in HEK cells using the perforated patch method, a technique that causes less perturbation of cellular contents than whole-cell recordings. As in oocytes, Na+ currents increased with kinase stimulation (with a representative example in Fig. 2D, and summary data in Fig. 2E; INa increased 14±1%; n=14 cells each), without a significant shift in the voltage dependence of channel gating (activation V½ was −38±1 mV before and −43±1 mV after PKA activation; for inactivation, V½ was −86±1 mV before and −85±1 mV after PKA activation; n=10, P>0.05 for both). Potentiation of INa was completely inhibited when cells were preincubated for 1 h with the cell membrane-permeant, specific PKA peptide inhibitor myristoylated PKI 14–22 amide 1 μM (current density was 423±1 pA/pF before and 410±2 pA/pF after PKA activation, data not shown; n=7), confirming that the increase in INa was mediated by PKA.
3.2. Intracellular distribution of Nav1.5 channels: ER and sub-membrane localization
Using both differential interference contrast (DIC) images (Fig. 3A) and exposure of cells to Di-8-ANEPPS (Fig. 3B) to delineate the plasma membrane, it is readily apparent that fluorescence intensity representing intracellular Nav1.5–GFP channels was greatest in the perinuclear region. Using a related Nav1.5–GFP construct, this was recently shown to represent accumulation of channel protein in the ER . In addition, intracellular channels were often detected just inside the plasma membrane in the sub-membrane space (illustrated in Fig. 6). These results are consistent with findings in cardiac myocytes that demonstrate accumulation of intracellular Na+ channels in both the ER  and caveolae  (lipid-rich invaginations of the plasma membrane that can pinch off to form subsarcolemmal vesicles [23–25]).
3.3. Quantitation of Nav1.5 trafficking: effect of light exposure
As detailed in the Methods, we utilized a spatial measurement, the green/red (G/R) ratio, to quantitate colocalization of GFP-tagged Nav1.5 channels (green) with the Di-8-ANEPPS-labeled plasma membrane (red). Unexpectedly, we found that frequent imaging of HEK cells at baseline promoted intracellular redistribution of Nav1.5–GFP channels. As shown in Fig. 4, there was a significant rise in the green/red (G/R) ratio when images were acquired every 1 or 2 min (+36±5% and +41±16% at 25 min, respectively [P<0.05 for both]; n=17 and 7 fields from 3 experiments for each), implying a continually-increasing number of Nav1.5–GFP at the plasma membrane under control, nonstimulated conditions. Because the cells under study stably expressed Nav1.5, these results are not compatible with the known behavior of these cells, with Na+ currents that are essentially constant over time. It is recognized that light-mediated excitation of fluorescent probes can produce photochemical effects capable of modulating biologic processes through complex mechanisms [26–28]. We hypothesized that this excitation was responsible for the perturbed baseline trafficking, and image acquisition frequency was reduced to 5 min intervals. Under these conditions (as shown in Fig. 4), the G/R ratio was minimally altered over time (+9±10% at 25 min [P=0.34]; n=11 fields from 3 experiments), with similar results when image frequency was further reduced to 10 min intervals (+11±6% at 30 min [P=0.4]; n=6 fields from 3 experiments). These results indicate that light exposure alters baseline trafficking and intracellular distribution of Nav1.5–GFP channels, presumably due to photochemical effects. Similar findings were obtained when the human β1 subunit was coexpressed with Nav1.5–GFP (data not shown).
3.4. Effect of PKA activation
To examine the effects of PKA activation on Na+ channel trafficking, HEK cells expressing Nav1.5–GFP were imaged every 5 min (to minimize light effects on baseline trafficking) in the absence or presence of PKA activators. Under these conditions, PKA stimulation promoted redistribution of channels, with movement towards the plasma membrane. This is reflected by the increased yellow color at the cell membrane when Di-8-ANEPPS images are superimposed in Fig. 5A, and the significant rise in G/R ratio in Fig. 5B and C (+45±16% at 25 min with PKA activation, compared to +9±10% under control conditions [P=0.01]; n=11 fields from 3 experiments for each), reflecting increased colocalization of the channel with the plasma membrane. Similar results were obtained when cells were imaged every 10 min, as shown in Fig. 5C. Of note, this effect of PKA was masked with frequent (every 2 min) imaging (Fig. 5C), during which baseline trafficking was disturbed. Because the G/R ratio is a spatial measurement, these findings cannot be attributed to photobleaching (which was nonetheless minimal in our experiments: total green signal intensity for a field of cells was 64,193±10,963 and 63,640±9980 AU at baseline and after 25 min of imaging every 5 min, respectively; for the red channel, similar values were 72,744±5231 and 75,507±7882 AU; n=6 fields each).
Additional experiments were performed to further confirm the role of PKA activation in Na+ channel redistribution. HEK cells expressing Nav1.5–GFP were exposed to 8-cpt-cAMP and forskolin in the absence of IBMX, a less specific activator of PKA [29,30]. In this setting, a similar increase in the G/R ratio was observed (+50±10% with PKA activation, compared to +15±10% under control conditions [P=0.03]; n=7 and 9 fields from 3 experiments for each). In addition, preincubation of cells with myristoylated PKI 14–22 amide (1 μM for 1 h) inhibited the increase in the G/R ratio caused by 8-cpt-cAMP, forskolin, and IBMX, as illustrated in Fig. 5D, confirming that these effects were mediated by PKA activation.
3.5. Intracellular redistribution of Nav1.5–GFP channels
In response to PKA activation, the pattern of GFP fluorescence within cells was altered in two distinct ways. First, areas of green fluorescence concentrated in the perinuclear region moved away from the nuclear membrane towards the plasma membrane, as demonstrated in selected Z-series images in Fig. 6A. Second, sub-membrane areas of green fluorescence merged with the plasma membrane region, reflected by the increased yellow color intensity at the membrane in the magnified views in Fig. 6B. In Fig. 6C, green and red color intensity along the arrows in Fig. 6B are quantitated. Peak intensity of green fluorescence (*) moves from a region just inside the cell membrane in the left panel, to a position that aligns with peak intensity of Di-8-ANEPPS (i.e., the plasma membrane) in the right panel. These altered spatial relationships support our quantitative analysis that PKA stimulation leads to redistribution of intracellular cardiac Na+ channels, with trafficking away from the ER and sub-membrane regions, towards the plasma membrane.
3.6. Confirmation of PKA-mediated trafficking of cardiac Na+ channels
Additional experiments were performed to confirm the results obtained using confocal microscopy, using a chemiluminescence-based assay of plasma membrane protein expression that utilizes an extracellular epitope in the protein under study . For these experiments, three copies of the hemagglutinin (HA) epitope were inserted into the extracellular loop of S5–S6 in Domain I of Nav1.5 (Nav1.5–HA). Expression of this mutant construct with hβ1 in Xenopus oocytes resulted in Na+ currents resembling wild-type, with preservation of the PKA response (+58±15%, n=5; data not shown). Similarly, robust expression (Fig. 7A) and Na+ currents (Fig. 7B) were evident in HEK cells (current density was 658±23 pA/pF at −10 mV; n=7), with Na+ channel protein expression detected by Western analysis of cell membrane fraction (data not shown). Using the chemiluminescence assay, plasma membrane expression of Na+ channels increased significantly when cells were exposed to PKA activators, as compared to control, unstimulated cells (Fig. 7C; P<0.01). These findings provide additional evidence for PKA-mediated trafficking of cardiac Na+ channels to the plasma membrane.
Our results provide direct evidence for PKA-mediated trafficking of cardiac Na+ channels, with redistribution from the ER and sub-membrane space towards and into the plasma membrane. In addition, they support the existence of multiple intracellular storage pools of channel protein that can be mobilized following a physiologic stimulus.
We used intact cells for these studies in order to preserve intracellular contents, including critical signaling components, given the disparate results of reports examining the effect of PKA activation on Nav1.5 currents during whole-cell recordings in mammalian cells [12,13]. For quantitative analysis during confocal imaging, we utilized a measurement (G/R ratio) that offered several advantages: 1) it could control for the complex variables associated with live-cell imaging (e.g., baseline trafficking of channels, cell movement); 2) data could be analyzed in an unbiased manner from all cells visible within a given field; and 3) it represents a spatial relationship and therefore would be insensitive to the effects of photobleaching. This approach was critical, as simple visual inspection of the cells is inherently flawed. The true proportion of overlapping green and red color is difficult to judge by visual evaluation, given that the relative dynamic range of both signals is highly variable. This produces a range of green-to-yellow-to-red, all of which represent “colocalized”, although only a small proportion may be perceived as such, based on the simple and flawed assumption that yellow is the criterion for colocalization. Furthermore, living cells undergo significant intracellular motion during imaging, producing a dynamic distribution problem that cannot be easily evaluated by simple visual observation. By using a quantitative colocalization algorithm based on objective signal threshholding criteria as described in the Methods, we were able to demonstrate the effects of PKA activation on channel distribution within the cells. It is possible that PKA-mediated effects on cell morphology (i.e., cell swelling) could alter intracellular channel redistribution of Na+ channels. We would anticipate that if the principal effect of PKA was to promote cell swelling, the concentration of channels near the plasma membrane would decrease from a dilution effect as the membrane and cytosol expand. The fact that our quantitative results from hundreds of cells demonstrate the opposite finding further supports enhanced trafficking of channels to the plasma membrane in response to PKA activation.
An unanticipated result in our study was the finding that frequent light exposure used for fluorescence excitation perturbed the baseline dynamic distribution of GFP-tagged channels. With increased excitation exposure, PKA activation appeared to lack a stimulatory effect because the light exposure caused PKA-independent redistribution of GFP. With less frequent imaging, intracellular distribution of channels at baseline was minimally affected, and a stimulatory response to PKA activation was observed. Imaging-induced intracellular photochemistry is complex and often overlooked during fluorescence imaging, particularly when experiments are performed with live cells and tissues. Potentially perturbing events include oxidative processes, generation of free radicals, and many other excited-state phenomena that may be expected during the requisite fluorescence excitation . Excitation of fluorescein derivatives are known to produce altered mitochondrial activity  and GFP and its variants also produce photochemical species  that may interfere with endogenous physiology. It is likely that these perturbations were responsible for the altered baseline dynamic distribution of GFP-tagged channels that masked the effect of PKA during frequent imaging. These observations have important implications for the use of fluorescent probes to investigate intracellular protein trafficking and mandate the necessity for critical control experiments to investigate the biologic effects of photochemistry for a given protein.
For Na+ channels, the concept of an intracellular storage pool is not a new one, as originally proposed by Schmidt and Catterall in the 1980s based upon biochemical studies in rat brain neurons . Recent immunocytochemistry data have provided evidence for the concentration of cardiac Na+ channels in the ER in cardiac myocytes . Transient expression of membrane proteins in heterologous cell expression systems typically leads to accumulation of intracellular protein in the ER, and we sought to reduce this factor by establishing stable expression of Na+ channels for our experiments. Additional evidence to support a role for the ER in intracellular storage is the requirement of a cytoplasmic ER retention sequence, and proximally-located phosphorylation sites, for the PKA response to potentiate Nav1.5 current . ER retention sequences play an important role in the trafficking of many plasma membrane proteins to the cell surface [32–35]. By binding to resident proteins in the ER, they serve as a checkpoint or brake for protein expression, which can be relieved when additional proteins bind the protein and suppress ER retention. We hypothesize that PKA-mediated phosphorylation of Nav1.5, and possibly other substrates, permits binding of an accessory protein to the I–II interdomain linker, thus allowing exit from the ER (and/or other compartments) with trafficking towards the plasma membrane. A similar effect was recently reported for the NMDA receptor [5,36]. PKA and protein kinase C-mediated phosphorylation of residues near an ER retention site in the NR1 subunit relieves ER retention of the protein and mediates forward trafficking to the plasma membrane.
Our finding of what appears to be an additional pool of Nav1.5 channels just beneath the cell surface is consistent with the concept that cardiac Na+ channels can partition in caveolae, with trafficking into the membrane upon PKA activation . Caveolae are small, lipid-rich invaginations in the plasma membrane known to contain multiple cell signaling molecules, including β-adrenergic receptors, and they participate in the trafficking of other membrane channel proteins [23–25]. They have also been shown to pinch off to form sub-membrane vesicles. Recently, Yarbrough and colleagues demonstrated in rat cardiac myocytes that inclusion of cardiac-specific anti-caveolin antibodies in the patch pipette prevented the isoproterenol-mediated increase in INa, and that cardiac Na+ channels were physically associated with caveolae . Based on these results, they proposed that caveolae are involved in the presentation of Na+ channels to the plasma membrane with β-adrenergic stimulation. In our experiments, we found a population of sub-membrane Na+ channels that redistributed into the plasma membrane with PKA activation. The location of these channels beneath the cell membrane and their translocation with PKA stimulation is consistent with an association with caveolae.
Of interest, PKA-mediated potentiation of Na+ current in HEK cells was rapid, occurring within minutes, consistent with other studies using mammalian (CHO) cells to express Nav1.5  and cardiac myocytes . The brisk time course also supports a role for caveolar storage of Na+ channels in close proximity to the plasma membrane. On the other hand, INa potentiation was slower in oocytes, with a continual increase for tens of minutes . For electrophysiology experiments in HEK cells, PKA activators were added to the bath using a rapid perfusion system, while in oocytes, a slower perfusion system was employed. Moreover, it is well recognized that the speed and/or potency of physiologic and pharmacologic effects can be reduced in oocytes, compared to mammalian cells [37–39]. It is likely that the yolk, insufficient perfusion, and factors unique to oocytes account for these discrepancies . The time course of PKA effect that we observed in HEK cells is also consistent with the recently reported kinetics of compartmentalized cAMP release and PKA stimulation in mammalian cells , which can vary between local subcellular domains and an entire cell, and by analogy between single and multicellular preparations. It is probable that this effect contributes to the slower time course observed in the multicellular preparations visualized during our confocal experiments. Moreover, during imaging experiments, PKA activators were by necessity slowly added to the experimental bath (to avoid washing away the cells that were being visualized), in contrast to the rapid perfusion used during electrophysiology experiments. Thus, it is likely that the variability in both experimental techniques (i.e., delivery of PKA activators) and the cellular preparations (HEK cells vs oocytes, and single vs multicellular preparations) studied account for the time course differences observed in our results.
While our experiments were conducted using recombinant Nav1.5, there is considerable evidence that the PKA-mediated trafficking we observed is also relevant for cardiac myocytes. In rat cardiac myocytes, the increase in INa amplitude with β-adrenergic receptor stimulation was accompanied by an increase in the number of functional channels , while antibodies to caveolin abolished Na+ current potentiation by isoproterenol . More recently, Baba and coworkers have shown that activation of PKA increased Na+ current in myocytes from both normal canine ventricle and myocardial infarct border zone . The PKA response was inhibited by pretreatment with chloroquine, which disrupts membrane vesicle trafficking. These findings further support a role for channel trafficking in PKA-mediated potentiation.
In conclusion, we have demonstrated that activation of PKA causes redistribution of cardiac Na+ channels in living cells from intracellular storage pools, with increased trafficking into the plasma membrane. These results provide strong support for regulated trafficking as a novel cellular mechanism to modulate cardiac Na+ current.
This work was supported by grants from the US Public Health Service (HL55665 and HL071002) and a postdoctoral fellowship grant from the American Heart Association, Southeast Affiliate (J.Z.). Confocal microscopy and image analysis were performed through the Vanderbilt Cell Imaging Shared Resource (supported by NIH grants CA68485, DK20593, DK58404, HD15052, DK59637 and EY08126). We thank Zaid Brifkani, Carrie Browning, and Xinran Hu for their excellent technical assistance.