ROMK1 (Kir1.1) Causes Apoptosis and Chronic Silencing of Hippocampal Neurons

H. Nadeau, S. McKinney, D. J. Anderson, and H. A. Lester

Division of Biology, California Institute of Technology, Pasadena, California 91125


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
CONCLUSION
APPENDIX A
APPENDIX B
REFERENCES

Nadeau, H., S. McKinney, D. J. Anderson, and H. A. Lester. ROMK1 (Kir1.1) Causes Apoptosis and Chronic Silencing of Hippocampal Neurons. J. Neurophysiol. 84: 1062-1075, 2000. Lentiviral vectors were constructed to express the weakly rectifying kidney K+ channel ROMK1 (Kir1.1), either fused to enhanced green fluorescent protein (EGFP) or as a bicistronic message (ROMK1-CITE-EGFP). The channel was stably expressed in cultured rat hippocampal neurons. Infected cells were maintained for 2-4 wk without decrease in expression level or evidence of viral toxicity, although 15.4 mM external KCl was required to prevent apoptosis of neurons expressing functional ROMK1. No other trophic agents tested could prevent cell death, which was probably caused by K+ loss. This cell death did not occur in glia, which were able to support ROMK1 expression indefinitely. Functional ROMK1, quantified as the nonnative inward current at -144 mV in 5.4 mM external K+ blockable by 500 µM Ba2+, ranged from 1 to 40 pA/pF. Infected neurons exhibited a Ba2+-induced depolarization of 7 ± 2 mV relative to matched EGFP-infected controls, as well as a 30% decrease in input resistance and a shift in action potential threshold of 2.6 ± 0.5 mV. This led to a shift in the relation between injected current and firing frequency, without changes in spike shape, size, or timing. This shift, which quantifies silencing as a function of ROMK1 expression, was predicted from Hodgkin-Huxley models. No cellular compensatory mechanisms in response to expression of ROMK1 were identified, making ROMK1 potentially useful for transgenic studies of silencing and neurodegeneration, although its lethality in normal K+ has implications for the use of K+ channels in gene therapy.


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
CONCLUSION
APPENDIX A
APPENDIX B
REFERENCES

Potassium ion selective channels establish the neuronal resting membrane potential (RMP) and restore it after firing. We describe a method for expressing a new set of K+ channels in neurons, to alter their excitability. Neuronal RMP is often 10-20 mV depolarized relative to the K+ reversal potential, and thus more open K+ channels are expected to hyperpolarize the cell. Moreover, more open channels lead to a reduction in input resistance, rendering excitatory synaptic currents less effective in depolarizing the cell to threshold. In brief, a change in input resistance due to more open K+ channels can abolish action potential firing ("silence" the cell).

There are several reasons to silence neurons in vitro and in vivo. Selectively targeting pathways or populations of neurons has revealed the importance of interneuronal communication in developing (Murakami et al. 1992) and adult (de la Cruz et al. 1996) systems. Botulinum toxin is used to lessen muscle spasms in patients with cerebral palsy (Flett et al. 1999) and spinal cord injuries (Al-Khodairy et al. 1998), and targeted lesions are often the only possible treatment for those with intractable epilepsy (Jallon 1997; Nayel et al. 1991). Control of excitability may also be important for lessening neurological damage following ischemic injury or in degenerative disease (Rodriguez et al. 1998). A genetic approach has two major advantages over toxins and surgery: one, it can target a pathway that is not fully understood; and two, it has the potential to be fully inducible and reversible over a time course of hours.

However, to evaluate genes as silencing candidates, it is important to be able to translate alteration of excitability seen in culture into in vivo long-term behavior. We use an HIV-based lentiviral vector to create an in vitro model of transgenesis, with a K+ channel as the candidate silencer. Neurons are transduced soon after plating and allowed to grow and develop for days to weeks with unopposed channel expression. The viral genome is integrated into its host at low copy number, leading to stable expression levels and no interference with host protein synthesis. Efficacy and lack of toxicity of the viral vector itself are established, and no inactivation or down-regulation of the channel is observed over a 3-wk period. However, the chronic efflux of K+ leads to apoptotic cell death unless counteracted by a raised K+ concentration in the culture medium. This imposes limits on the possible in vivo use of ROMK1, and perhaps of other K+ channels.


    METHODS
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
CONCLUSION
APPENDIX A
APPENDIX B
REFERENCES

Molecular biology

The Xba I fragment of pEGFP (Clontech, Palo Alto, CA), containing the complete enhanced green fluorescent protein gene, was inserted into pTRE (Clontech) in the correct orientation to give pTRE-EGFP. This was cut with EcoR I and Age I, and a 33-bp polylinker containing Pac I and Swa I sites

5'AATT CCCCC TTAATTAA CTAG ATTTAAAT CCCA

3' GGGGG AATTAATT GATC TAAATTTA GGGTGGCC

was linked to the sticky ends. The cap-independent translation enhancer (CITE) (500 bp from encephalomyocarditis virus) was amplified by polymerase chain reaction (PCR) (High Fidelity PCR Kit, Boehringer Mannheim, Indianapolis, IN) from pCITE-2a (Novagen, Madison, WI) and inserted in frame into the Age I-Nco I sites. PCR was verified by complete sequencing on both strands. The fragment including the polylinker and CITE-EGFP was then subcloned into the EcoR I-Not I sites of pCITE-2a to give [pCITE-(CITE-EGFP)].

The ROMK1 complete cDNA (Ho et al. 1993) (1.3 kb) plus 0.9 kb of 5' untranslated sequence was excised from pSport (Life Technologies, Gaithersburg, MD) as a single Mlu I fragment and inserted into the BssH II site of pNEB193 (New England Biolabs, Beverly, MA) to give pNEB193-ROMK1; the orientation in which the Pac I site was located on the 3' end of the gene was selected.

To create lentiviral constructs, sequences were cloned into the plasmid pHR' (gift of Didier Trono, The Salk Institute), which contains a human cytomegalovirus (CMV) promoter. The 1.3-kb CITE-EGFP was removed from pCITE-(CITE-EGFP) with BamH I and Xho I and ligated to the corresponding sites of pHR' to give pHR'CITE-EGFP. The EcoR I and Pac I sites on the 5' end of this fragment serve as a unique polylinker in this vector; ROMK1 was inserted into the EcoR I and Pac I sites after removal from pNEB193-ROMK1 to give pHR'ROMK1-CITE-EGFP (Fig. 1A).



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Fig. 1. ROMK1 in the viral plasmid pHR'. 5' and 3'LTR, HIV long terminal repeats; CMV, cytomegalovirus immediate early promoter; UTR, 0.9 kb of ROMK1 untranslated sequence. A: bicistronic vector, ROMK1-CITE-EGFP. B: EGFP-ROMK1 fusion protein. Inset, above: detail of the fusion showing Mlu I site between the last Lys of EGFP and the first Met of ROMK1.

The control construct, encoding for EGFP alone, was constructed by subcloning the Xba I fragment of pEGFP into pBluescript (Stratagene, La Jolla, CA). The gene was then excised with BamH I and Xho I and ligated into the same sites of pHR'.

To generate the EGFP-ROMK1 fusion protein, the EGFP gene minus the final stop codon was amplified from pEGFP by PCR, generating BamH I and Mlu I ends; this was inserted into the corresponding sites of pHR' to give pHR'EGFPnostop. The first 500 bp of ROMK1 was also amplified by PCR, giving an Mlu I-Bgl II fragment. The remaining 1.7 kb was excised from pSport with Bgl II and Xho I, and a three-way ligation between these two fragments and pHR'EGFPnostop (cleaved with Mlu I and Xho I) produced the final construct, pHR'EGFP-ROMK1 (Fig. 1B). All PCR products were verified by complete sequencing, and ligations were verified by restriction digest and partial sequencing.

Generation of lentiviruses

Plasmids were amplified using Maxi and Mega kits from Qiagen (Valencia, CA). The plasmids pHR'ROMKI-CITE-EGFP, pHR'EGFP-ROMK1, pHR'EGFP, or pHR' alone (which encodes LacZ) were cotransfected into 293T cells with the plasmids pMD.G and pDelta R8.9 in the ratios published (Naldini et al. 1996). Transfection was performed in 15-cm tissue culture dishes (Falcon, Oxnard, CA) at 80-90% confluence, using the cationic lipophilic reagents Superfect or Effectene (Qiagen). A total of 20 µg of plasmid DNA was added per 15-cm dish when Superfect was used; a total of 4 or 8 µg was added with Effectene. In the latter case, the ratio of Enhancer to microgram of plasmid DNA was always maintained at 8:1. Supernatant was harvested at 48, 72, and 84 h after transfection and spun at 1,000 g for 5 min to remove cellular debris. It was then passed through a 0.45 micron filter and either subjected to two 15-min spins at 1,000 g in a Centricon-500 unit (Millipore, Bedford, MA) (ultrafiltered virus) or spun for 1.5 h at 4°C in a Beckman ultracentrifuge, using an SW41.1 Ti swinging-bucket rotor at 20,000 rpm (ultraconcentrated virus). Virus from either stock was assayed for infectivity on 293 T or Chinese hamster ovary cells. Ultrafiltered virus averaged 5 × 106 transducing units (TU)/ml, ultraconcentrated, 5 × 107. The former was stored in 1 ml aliquots and the latter in 50 µl aliquots, all at -80°C.

Cell culture

Pregnant Wistar rats were euthanized by inhalation of CO2 at day 18 of gestation. Embryos were immediately removed by caesarian section, and hippocampi rapidly extracted under stereomicroscopic observation under sterile conditions, cut into 1 mm pieces, and digested with 0.25% trypsin and 0.25 mg/ml DNAse (Sigma, St, Louis, MO) at 36°C for 15 min. The pieces were then gently rinsed in Hanks' balanced salt solution without Ca2+ or Mg2+ (HBSS, Life Technologies), washed twice in plating medium, and gently triturated in 1 ml of plating medium with five passes through the 0.78 mm opening of a tip of a P-1000 Pipetman. Suspended cells were removed with a Pasteur pipette, and the remaining pieces triturated once more. The resulting suspensions were gravity-filtered through a 70-µm nylon mesh to remove large debris, and centrifuged for 2 min at 150 g to pellet the cells, which were resuspended by trituration as above. Approximately 35,000 cells were plated in an area 15 mm in diameter at the middle of a 35-mm plastic culture dish that had been coated with poly-D-lysine (PDL) and laminin. Cultures were maintained at 36°C in a 5% CO2 incubator. One half volume of medium was changed twice weekly with culture medium. Plating and feeding medium was Neurobasal with B27 supplement, with 500 µM Glutamax, 25 µM glutamate, and 5% horse serum (Life Technologies); [K+] in this medium is 5.4 mM.

HEK 293 cells were maintained with weekly passages in DME high glucose medium (GIBCO) supplemented with 10% fetal bovine serum, 200 µM glutamine, and penicillin/streptomycin. Transfections were performed with Effectene (Qiagen) in 35-mm dishes according to the manufacturer's instructions.

Infection of hippocampal neurons

Cells were infected 1-3 d after plating by the addition of 20-30 µl of ultraconcentrated or 200-300 µl of ultrafiltered virus to the medium. Viral supernatant was not washed off, although cells continued to be fed on a weekly basis. Stocks of virus were pooled so that each dish in a preparation received the same concentration. For "high K+" cells, supplementary KCl was added to a total concentration of 15.4 mM 12-24 h after infection. At least 40 h were allowed to elapse before assaying for EGFP expression. Control neurons were matched for age (to within 1 d) and for time since application of drugs and/or EGFP-only virus. All controls were from separate dishes; nonfluorescent cells from ROMK1 dishes were never used as controls because of the difficulty of excluding faint fluorescence. Sixteen of thirty-two high K+ controls were EGFP-infected and 16/32 were uninfected; data from these two groups were pooled when no effects of infection were detected on resting membrane potential, spike threshold, input resistance, or response to Ba2+. Similarly, 3/9 low K+ controls were EGFP-infected, 6/9 uninfected, and the data pooled. Data from cells in high versus low K+ were never pooled. When pooled data were averaged, the control and ROMK1 groups contained equal proportions of neurons with identical ages postinfection and postplating. All data presented for ROMK1-infected neurons are from those infected with the bicistronic message; the fusion protein was used only in localization studies and HEK cell recordings, primarily because its fluorescent signal was fainter than that of the cytosolic EGFP. EGFP was visualized for electrophysiology by fluorescence microscopy on a Nikon inverted microscope illuminated by a 100 W Hg lamp, using either a EndowGFP double band-pass filter (exciter 470/40; dichroic 495 LP; emitter 525/50) or a HiQFITC band-pass/longpass filter (exciter 480/40; dichroic 505 LP; emitter 535/50) (Chroma Technologies, Brattleboro, VT). The objective was a 40× air with NA = 0.5 (Nikon, modified by Modulation Optics Inc., Greenvale, NY). Confocal images of fixed specimens stored in phosphate-buffered saline (PBS) were obtained on a Zeiss LSM-510 using a laser tuned to 488 nM for FITC and EGFP and a 40× Plan-Neofluar water immersion lens with NA = 0.9 (Zeiss). Beta-galactosidase was visualized by standard methods after fixation for 2 min in 50/50 methanol/acetone. No viral toxicity was observed with either control construct (EGFP or LacZ).

Whole-cell recording

All recordings were performed at room temperature. For hippocampal neurons, borosilicate Omega -dot glass capillaries (Sutter Instruments, Novato, CA) were pulled to a tip resistance of 5-10 MOmega and filled with either a Mg2+-containing internal solution consisting of (in mM) 100 K-gluconate, 10 HEPES, 3 phosphocreatine, 1.1 EGTA, 3 MgATP, 0.2 NaGTP, 5 MgCl2, 0.1 CaCl2; or a Mg2+-free internal solution, 100 K-gluconate, 10 HEPES, 1.1 EGTA, 0.1 CaCl2, both adjusted to pH 7.2 with KOH and 250 mOsm with sucrose. The bath solution contained (in mM) 110 NaCl, 10 HEPES, 5.4 KCl, 1.8 CaCl2, 0.8 MgCl2, 10 glucose, adjusted to pH 7.4 with NaOH (EK = -76 mV at 25°C). Calculated junction potential for these solutions is 14 mV (Clampex 8.0), and all reported membrane potentials are corrected for this value. Tetrodotoxin (TTX) was bath-applied to a final concentration of 1 µM or perfused at 500 nM; Ba2+ (500 µM), Co2+ (1 mM), and a "hippocampal cocktail" consisting of bicuculline (10 µM), APV (50 µM), and CNQX (20 µM) (all from RBI, Natick, MA) were perfused continually through flow pipes of 250 µm internal diameter mounted ~500 µm from the recorded cell. The dish was washed with 4-6 ml of control saline between recordings. For HEK 293 cells, capillaries were pulled to a tip resistance of 2-5 MOmega and filled with an internal solution containing (in mM) 130 KCl, 0.8 MgCl2, 5 EGTA, 5 MgATP, 10 HEPES (pH to 7.2 with KOH); the bath solution consisted of (in mM) 137 NaCl, 10 HEPES, 4.0 KCl, 1.8 CaCl2, 1.0 MgCl2, 10 glucose, adjusted to pH 7.4 with NaOH (EK = -91 mV at 25°C). Junction potential with this solution is 4.5 mV.

Signals were recorded with an Axopatch 1D amplifier (Axon Instruments, Foster City, CA) and sampled by a Digidata 1200 at 20 kHz (100 kHz for transients) to a Pentium PC. Series resistance was not compensated but was monitored throughout the recording. For I-V analysis, series resistance was compensated off-line (Nadeau and Lester 2000; Traynelis 1998). Current and voltage commands and data acquisition were performed using PCLAMP6.0 and 8.0. Data were analyzed with AxoGraph 3.5 (Axon); individual compiled modules were written using CodeWarrior Pro (Metrowerks Software). Cells were eliminated from the analysis if series resistance changed by more than 20% during the course of the recording, if the cell spontaneously depolarized, or if the membrane capacitance changed in either direction by more than 10%. Data from cells in TTX and cocktail were pooled for many analyses as there were no detectable differences in membrane potential or effects of ROMK1 expression or blockade. Data on threshold and silencing were unavailable for cells with TTX bath application (n = 10 ROMK1 cells, 5 high K+ controls).

Immunocytochemistry

TUNEL staining was performed with the In Situ Cell Death Detection Kit-POD (Boehringer) according to the manufacturer's instructions; the horseradish peroxidase (HRP)-conjugated secondary was developed with nickel-diaminobenzidine reagent and visualized under brightfield. Antibody staining was with polyclonal rabbit anti-ROMK1 (Alomone Labs, Jerusalem, Israel). Cultured cells were fixed for 2 min in methanol/acetone, permeabilized for 2 min on ice with 1% Triton X-100, and preincubated for 30 min in PBS with 5% goat serum. Primary antibody was diluted 1:50 in the same solution and incubated with gentle shaking for 2 h at room temperature or overnight at 4°C. The dish was washed five times and incubated with fluorescent secondary antibody (Cy-3 conjugated goat anti-rabbit, Jackson ImmunoResearch, West Grove, PA) for 60 min at 37°C and visualized with a Texas Red filter (exciter 560/55; dichroic 595 LP; emitter 645/75) (Chroma). Omission of primary antibody led to weak, nonspecific staining. Fluorescence was quantified with NIH Image using confocal images to distinguish membrane-bound from cytoplasmic fluorescence.

Pharmacology

Drugs were added to infected neurons 12-24 h after application of virus. All were dissolved in stock solutions in DMSO in the following concentrations (in mM): 10 BayK 8644 (Calbiochem, La Jolla, CA); 10 nifedipine (ICN Biomedicals, Aurora, OH); 2 thapsigargin (RBI, Natick, MA); 100 8-4(chlorophenylthio)cAMP (cpt-cAMP, Boehringer). Brain-derived neurotrophic factor (BDNF) (Sigma) was dissolved in PBS containing 0.1% bovine serum albumin to 10 µg/ml and stored in aliquots at -20°C.


    RESULTS
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
CONCLUSION
APPENDIX A
APPENDIX B
REFERENCES

Transient transfection of bicistronic and fusion vectors

Transfected into HEK 293 cells, the vectors pHR'ROMK1-CITE-EGFP and pHR'EGFP-ROMK1 produce weakly inwardly rectifying currents that are blockable by 500 µM Ba2+ in a time- and voltage-dependent manner. The block is reversible on Ba2+ wash-out and reverses at EK after leak subtraction (Fig. 2). The bicistronic message produces a strong fluorescent signal throughout the cell, while the fusion protein appears as a weaker, punctate fluorescence mostly directed to the plasma membrane and completely excluding the nucleus (data not shown; see Fig. 6 for distribution in neurons).



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Fig. 2. Detection of ROMK1. A: voltage step protocol, with test potentials between -140 and +60 mV (holding at -80 mV) for the detection of functional ROMK1. B: HEK 293 cell transfected with ROMK1-CITE-EGFP. C: the same cell with constant perfusion of 500 µM Ba2+. D: Ba2+ washout. E and F: a mock-transfected cell with and without Ba2+ showing some nonspecific leak currents. G: mean ± SE IV curves: filled circles, ROMK1 (n = 6, 3 transfected with ROMK1-CITE-EGFP and 3 with the EGFP-ROMK1 fusion protein); filled triangles, controls (n = 5); open squares, difference. When error bars do not appear, they are smaller than the symbols. The total steady-state current in ROMK1 cells reverses at -82 mV; when the control trace is subtracted, Vrev goes to -91 mV, EK in this medium.

Effects ROMK1 on neuronal morphology and survival

Hippocampal neurons infected with control virus, bearing EGFP only, become visibly fluorescent after 24-36 h and increasingly so for several days thereafter. Cell morphology, processes, and underlying glia are unchanged. Infected cells can be maintained and recorded from for up to 6 wk; their electrophysiological properties are identical to those of normal cells (Fig. 3, A and B; Table 2). Hence, there is no viral toxicity detected within the sensitivity of our experiments.



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Fig. 3. Appearance of lentivirally infected hippocampal neurons. Scale bars are 20 µm. A and B: neurons infected with EGFP alone, 14 days in culture (dic), 12 days post infection (dpi). Infected cells are bright under phase contrast and show normal electrophysiological properties. C and D: neurons infected with ROMK1-CITE-GFP, 14 dic, 3 dpi. The one well-labeled neuron is flattened, with cytoplasmic blebbing (white arrow); neurons that appear healthy under phase contrast fluoresce weakly or not at all (red arrow). There are two apparently normal astrocytes with high levels of expression (yellow arrow and lower right). E and F: TUNEL-DAB staining of neurons infected with ROMK1-CITE-EGFP, 24 and 48 h post infection. No apoptotic cells are apparent at 1 d, but nearly all nuclei are labeled by 2 dpi. G: high-power image from the same dish as in F, showing labeled nuclei of apoptotic neurons (arrows). The remains of a ruptured cell, perhaps neuronal, are visible at center bottom. Many of the small, round glia are not labeled. H: at 4 dpi, most of the neurons have disappeared from the dish (14 dic, 4 dpi, unstained). I and J: hippocampal neurons infected with ROMK1-CITE-EGFP, supplemented with KCl to 15.4 mM 12 h after infection; 16 dic, 14 dpi. Many neurons, appearing round in the left panel, are both healthy and green (arrows); just above the neurons is a fluorescent glial cell. The location of the flat glia on a different focal plane than the neurons allows them to be easily distinguished under phase-contrast microscopy.

In contrast, cells infected with ROMK1-CITE-EGFP show no healthy green neurons at 48 h. Expression is limited to astrocytes, possibly activated microglia, and dead or dying cells of varying morphologies. Many dead cells are shrunken and floating, a classic indicator of apoptosis (Gibson 1999) (Fig. 3, C and D). TUNEL staining (Villalba et al. 1997) reveals no apoptotic cells 24 h post infection, but nearly 100% of the neurons are apoptotic at 48-72 h (Fig. 3, E-G). By the fourth to fifth day, few neurons remain in the dish (Fig. 3H). Glial cells can maintain stable infections with ROMK1-CITE-EGFP for weeks or months, as can CHO or HEK-293 cells. The latter may be serially passaged indefinitely without visible change in the proportion of fluorescent cells. Thus cultured hippocampal neurons but neither glia nor clonal cell lines appear susceptible to overexpression of ROMK1.

Channel block by inorganic ions is ineffective at rescuing ROMK1-expressing neurons. Dishes supplemented with 200-500 µM BaCl2 show the same pattern of apoptosis as untreated cultures; however, the blockade at these concentrations affects mainly inward and not outward K+ currents (Ho et al. 1993). Higher concentrations of Ba2+ permit <10% of ROMK1-expressing neurons to survive but are toxic to glia and hence lead to massive deterioration in all cells.

Elevated K+ prevents apoptosis

We tested the hypothesis that K+ loss through ROMK1 causes apoptosis. When the neuronal growth medium K+ concentration is increased to 15.4 from the usual 5.4 mM (high K+), which shifts EK from -79 to -51 mV at 36°C, fluorescence microscopy of ROMK1-CITE-EGFP infected cells at 48-72 h reveals 50-90% green neurons with normal morphology (Fig. 3, I and J). Smaller elevations of K+ levels, to 9.4, 11.4, and 13.4 mM, are insufficient to maintain healthy infected cells (data not shown). Importantly, increased K+ has no visible effect on the fluorescence of EGFP-only cells. ROMK1-expressing neurons can be maintained with weekly feedings of medium containing 15.4 mM K+ for >3 wk. This life span is similar to that of controls in high K+. Thus this elevation of K+ is sufficient to prevent apoptosis and to restore ROMK1-infected neurons to an apparently normal state of health.

The results are consistent with a specific action of K+: activation of apoptotic pathways due to K+ efflux (Yu et al. 1997). However, they may be equally explained by nonspecific trophic actions of chronic depolarization and Ca2+ entry. The use of Ca2+ agonists and antagonists is necessary to distinguish these mechanisms.

The protective effect is specific to K+

Unlike many neuronal cells in culture, hippocampal neurons normally are not dependent on sustained depolarization for survival. They may be grown in media containing as little as 2.5 mM K+ or in 1 µM TTX, a concentration sufficient to block all action potentials (Table 1). On the other hand, they are highly sensitive to excitotoxicity, and increased current through L-type Ca2+ channels is harmful (Porter et al. 1997). It is therefore unlikely that any amount of ROMK1-mediated hyperpolarization and altered membrane conductance could lead to loss of Ca2+ sufficient to cause the rapid, total cell death that we observe. To eliminate this possibility, however, we treated infected dishes with combinations of trophic factors and Ca2+ agents: cpt-cAMP (0.1-0.5 mM); thapsigargin (100 nM), which causes the release of intracellular Ca2+ stores; BayK 8644 (1 µM), an L-type Ca2+ channel agonist; BayK 8644 plus nifedipine (10-100 µM), an L-type antagonist expected to counteract the effects of BayK; glutamate (40 µM); or BDNF (20 ng/ml).


                              
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Table 1. Survival of ROMK1-infected neurons exposed to pharmacological agents

None of these agents are able to maintain ROMK1-infected neurons (Table 1). Neurons in normal K+ exposed to agents that increase intracellular Ca2+ show 100% apoptosis by 48 h, as in untreated cells (Fig. 4A). Neither EGFP-infected controls nor ROMK1 cells in high K+ are harmed by these drugs. Ca2+ antagonists reverse the beneficial effects of high K+ only slightly.



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Fig. 4. Elevation of intracellular Ca2+ does not prevent cell death. A: TUNEL-DAB staining of hippocampal neurons infected with ROMK1-CITE-EGFP and supplemented with 0.5 mM cpt-cAMP, 48 h post infection. Nearly 100% of neurons (black arrows) are shrunken and stained positive; a glial cell (white arrow) is healthy. B: distorted neurons seen 5 dpi in a ROMK1-infected dish supplemented with cpt-cAMP. These cells are detached from the substrate and depolarized.

There is a difference in appearance between the ROMK1-infected cells with and without Ca2+ elevation. In the former case, nearly every neuron in the dish is fluorescent, but their morphologies are highly bizarre; they are completely depolarized, and they detach from the dish (Fig. 4B). In the latter case, the neurons disappear before many distorted forms are seen. This suggests that Ca2+ elevation is acting to prevent phagocytosis by astrocytes or microglia, without reversing the lethal phenotype caused by the channel; this could result either from effects of elevated Ca2+ on the apoptotic process (Bratton et al. 1999) or from direct effects on the glia.

These data point to K+-efflux mediated apoptosis as the cause of cell death in long-term ROMK1 expression. Loss of K+ may impose a metabolic burden on the cell, instead of or in addition to triggering apoptotic pathways; this is a topic for future study.

K+ loss is a critical feature of ROMK1 expression that restricts its applicability for gene therapy or transgenics. However, it may still possess properties of a useful silencing gene, and understanding its effects can lead to improvements in silencing strategies. In addition, outward ROMK1 currents may not occur in all neurons at all ages, especially those subjected to chronic electrical input and depolarization. It is therefore informative to examine the properties of cells maintained in high K+, where ROMK1-infected cells have the same growth patterns, morphology, and life span as matched EGFP-infected controls.

Electrophysiology: Expression levels and localization

Because this study concerns the effects of a new conductance on encoding, an accurate description of membrane parameters is important. Many neurons in culture can be successfully described by a single capacitance, input resistance, and series (pipette) resistance. The resistance of the proximal dendrites is large enough that little attenuation occurs in the signal as it travels to the soma, and the soma resistance is itself large enough to overcome most of the artifacts caused by the series resistance (typical values of input and series resistance are 1000 and 10 MOmega , respectively). However, for cells infected with a K+ channel, this may no longer be the case: input resistance may decrease two- to fivefold, with a corresponding increase in artifacts. We therefore use a two-compartment model to provide better exponential fits and the ability to distinguish somatic conductances from poorly space-clamped conductances on the distal dendrites (APPENDIX B; see also Nadeau and Lester 2000).

ROMK1-infected and control cells were similar in age and size of both the proximal and distal compartments (Table 2); the apparently small size of CD in the low K+ controls is due to a preponderance of young cells in this group [only 2 of 9 were over 7 days in culture (dic)]. The cells are somewhat smaller than in other studies of hippocampal neurons (Mennerick et al. 1995), which primarily reflects our inclusion of young cells, although some stunting due to high K+ may also occur.


                              
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Table 2. Characteristics of cells used in the final data analysis

Blockade by 500 µM Ba2+ was used to quantify expression. The honeybee toxin tertiapin (Jin and Lu 1998) has been proposed as a specific blocker for inward rectifier K+ channels, but it is sensitive to oxidation and its effects on neurons are unknown. Application of fully oxidized tertiapin at a concentration of 2 kD (kD = 7.7 µM) led to increased leak in 2/3 ROMK1 cells and 3/3 controls (not shown). Washout was slow and incomplete. While a stable version of the venom has recently been synthesized (Jin and Lu 1999), its effects on normal neurons will have to be investigated before it becomes a useful tool.

On the other hand, inhibition of the channel by submillimolar Ba2+ is well characterized, rapid, and easily reversible (Choe et al. 1998; Ho et al. 1993). Its only drawback is incompleteness of block at depolarized potentials, following the Woodhull equation
<IT>C</IT>(<IT>V</IT>)<IT> = 1 − </IT><FR><NU><IT>I</IT><SUB><IT>Ba</IT></SUB></NU><DE><IT>I</IT></DE></FR><IT>=1−</IT><FR><NU><IT>1</IT></NU><DE><IT>1+</IT><FR><NU>[<IT>Ba</IT>]</NU><DE><IT>K</IT><SUB><IT>d</IT><IT>0</IT></SUB></DE></FR><IT> exp</IT><FENCE>−<FR><NU><IT>&dgr;</IT><IT>zVF</IT></NU><DE><IT>RT</IT></DE></FR></FENCE></DE></FR>

<IT>I</IT><IT>−</IT><IT>I</IT><SUB>Ba</SUB><IT>=</IT><IT>I</IT><FENCE><IT>1−</IT><FR><NU><IT>1</IT></NU><DE><IT>1+</IT><FR><NU>[<IT>Ba</IT>]</NU><DE><IT>K</IT><SUB><IT>d</IT><IT>0</IT></SUB></DE></FR><IT> exp</IT><FENCE>−<FR><NU><IT>&dgr;</IT><IT>zVF</IT></NU><DE><IT>RT</IT></DE></FR></FENCE></DE></FR></FENCE> (1)
where V represents membrane potential, z is the valence of the blocking ion, delta  is the electrical distance, Kd0 is the required [Ba2+] for half block at V = 0 mV, F is the Faraday constant, R is the gas constant, and T is the absolute temperature (here and elsewhere, the subscript Ba indicates quantities measured in Ba2+). Previously reported values of Kd0 = 10 mM and delta  = 0.41 (Loffler and Hunter 1997) give blocked fractions of 0.85 and 0.25 at -150 and -60 mV, respectively.

Total blocked current at a holding potential of -130 mV (corresponding to a membrane potential of -144 mV) (Fig. 5A) shows an upward trend with time in ROMK1-infected neurons, with levels of expression peaking at 6-7 d and remaining steady thereafter (Fig. 5B). In low K+ controls, this concentration of Ba2+ blocks no native channels, but after several days in high K+ up-regulation of native inwardly rectifying K+ channels (Guo et al. 1997; Knutson et al. 1997) causes high K+ controls to exhibit a smaller but significant Ba2+ block. It is not certain that the up-regulated channels are members of the Kir superfamily, but for convenience, we shall abbreviate them as EIRKs, for "endogenous inwardly rectifying K+ " channels. We show (APPENDIX A, Fig. 8) that they display strong inward rectification. Levels of this channel(s) remain essentially constant over the period studied.



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Fig. 5. Development of Ba2+-blockable currents in ROMK1 cells and controls. A: voltage clamp currents for a step from -80 to -130 mV (corresponding to -94 to -144 mV with junction potential correction). There is a large current blocked at this voltage for a ROMK1-infected cell (top); a smaller current in high K+ controls (EGFP infected) after 5 d in 15.4 mM K+ (second from top), after 14 d (third), and after 21 d (bottom). The lowest trace is before Ba2+; all upper traces are in continuous Ba2+ perfusion. In the upper 3 images, the two traces are <3 s apart. The bottom trace illustrates the slow, stepwise block that occurs in a fraction of high K+ controls at these late time points; it is accompanied by substantial membrane hyperpolarization (15 mV for the cell illustrated) (see discussion of RMP change, in text, and APPENDIX A for details). B: currents and Ba2+-blockable fraction at -144 mV in control and ROMK1 cells (n = 6-15 for each point). Ba2+ block reaches a plateau in ROMK1 cells at 5-6 d (solid circles), but total currents (I144) peak at ~12 d (solid squares), representing similar reactions to high K+ as in control cells (open squares). The apparent sharp increase in currents in controls results from 2 cells and is not statistically significant. Blockable current in controls remains roughly constant in controls (open circles) as long as the Ba2+ application is rapid (on the order of ms); after 2-3 wk in high K+, more slowly blocked currents also appear (dotted line, filled diamonds).

With Mg2+-free internal solution, the mean current at this voltage is 1.75-fold that is seen with internal solution containing Mg2+, ATP, and GTP (n = 5 for Mg2+-free cells, P < 0.15, t-test), consistent with suppression of ROMK1 by cytoplasmic ATP (Ho 1993). If blockable currents in control cells are subtracted from those in ROMK1 neurons (this slightly underestimates the amount of ROMK1 expressed, see APPENDIX A), the resulting average expression level is approximately one quarter of that previously measured with adenovirus-mediated G-protein-activated inward rectifier K+ channel expression (Ehrengruber et al. 1997). The maximum level in the present experiments equals the mean of 40 pA/pF as reported by Ehrengruber et al.; a level this high represents an unusual situation for a retrovirus and is probably due to positional effects (Schubeler et al. 1998). However, even with retroviral vectors, current densities of >12 pA/pF were not unusual, representing 11% of all infected cells.

We identify a subgroup of infected neurons that do not fire action potentials at the maximum tested level of current injection (200 pA), but exhibit normal firing patterns after blockade by Ba2+. These are designated "completely silenced cells" and show significantly greater blockable currents than the mean of all ROMK1-infected neurons; these cells illustrate the effects of the maximal level of expression achievable with lentiviral transduction.

Localization was investigated by antibody staining, confocal imaging of EGFP-ROMK1 fusion expression, and two-compartment calculation of currents (Fig. 6). No specific localization pattern was identified, consistent with simple diffusion of proteins throughout the cell.



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Fig. 6. Localization of ROMK1 in infected neurons. A: confocal image of cells 14 dic, 3 dpi with EGFP-ROMK1 fusion protein containing the 3' UTR. The channel is clearly targeted to the membrane, and somatic staining is very bright. Dendritic staining is also present. This pattern agrees with that seen with antibody staining (not shown). Because each channel is associated with one GFP molecule, relationship of functional channel to fluorescence is quantitative. B: membrane fluorescence shown as a function of distance from the soma for 3 representative cells. Fluorescence was quantified throughout the cell body and the length of the processes by tracing individual dendrites (dashed lines in A); care was taken to clearly identify each fluorescent process with its parent neuron. A value of 255 represents no fluorescence. Integrated total fluorescence shows little variation from cell to cell: 81 ± 2% within 1 µm of the soma, n = 5. C: the time course of the conductance change reveals its localization (see APPENDIX B). Plots show the time course of the current block that occurs with Ba2+ wash-in (block is defined as the difference of currents before and after Ba2+). Voltage step is from -94 to -104 mV. The plot labeled "ROMK1" is the average of 7 neurons with mean block of 30.5 pA; "control" represents 1 cell with the largest block observed in EGFP-only neurons. Simulated cells with the same passive membrane properties illustrate the time courses expected if the conductance is purely somatic (all soma, solid line); purely dendritic (all dendrite, dashed line); or a mixture of the two (50/50, dots). Block in ROMK1 neurons is consistent with a predominantly somatic conductance, while the control neuron shows a mixed pattern.

Effects of ROMK1 on encoding properties: Change in RMP with Ba2+

Full I-V relations with and without Ba2+ demonstrate voltage dependent block in both ROMK1 and control neurons (Fig. 7). While EIRK complicates the picture at hyperpolarized potentials, blockable currents near typical RMP values of -60 to -65 mV are due almost entirely to ROMK1, giving an I-V similar to that seen in HEK cells (Figs. 7E and 8). Therefore, a significant change in RMP is expected in expressing cells with the addition of Ba2+, but not in controls. This depolarization can be predicted, with two assumptions: that Na+ and K+ are the only ions contributing to RMP, and that Na+ conductance does not change with Ba2+. Then
&Dgr;<IT>V<SUB>m</SUB></IT><IT>=</IT><FR><NU><IT>G<SUB>K</SUB></IT><IT>−</IT><IT>G</IT><SUB><IT>KBa</IT></SUB></NU><DE><IT>G</IT><SUB><IT>TOT</IT></SUB></DE></FR> (<IT>E<SUB>K</SUB></IT><IT>−</IT><IT>V</IT><SUB><IT>m</IT><IT>Ba</IT></SUB>)<IT>C</IT>(<IT>V<SUB>m</SUB></IT>)<IT>=</IT><FR><NU><IT>G</IT><SUB><IT>ROM</IT></SUB></NU><DE><IT>G</IT><SUB><IT>TOT</IT></SUB></DE></FR> (<IT>E<SUB>K</SUB></IT><IT>−</IT><IT>V</IT><SUB><IT>m</IT><IT>Ba</IT></SUB>)<IT>C</IT>(<IT>V<SUB>m</SUB></IT>) (2)
where Delta Vm is the shift in RMP, EK is the K+ reversal potential, GK represents K+ conductance, and GTOT is the total conductance, and C(Vm) is the Woodhull equation Eq. 1 giving the fractional Ba2+ block at RMP. Using this formula with values of C(V)GROM obtained from block at -55 mV predicts Delta Vm = -8.2 ± 0.4 for all cells <5 dpi. The observed value in this group is -8.4 ± 0.5 mV (n = 6), giving excellent agreement between the observed and theoretical values. All cells in this age group show a change in RMP; the minimum and maximum are -7 and -10 mV, respectively. Control cells do not change by more than 1 mV in either direction (n = 6; -1 ± 1 mV, mean ± SE). Ba2+ blockade restores RMP of ROMK1 neurons to the level seen in low K+, rather than high K+, controls (Table 2). This suggests that less EIRK is present in ROMK1-expressing neurons than in controls (discussed in detail in APPENDIX A).



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Fig. 7. Detection of ROMK1 in neurons. Representative traces and mean ± SE IV curves for the voltage protocol as in Fig. 2, before (left) and during (center) application of Ba2+. The right traces are after Ba2+ washout. A: a ROMK1-infected neuron, showing a large Ba2+-blockable conductance at negative potentials. B: a ROMK1 neuron with 1 µM TTX in the bath, again showing the blocked conductance that is restored with Ba2+ washout. C: an EGFP high K+ control, showing little current at negative potentials and little Ba2+ block. D: a high K+ control in 1 µM TTX. E: mean ± SE of Ba2+ blockable currents in high K+ controls (filled triangles, n = 10, cells >= 6 and <18 dpi only), ROMK1 neurons (filled circles, n = 20, cells >= 6 and <18 dpi only), and the difference (open squares). See APPENDIX A for a discussion of the Na+ current seen in TTX. In neuron recording medium, Erev = -72 mV and EK = -76 mV. The control current reverses at -45 mV, and subtracting this from the ROMK1 trace changes Erev only slightly, to -75 mV; this indicates that the control currents are not all nonspecific leak, but contain a K+-specific component. In controls >= 18 dpi, the reversal potential shifts to -59 mV (not shown). The deviation of Erev from EK at these later times results from Na+ permeable channels that are blocked by Ba2+ (see APPENDIX A).



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Fig. 8. There is little EIRK conductance at threshold potentials. The slope of the change in membrane potential Vm in response to current injections Iinj gives the neuron's input resistance; in a representative ROMK1 cell (filled circles); this increases with the addition of Ba2+ (filled squares), but not to control levels due to the incompleteness of the block. Fit equations: no Ba2+, Vm = -70.458 + 0.098545Iinj; Ba2+, Vm = -64.595 + 0.15764Iinj, R2 = 0.99 for both. In a control cell with the maximum observed EIRK current (105 pA of Ba2+-blockable current at -144 mV), the resistance remains essentially constant in Ba2+ (open circles, no Ba2+; open squares, Ba2+) throughout the range of voltages from -75 to -20 mV.

With increasing periods, postinfection changes in RMP become smaller, even in cells expressing 20-40 pA/pF of Ba2+ blockable current at hyperpolarized potentials. At 13 dpi, average Delta Vm has fallen to -5.1 ± 1.0 mV (n = 12), even as ROMK1 expression has increased (Fig. 5B). There are two identifiable reasons for this. First, VmBa is hyperpolarized by 1 mV, from -58 ± 1 to -59 ± 2 mV, making it closer to EK; the predicted change has now decreased slightly, to -7.0 ± 1.6 mV.

In addition, there is now a discrepancy between the predicted and observed values. Its origin is a hyperpolarization resulting from Ba2+ wash-in in neurons in long-term high K+; this means that our assumption that GNa does not change with Ba2+ is no longer correct. At 13 dpi, the mean in controls is 1.5 ± 1 mV, n = 10. Subtracting this from the mean given by Eq. 2 gives an adjusted value of -5.5 ± 1.9 mV, well in line with the observations.

By 18-21 dic, average RMP change in ROMK1 cells has fallen to -2.4 ± 0.8 mV (n = 12). This can be almost entirely explained by proximity of VmBa to EK: cells are now hyperpolarized to -66 ± 1 mV, and the predicted change is -3.5 ± 0.6 mV. The discrepancy of 1.8 ± 0.8 mV results from 4/12 cells that show no depolarization or even hyperpolarization in Ba2+ (2 cells with hyperpolarization, 1.9 and 3.2 mV).

In controls (n = 7), hyperpolarization is also seen only in a subset of cells, always accompanied by the slow, stepwise block shown in the last panel of Fig. 5A. Mean change in RMP is 4.66 ± 2.2 mV, range -1.2 to 15 mV, with no hyperpolarization seen in two cells and >= 10 mV in two cells. This may reflect different subpopulations of neurons with differing responses to elevated K+ and is a topic for future study. The degree of hyperpolarization does not appear to correlate with level of ROMK1 expression, but the numbers of neurons displaying this phenomenon are too small for meaningful statistics.

These compensatory channels may explain the extended lifetime of infected cells washed back into normal K+ after >7 dpi: 4-5 d versus <48 h for cells infected in low K+. However, despite the disappearance of membrane hyperpolarization, silencing is not eliminated: the effects of the channel on excitability increase with increasing levels of ROMK1 expression and are only partially dependent on RMP.

Three changes are responsible for silencing

In the cells we studied, spiking elicited by current injection shows a pattern typical of hippocampal neurons (Fig. 9A). There is a definite threshold potential for the occurrence of spikes (threshold was taken to be the point at which the time derivative of the current exceeded 10 V/s). In most cells, accommodation of firing rates occurs during a depolarization lasting 800 ms. Higher currents increase the firing rate, but beyond a maximum level of ~20 Hz, spikes broaden and firing rates decrease. Plots of spike frequency versus current injection reveal that the addition of Ba2+ to infected cells changes the threshold of spike response without affecting the overall shape of the frequency-versus-current relation (Fig. 9B). We define this shift, with the dimensions of pA, as the measure of "silencing," S. 



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Fig. 9. Excitability changes identified by action potential analysis. A: spiking in response to current injection. Left: the upper trace shows a ROMK1-infected cell subjected to a series of depolarizing current steps in 20 pA intervals, from -20 to 160 pA. Lower trace: the same cell during application of Ba2+; the membrane potential is slightly depolarized, the input resistance is increased, and a classic pattern of spiking is displayed. Right: control cells show very little difference without (above) and with (below) Ba2+ perfusion. Infected cells display reduced input resistance with and without Ba2+, as evidenced by smaller changes in membrane potential between each 20 pA step as compared with controls. B: spike frequency versus current injected for 2 representative ROMK1 cells and a high K+, EGFP-infected control. Cell 1 (green, circles) shows a similarly shaped spike-versus-current curve with (open symbols) and without (solid symbols) perfusion of 500 µM Ba2+. The shift in the curve is our definition of silencing, S. Cell 2 (red, squares) fails to fire without Ba2+ even at the highest level of current injected (200 pA) and is designated a completely silenced cell. The value of S for such a cell can be estimated by calculating the expected threshold based on the membrane potential and input resistance (see RESULTS). The control cell (black, dashed lines) shows little change in the firing peak with (open diamonds) and without (filled diamonds) Ba2+ perfusion; mean S < 0 pA for controls (see Fig. 11).

S can be broken down into three components, indicating the amount of current necessary to overcome the ROMK1-induced hyperpolarization Delta Vm = Vm - VmBa, the increased membrane conductance Delta G = G - GBa, and the change in threshold Delta T = T - TBa
<IT>S</IT><IT>=</IT>(<IT>T</IT><SUB><IT>Ba</IT></SUB><IT>−</IT><IT>V</IT><SUB><IT>m</IT><IT>Ba</IT></SUB>)(<IT>&Dgr;</IT><IT>G</IT>)<IT>+</IT><IT>G</IT>(−<IT>&Dgr;</IT><IT>V<SUB>m</SUB></IT><IT>+&Dgr;</IT><IT>T</IT>) (3)
(Fig. 10). Each of these contributions to the curve shift can be determined from a Hodgkin-Huxley (HH) model; the only necessary measurements are cell conductance with and without Ba2+ (corresponding to the amount of ROMK1 present), RMP, and spike threshold in Ba2+. Then the G's in Eq. 3 are known; the RMP shift is given by Eq. 2, and the threshold change results from the number of extra Na+ channels that will have to open to compensate for the increase in GK. If the Na+ conductance is assumed to exhibit a voltage dependence of the form (Hille 1992)
<IT>G</IT><SUB><IT>Na</IT></SUB>(<IT>V</IT><IT>+&Dgr;</IT><IT>V</IT>)<IT>=</IT><IT>G</IT><SUB><IT>Na</IT></SUB>(<IT>V</IT>)<IT> exp</IT>(<IT>&Dgr;</IT><IT>V</IT><IT>/3.9</IT>) (4)
then the extra needed Na+ conductance will balance a new K+ conductance when
&Dgr;<IT>G<SUB>K</SUB></IT><IT>=</IT><IT>G</IT><SUB><IT>Na</IT></SUB>(<IT>V</IT><SUB><IT>T</IT><IT>Ba</IT></SUB>)<IT> exp</IT>(<IT>&Dgr;</IT><IT>T</IT><IT>/3.9</IT>)<IT>−1</IT>

&Dgr;<IT>T</IT><IT>=3.9 ln </IT><FENCE><FR><NU><IT>&Dgr;</IT><IT>G</IT><SUB><IT>K</IT></SUB></NU><DE><IT>G</IT><SUB><IT>Na</IT></SUB>(<IT>V</IT><SUB><IT>T</IT><IT>Ba</IT></SUB>)</DE></FR><IT>+1</IT></FENCE><IT>=3.9 ln </IT><FENCE><FR><NU><IT>G</IT><SUB><IT>ROM</IT></SUB></NU><DE><IT>G</IT><SUB><IT>Na</IT></SUB>(<IT>V</IT><SUB><IT>T</IT><IT>Ba</IT></SUB>)</DE></FR><IT>+1</IT></FENCE><IT>=3.9 ln </IT><FENCE><FR><NU><IT>2</IT><IT>G</IT><SUB><IT>ROM</IT></SUB></NU><DE><IT>G</IT>(<IT>V</IT><SUB><IT>T</IT><IT>Ba</IT></SUB>)</DE></FR><IT>+1</IT></FENCE> (5)
since at threshold GNa = GK. Equation 5 predicts the observed threshold change very well for a majority of neurons: for ROMK1 cells <18 dic that fire both before and after Ba2+ (n = 30), a least-squares fit to the data gives Eq. 5 with a slope of 3.80 rather than 3.9. Beyond this age, compensatory channels confound the picture, giving a greater Na+ channel conductance than that predicted by the HH equations (see APPENDIX A). Observed spike thresholds are -50 ± 2 mV for ROMK1 cells, -53 ± 1 mV for ROMK1 cells in Ba2+, and -59 ± 3 mV for high K+ control cells. This change corresponds to a Ba2+-induced conductance decrease of approximately 50%.



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Fig. 10. Addition of a new conductance affects excitability. In a normal neuron (left), the current I required to induce firing is a product of the whole-cell conductance G and the voltage difference V between RMP Vm and spike threshold T. Right: a ROMK1-infected cell must overcome 3 factors to fire: RMP is now a more hyperpolarized V'm; spike threshold is at a higher value T'; and the whole-cell conductance is increased, so that more current is needed to change Vm.

Since cells must fire both with and without Ba2+ to yield values of S, the completely silenced cells are excluded from this analysis. Nevertheless, even neurons expressing an average amount of ROMK1 have significant Ba2+blockable conductance at depolarized potentials, and the effects on firing are well predicted by this simple ohmic model (Fig. 11).



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Fig. 11. Theoretical prediction for S based on Hodgkin-Huxley models. A: the straight line is the theoretical value based on calculated change in threshold; the filled circles are ROMK1-infected cells of all ages that fire with and without Ba2+ (n = 38); the open diamonds are controls (n = 16); and the open squares are extrapolations for completely silenced cells (n = 9). The crossed squares give the minimum S possible for the completely silenced cells, based on the assumption that they will fire at the very next level. For ROMK1 cells, S/Spredicted = 0.72 ± 0.12; mean ± SE observed, 29.4 ± 4.2 pA; predicted based on observed threshold change, 36.7 ± 6.4; based on theoretical threshold change, 36.1 ± 5.8. For controls, mean ± SE observed, -10 ± 7 pA; predicted based on observed conductance change, -5 ± 5 pA; predicted based on theoretical threshold change, 17 ± 11 pA. B: total S in pA plotted versus Ba2+-blockable conductance. Experimental values for ROMK1 cells(filled circles) and controls (filled triangles), theoretical values for ROMK1 cells (open circles) and controls (open triangles), and theoretical extrapolations for completely silenced cells (gray diamonds). C: standardized residuals of (B) plotted against predicted S (left) and Ba2+-blockable conductance (right). Negative values indicate that S is overestimated. Many values cluster near 0, but for the highest values of both predicted S and conductance change, large discrepancies occur. This is because Na+ permeable channels make up a significant fraction of Ba2+-blockable current in some cells (see APPENDIX A).


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
CONCLUSION
APPENDIX A
APPENDIX B
REFERENCES

K+ channels have been proposed as candidates for transgenesis and gene therapy, because expression of several different types has been shown to decrease excitability in cultured neurons (Ehrengruber et al. 1997; Johns et al. 1999). In all of these experiments, however, the channels were active only in the presence of agonist or inducer. Expression was activated during or immediately before electrophysiological recording, so that long-term health effects of the gene or neuronal compensatory mechanisms could not be identified. Furthermore, adenovirus expression levels are many times those seen in transgenic animals; it is unknown whether a small change in K+ conductance will have equally profound effects. In fact, mice transgenic for Shaker (AKv1.1a) (Sutherland et al. 1999) show a hyperexcitable phenotype, with spontaneous EEG discharges and lowered seizure thresholds, in apparent contradiction to the above results and underlining the complexity of the problem.

We begin to address some of these issues by examining chronic, unopposed K+ conductance expressed at low levels from a viral vector with minimal toxicity. The weak inward rectifier ROMK1 results in apoptosis in 100% of dissociated hippocampal neurons, independent of age. Cell death cannot reliably be prevented by any pharmacological agents other than K+ that we have studied. Ca2+ channel agents and growth factors can block apoptosis, but the resulting neurons are depolarized and morphologically abnormal. Accordingly, K+ rescue is not antagonized by Ca2+ channel blockers.

Although increasing K+ to 15.4 mM is sufficient to permit survival of nearly all ROMK1-expressing cells, this manipulation has profound effects, especially after prolonged times. EGFP-infected and uninfected cells up-regulate native inward rectifiers, which we have termed EIRKs in this paper, so that they are hyperpolarized by 5 ± 2 mV when recorded in normal K+ medium. They also show a threefold increase in current at -144 mV and a slightly more than twofold decrease in input resistance at -94 mV relative to neurons in normal K+. After more than 2 wk in culture, additional channels begin to appear, some of which (a) are permeable to Na+ and (b) lead to hyperpolarization on Ba2+ application (details in APPENDIX A).

Application of 500 µM Ba2+ leads to block of EIRK and ROMK1 at hyperpolarized potentials, while at more depolarized potentials near threshold, the EIRK conductance disappears and a smaller fraction of ROMK1 is blocked. However, the blockade is sufficient to restore the membrane potential, spike threshold, and whole-cell conductance of ROMK1 expressing cells to near control values while they are in Ba2+.

Observed reduction in excitability is essentially ohmic and is due to three factors: decreased membrane resistance near RMP (1.5- to 5-fold), raised action potential threshold (2.6 ± 0.5 mV), and membrane hyperpolarization (7 ± 2 mV for all ROMK1 cells relative to precisely age-matched controls). The latter two factors are directly related to the change in resistance, identifying a single factor that is necessary for silencing and that can be used to predict neuronal response to any foreign channel.

The observed results confirm that ROMK1 is functional in the infected neurons studied and that changes in resting excitability are due to a Ba2+-blockable K+ conductance. The predictability of these changes, and the normal firing patterns of infected cells in Ba2+ even after weeks of infection, suggest that nonspecific metabolic effects of the channel are minimal in 15.4 mM K+. As few as 150 open ROMK1 channels can result in silencing of 20 pA, while higher numbers (up to 1,000) lead to neurons that cannot fire in the absence of Ba2+.


    CONCLUSION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
CONCLUSION
APPENDIX A
APPENDIX B
REFERENCES

What is the future of K+ channels as silencing agents? Our results confirm previous acute experiments showing hypoexcitability in response to a foreign K+ channel, but underline the importance of long-term expression to identify adverse affects that may occur in transgenic animals or in gene therapy. A transgenic silencing experiment may be regulated by means of inducible promoters with adjustable levels of induction, such as the tetO-CMV promoter (Huang et al. 1999) and ideally would be fully reversible. However, the time scale of induction and reversal with even the best genetic systems is days to weeks (Chen et al. 1998), several times longer than the life span of neurons expressing even the lowest levels of ROMK1. This channel therefore does not provide an alternative to all-or-nothing lesions or knockouts, at least in adult neurons.

A weak inward rectifier was chosen for these experiments partially because of its expected ability to hyperpolarize neurons with outward current, but the demonstrated minor role of membrane potential in silencing suggests that this type of channel is not the best. A strong inward rectifier may provide all of the silencing with less or none of the K+ loss; experiments to test long-term expression of such channels are therefore the next step.

There are also neuronal subtypes and developmental stages that require high K+ medium in dissociated culture, and analogously, high levels of electrical input in vivo: cerebellar granule cells (Galli et al. 1995) and retinal cells (Araki et al. 1995) are prime examples. As long as these depolarizing conditions exist, such cells may be silenced but not killed by the presence of ROMK1, as are hippocampal neurons in high K+. The channel may therefore be a useful tool for studying the dependence of neuronal migration and differentiation on excitability. Whether cells that require elevated K+ are able to survive ROMK1 expression in vitro may be easily tested before carrying out transgenic experiments. The increasingly understood link between apoptotic pathways and K+ loss (Padmanabhan et al. 1999; Pike et al. 1996) may also make ROMK1 an excellent model of neurodegeneration.

Finally, our electrophysiological results identify the factors important for electrical silencing and suggest alternative methods of achieving this goal. K+ may be too intimately connected with the cell cycle to allow its balance to be altered, but any ion channel that doubles a neuron's input conductance will be a significant silencing agent, as long as its reversal potential is more negative than RMP. An example would be Cl- channels, which play important inhibitory roles in vertebrate and invertebrate nervous systems.


    APPENDIX A: Effects of high K+ culture conditions
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
CONCLUSION
APPENDIX A
APPENDIX B
REFERENCES

A variety of new or up-regulated channels and functional changes were identified in hippocampal neurons in response to chronic application of 15.4 mM K+. Apart from EIRK, these effects do not alter our analysis of ROMK1, as they occur to the same extent in control and ROMK1 neurons. Nevertheless, they are important for situations in which these cells may be exposed to depolarizing conditions.

Synaptogenesis, cation channels, and TTX-insensitive Na+ current

A lack of synaptogenesis due to prolonged elevation of K+ has been seen in neocortical neurons (Baker et al. 1991). We observe a similar effect here, where no postsynaptic potentials or currents are resolvable in high K+ control cells even after 14-21 dic. An identical picture occurs in the ROMK1 cells, not reversed by Ba2+ application (Fig. A1). The mechanisms responsible for this synaptic silence may be different in the two cases: in the ROMK1 cells, high-input conductance may shunt synaptic input. However, effects of ROMK1 on synapses are experimentally inaccessible under these conditions.



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Fig. A1. Elevated K+ disrupts synapse formation. 10-s voltage-clamp traces (Vhold = -74 mV) showing (A) normal distribution of synaptic currents in a hippocampal neuron 14 dic; B: complete suppression of currents in an uninfected neuron 15 dic, switched from 5.4 to 15.4 mM K+ at 1 dic; C: a similar lack of events in a ROMK1-infected cell, before and after perfusion of Ba2+.

The neurons also show the development of at least one Ba2+-blockable nonspecific or cation current, so that by 13 dic, Ba2+ wash-in leads to membrane hyperpolarization. The effect of Ba2+ on neurons in long-term high K+ is complex and follows at least two separate time courses (see Fig. 5A): a rapid hyperpolarization, followed by a much slower increase in membrane resistance at voltages near threshold (more positive than -60 mV). On Ba2+ washout, the membrane potential recovers rapidly, but the resistance remains at this higher value throughout the time courses observed (1-2 min). There is no apparent difference between ROMK1-infected cells and controls.

Additionally, both the ROMK1 and high K+ control neurons show a TTX-insensitive Na+ conductance that becomes apparent with the application of 500 µM Ba2+ (Fig. 7, B and D); this has also been noted in brainstem motor neurons raised in elevated K+ (Eustache and Gueritaud 1995). The conductance is not altered by application of Co2+ and is therefore not Ca2+ dependent. It is slightly but not significantly larger in high K+ controls (38 ± 6 pA/pF, n = 4, occurrence in 4/5 cells in TTX) than in ROMK1 cells (25.4 ± 3.7 pA/pF, n = 9; occurrence in 9/10 cells in TTX; P = 0.2). This indicates that it is a response to the elevated K+, not to ROMK1, and may in fact occur to a slightly lesser degree in the latter. Nevertheless, ROMK1 cells and not controls are able to fire spikes in the presence of 1 µM TTX, 1 mM Co2+, and Ba2+ (Fig. A2).



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Fig. A2. High K+ affects Na+ channels. A ROMK1 infected hippocampal neuron, 13 dic, 12 dpi, showing response to current injections in the presence of 1 µM TTX and 1 mM Co2+(left), and 1 µM TTX, 1 mM Co2+, and 500 µM Ba2+ (right). Very little change is seen in control traces on Ba2+ application, and spiking is never present (not shown).

EIRK up-regulation: Less in ROMK1 neurons

The only adaptive change that differed between ROMK1 and control neurons involved the inwardly rectifying K+ conductance referred to in this paper as EIRK. As noted in the discussion of RMP, Ba2+ blockade of ROMK1 restores infected neurons to a state more like that of low K+ controls than high K+ controls. So is it correct to subtract the high K+ control I-V from that of the ROMK1 cells to quantify expression levels at very negative potentials, or is all current in ROMK1 cells due to ROMK1?

ROMK1-expressing neurons will not necessarily up-regulate EIRK as do controls, because ROMK1 prevents the hyperexcitability caused by high K+ that presumably potentiates development of native inward rectifiers (Fig. A3). It is possible to distinguish the two types of conductance by their sensitivity to Ba2+ at -144 mV. This will provide an estimate of the percentage of Ba2+-blockable conductance that is due to EIRKs in ROMK1 cells.



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Fig. A3. ROMK1 suppresses some effects of high K+. Hippocampal neurons 18 dic, 5 d after application of virus and supplemental K+. Left: control high K+ cells show unstable RMP and spontaneous firing. Right: this phenotype is suppressed to a large degree in a ROMK1-infected cell, but application of Ba2+ (below) leads to uncontrollable spiking as in the control case.

Examination of the currents at -144 mV (Tables 2 and A1) reveals that the Ba2+-blockable current in completely silenced cells is significantly greater than in all ROMK1 cells, but the residual (nonblockable) current is not. Further, the percentage of current that is blockable increases with increasing expression of ROMK1. This suggests that the fractional block of EIRK differs from that of ROMK1, and that as ROMK1 expression levels increase, the percentage of current due to EIRKs decreases proportionately. If the current at -144 mV in a high K+ control is assumed to be made up of IEIRK and a nonblockable part I0, then the fraction blocked is given by
<IT>I</IT><IT>144−</IT><IT>I</IT><IT>144</IT>(<IT>Ba</IT>)<IT>≡&bgr;</IT><IT>I</IT><SUB><IT>EIRK</IT></SUB>

<FR><NU><IT>I</IT><IT>144−</IT><IT>I</IT><IT>144</IT>(<IT>Ba</IT>)</NU><DE><IT>I</IT><IT>144−</IT><IT>I</IT><SUB><IT>0</IT></SUB></DE></FR><IT>=&bgr;</IT> (A1)
using the value of I0 from the low K+ controls, -78 ± 5 pA, gives beta  = 0.32 ± 0.12. Then the currents in ROMK1-infected cells are
<IT>I</IT><IT>144=</IT><IT>I</IT><SUB><IT>0</IT></SUB><IT>+</IT><IT>I</IT><SUB><IT>EIRK</IT></SUB><IT>+</IT><IT>I</IT><SUB><IT>ROM</IT></SUB>

<IT>I</IT><SUB><IT>ROM</IT></SUB><IT>=</IT><FR><NU><IT>I</IT><IT>144−</IT><IT>I</IT><IT>144</IT>(<IT>Ba</IT>)<IT>−&bgr;</IT>(<IT>I</IT><IT>144−</IT><IT>I</IT><SUB><IT>0</IT></SUB>)</NU><DE><IT>&agr;−&bgr;</IT></DE></FR> (A2)
where alpha  is the fraction of ROMK1 blocked at -144 mV. Using Kd0 = 10 mM and delta  = 0.41 gives alpha  = 0.82, yielding estimated EIRK ± SE currents for control, all ROMK1, and completely silenced cells of 175 ± 34, 122 ± 37, and 60 ± 30, respectively (P < 0.002 for control and all ROMK1 distributions; P < 0.0001 for controls and completely silenced cells). This validates the hypothesis that sufficient ROMK1 may prevent EIRK expression, but in the majority of cells, the effect is only partial. While not crucial to a study of silencing, this analysis is important because it indicates that ROMK1 expression occurs early enough and strongly enough to alter the neurons' compensatory behavior.


                              
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Table A1. Ba2+-sensitive current and its effect on membrane properties


    APPENDIX B: Two-compartment localization of currents
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
CONCLUSION
APPENDIX A
APPENDIX B
REFERENCES

Electrophysiological data from all cultured neurons in our experiments, regardless of age, could be fit to a semi-empirical model with five experimentally measured parameters that describe the time dependence of the current in response to a voltage step V0
<IT>I</IT>(<IT>t</IT>)<IT>=</IT><IT>I</IT><SUB><IT>ss</IT></SUB><IT>+</IT><IT>A</IT><SUB><IT>1</IT></SUB><IT> exp</IT>(−<IT>t</IT><IT>/&tgr;<SUB>1</SUB></IT>)<IT>+</IT><IT>A</IT><SUB><IT>2</IT></SUB><IT> exp</IT>(−<IT>t</IT><IT>/&tgr;<SUB>2</SUB></IT>) (B1)
where Iss is the steady-state current; A1 and tau 1 are the amplitude and time constant of the faster component, and A2 and tau 2 are the amplitude and time constant of the slower component. Such a model fits the observed data to within experimental accuracy; a single exponential is insufficient, while the addition of other parameters does not improve the fit.

A current of this form is consistent with a rapidly charging cell body and distal dendrites that charge more slowly (details in Nadeau and Lester 2000). The dendrites are separated from the soma by a resistance RC = 175 ± 25 MOmega for all ROMK1 cells, and this resistance is assumed to be a property of the cell's anatomy that does not change on Ba2+ wash-in. The voltage at the soma as a function of time is then given by
<IT>V</IT><SUB><IT>soma</IT></SUB>(<IT>t</IT>)<IT>=</IT><IT>V</IT><SUB><IT>0</IT></SUB><IT>−</IT><IT>R<SUB>S</SUB>i</IT><SUB><IT>ss</IT></SUB><IT>−</IT><IT>R<SUB>S</SUB>A</IT><SUB><IT>1</IT></SUB><IT> exp</IT>(−<IT>t</IT><IT>/&tgr;<SUB>1</SUB></IT>)<IT>−</IT><IT>R<SUB>S</SUB>A</IT><SUB><IT>2</IT></SUB><IT> exp</IT>(−<IT>t</IT><IT>/&tgr;<SUB>2</SUB></IT>) (B2)
while at the dendrites the charging is delayed
<IT>V</IT><SUB><IT>dend</IT></SUB>(<IT>t</IT>)<IT>=</IT><FR><NU><IT>1</IT></NU><DE><IT>1+</IT>(<IT>R<SUB>C</SUB></IT><IT>/</IT><IT>R<SUB>D</SUB></IT>)</DE></FR> <FENCE><IT>V</IT><SUB><IT>0</IT></SUB><IT>−</IT><IT>R<SUB>S</SUB>i</IT><SUB>ss</SUB><IT>−</IT><FENCE><FR><NU><IT>R<SUB>S</SUB>A</IT><SUB><IT>1</IT></SUB></NU><DE><IT>1−</IT><FR><NU><IT>&agr;</IT></NU><DE><IT>&tgr;<SUB>1</SUB></IT></DE></FR></DE></FR></FENCE><IT> exp</IT>(−<IT>t</IT><IT>/&tgr;<SUB>1</SUB></IT>)<IT>−</IT><FENCE><FR><NU><IT>R<SUB>S</SUB>A</IT><SUB><IT>2</IT></SUB></NU><DE><IT>1−</IT><FR><NU><IT>&agr;</IT></NU><DE><IT>&tgr;<SUB>2</SUB></IT></DE></FR></DE></FR></FENCE><IT> exp</IT>(−<IT>t</IT><IT>/&tgr;<SUB>2</SUB></IT>)</FENCE> (B3)
Here RD is the resistance of the dendrites, and the parameter alpha  is
&agr;=<FR><NU><IT>A</IT><SUB><IT>1</IT></SUB><IT>&tgr;<SUB>2</SUB>+</IT><IT>A</IT><SUB><IT>2</IT></SUB><IT>&tgr;<SUB>1</SUB></IT></NU><DE><IT>A</IT><SUB><IT>1</IT></SUB><IT>+</IT><IT>A</IT><SUB><IT>2</IT></SUB></DE></FR> (B4)
If tau 1 tau 2, which is the case in these experiments (tau 1 = 0.57 ± 0.04 ms, tau 2 = 3.0 ± 0.2 ms for all ROMK1 cells and similar in controls), then at t = tau 1 the soma has charged appreciably and the dendrites have not. The conductance change of the soma in Ba2+ is then proportional to the current change at this early time
<IT>G</IT><SUB><IT>soma</IT></SUB><IT>−</IT><IT>G</IT><SUB><IT>somaBa</IT></SUB><IT> ∝ </IT><IT>I</IT>(<IT>&tgr;<SUB>1</SUB></IT>)<IT>−</IT><IT>I</IT>(<IT>&tgr;<SUB>2</SUB></IT>) (B5)
Since the total conductance change is given by
<IT>G</IT><SUB>TOT</SUB>−<IT>G</IT><SUB><IT>TOTBa</IT></SUB><IT>=</IT><FR><NU><IT>I</IT><SUB><IT>ss</IT></SUB></NU><DE><IT>V</IT><SUB><IT>0</IT></SUB></DE></FR><IT>−</IT><FR><NU><IT>I</IT><SUB><IT>ssBa</IT></SUB></NU><DE><IT>V</IT><SUB><IT>0</IT></SUB></DE></FR><IT>,</IT> (B6)
the relative contributions of the soma and dendrites to the conductance change in Ba2+ can be evaluated.


    ACKNOWLEDGMENTS

We thank B. Khakh, G. Greif, J. Pine, and C. Lindensmith for useful suggestions and discussions.

This work was supported by Burroughs-Wellcome and by National Institute of Mental Health Grant MH-49176.


    FOOTNOTES

Address for reprint requests: H. Nadeau, Caltech Biology 156-29, 1200 E. California Blvd., Pasadena, CA 91125 (E-mail: nadeau{at}cco.caltech.edu).

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Received 27 March 2000; accepted in final form 4 May 2000.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
CONCLUSION
APPENDIX A
APPENDIX B
REFERENCES

0022-3077/00 $5.00 Copyright © 2000 The American Physiological Society