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Received 24 November 1997; accepted 10 February 1998.
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ABSTRACT |
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INTRODUCTION |
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As in non-neuronal tissues, KATP channels seem to operate with distinct characteristics in neurons of diverse regions of the mammalian brain, such as the neocortex, hippocampus, substantia nigra, locus coeruleus or hypothalamus (see Röper & Ashcroft, 1995; Trapp & Ballanyi, 1995; Ballanyi, Doutheil & Brockhaus, 1996; Fujimura, Tanaka, Yamamoto, Shigemori & Higashi, 1997, for references). For identification of the functional and molecular properties of metabolism-gated K+ channels in the brain, knowledge is required of the individual expression and constitution of the SUR and Kir subunits in a given neuron. By combination of patch-clamp techniques and molecular genetic characterization at the single cell level (van Gelder, von Zastrow, Yool, Dement, Barchas & Eberwine, 1990; Eberwine et al. 1992; Lambolez, Audinat, Bochet, Crépel & Rossier, 1992; Jonas et al. 1994), cytoplasm can be harvested from individual cells after their electrophysiological characterization, and expression of mRNA can be analysed.
We have employed the experimental approach of single cell antisense RNA (aRNA) amplification to detect the presence or absence of several mRNA species from a single cell and to characterize for the first time the molecular identity of neuronal KATP channels. For this purpose, we have used dorsal vagal neurons (DVNs), since our previous studies have suggested that sustained sulphonylurea-sensitive hyperpolarizations induced by oxygen or glucose depletion (Trapp & Ballanyi, 1995; Ballanyi et al. 1996) or by dialysis of cellular constituents upon establishing the whole-cell configuration (Trapp, Ballanyi & Richter, 1994), are mediated by KATP channels in these cells. The results indicate that these neuronal KATP channels are likely to be constituted from SUR1 receptors and Kir6.2 subunits.
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METHODS |
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Preparation and solutions
Experiments were performed on 16- to 22-day-old rats of either sex anaesthetized with ether and decapitated at the cervical spinal level. Brainstem slices 400 µm thick were obtained as previously described (Ballanyi et al. 1996). The recording chamber (volume 3 ml) was superfused with standard saline (flow rate 5 ml min-1) of the following composition (mM): 118 NaCl, 3 KCl, 1 MgCl2, 1·5 CaCl2, 25 NaHCO3, 1 NaH2PO4 and 10 glucose. The pH was adjusted to 7·4 by gassing with 95 % O2 and 5 % CO2. Superfusion of hypoxic standard solution, gassed with 95 % N2 instead of O2, led to tissue anoxia as described elsewhere (Trapp & Ballanyi, 1995). All drugs were purchased from Sigma (Deisenhofen, Germany) and were added to the superfusion fluid.
Electrophysiological recordings
Standard whole-cell recordings were done at 30°C using an EPC9 amplifier and Pulse software (HEKA, Lamprecht, Germany). The patch-pipette solution contained (mM): 140 KCl, 1 Na2-ATP, 2 MgCl2, 1 EGTA, 5 Hepes, 0·1 cAMP; pH adjusted to 7·4 with KOH. Membrane resistance (Rm) was measured by injection of hyperpolarizing current pulses (duration 500 ms, amplitude -10 to -100 pA). Single-channel activity was analysed in the cell-attached configuration at room temperature (20-23°C). The fire-polished pipettes (filled with a solution containing (mM): 140 KCl, 2 CaCl2, 0·3 MgCl2, 5 EGTA, 10 Hepes; pH adjusted to 7·2 with KOH) had a DC resistance of 5-7 M
. Seals were accepted with a resistance > 5 G. In most recordings, the pipette was held at 0 mV, no corrections for liquid-junction potentials were done. Analysis of single-channel data, low-pass filtered at 2 kHz, was performed using TAC (HEKA) or IgorPro (Wavemetrics, Lake Oswego, OR, USA) software.
Molecular biology
aRNA amplification from single DVNs and surrounding glial cells of the medullary slices was performed similarly to the detailed procedure described by Eberwine et al. (1992). In brief, 2·5 mM dNTPs, 2 ng µl-1 T7-oligo-d(T)24 primer, and 0·5 U µl-1 avian myeloblastosis virus (AMV) reverse transcriptase (Boehringer Mannheim, Germany) were either added to the internal solution before recording or were mixed in an Eppendorf tube with the aspirated cell contents and incubated at 37°C for 60-90 min. After harvesting of the cytoplasm, second strand synthesis was performed with T4 DNA polymerase-Klenow fragment (1 U µl-1 each), followed by treatment with S1 nuclease (1 U µl-1) and subsequently Klenow enzyme (1 U µl-1) to remove hairpin loops and to produce blunt ends on the the cDNA. After aRNA amplification with 10-100 U µl-1 T7 RNA polymerase, a second round of amplification, including the final synthesis of double-stranded cDNA, was conducted to yield adequate amounts of template for the expression profiling of different mRNAs from a single cell. All amplification steps were conducted under RNase-free conditions. In analogy to the rat SUR1 gene, the SUR2 gene may contain multiple introns. In contrast, rat Kir6.1 and rat Kir6.2 are likely to be intronless and, therefore, amplification from genomic sequences cannot be distinguished from cDNA. To rule out contamination generally from genomic DNA, amplified aRNA was incubated after each amplification with RNase-free DNase I at 37°C for 1 h.
Polymerase chain reaction (PCR) analysis of Kir6.1 and Kir6.2 channel- and SUR1 and SUR2 receptor-core fragments used primers based on the sequences of the rat genes for Kir6.1 (Inagaki et al. 1995), Kir6.2 (Tokuyama et al. 1996), SUR1 (Aguilar-Bryan et al. 1995), and SUR2 (Inagaki et al. 1996). Sense and antisense primers were chosen to specifically amplify fragments of 539-865 bp in length (base location indicated): rKir6.1 (Genbank accession no. D42145, 865 bp) sense primer 1765'-GAAGATGCTGGCCAGGAAGAG-3', antisense primer 10415'-CAGCCACTGACCTTGTCAACC-3'; rKir6.2 (no. U44897, 553 bp) sense primer 1295'-GGAGAGGAGGGCCCGCTTCGTGTC-3', antisense primer 6815'-GGCGCTAATGATCATGCTTTTTCGGAGGTC-3'; rSUR1 (no. L40624, 539 bp) sense primer 29265'-GCAGCCGAGAGCGAGGAAGATGA-3'; antisense primer 34645'-ACAGCCAGGGCGGAGACACAGAGTA-3'; rSUR2 (no. D83598, 603 bp) sense primer 13685'-CGCGGCGGTCATCGTGCTC-3', antisense primer 19705'-CGCCGCGCCTGCTCGTAGTT-3'.
PCR reactions were run with both taq (Perkin Elmer, Weiterstadt, Germany) and proofreading (pfu; Stratagene, La Jolla, CA, USA) polymerase for thirty-five cycles of 95°C denaturating, 53-56°C annealing, and 72°C extension for 1·5 min each, with a final extension of 15 min at 72°C. Amplified fragments were purified from agarose gels, digested at terminal restriction sites or blunt ends produced, and ligated into the adequately digested pBluescriptSKII vector (Stratagene). Double-stranded sequencing of the PCR products was performed on both strands using the prism sequenase dye terminator kit on an automatic sequencer (both from Perkin Elmer).
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RESULTS |
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DVNs were characterized by spontaneous spike discharge at a frequency of 0·5-3 Hz and an A-type K+ current, causing delay of recovery to resting potential after termination of hyperpolarizing current pulses (Trapp & Ballanyi, 1995; Ballanyi et al. 1996). In an initial series of experiments, single-channel activity was recorded during block of aerobic metabolism with cyanide (CN-). In a further approach, cytoplasm of DVNs was harvested for mRNA analysis subsequent to whole-cell recording in cells that had been exposed to anoxia.
Single-channel recording
In the cell-attached configuration, spontaneous spike activity was revealed in 45 of a total of 107 patches. CN- at 1-2 mM led to reversible suppression of spike discharge in thirty-two of the spontaneously active cells (Fig. 1A). In fifteen of these DVNs, such 'functional inactivation' was accompanied by activation of a single-channel current with an amplitude of 4·6-5·5 pA (Figs 1 and 2). In twelve of these patches, opening of only one channel was observed (Figs 1B and 2) whereas in the remaining three samples, up to four channels were activated by CN-. In four of these patches tested, both CN--evoked suppression of spike activity and opening of single channels were reversed upon addition of 200 µM of the sulphonylurea tolbutamide (Fig. 1; Ashcroft & Ashcroft, 1990). Figure 1 also shows that washout of tolbutamide in the continuous presence of CN- led again to block of spontaneous action potentials and an increase in single-channel activity.
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Figure 1. Cyanide-induced activation of single KATP channels in a DVN A, in this cell-attached recording, bath application of cyanide (CN-) led to inactivation of spontaneous spike discharge and to opening of single KATP channels. Both these effects were reversed by the sulphonylurea tolbutamide. B, a similar experiment in a different cell showing spike and single KATP channel activities at a higher time resolution. c, closed state.
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The voltage dependence of CN--activated single channel currents was studied in four patches. As illustrated in the example of Fig. 2A, the polarity of the current reversed at a potential of between 80 and 100 mV, consistent with the finding of a reversal potential of between -80 and -100 mV during anoxia-induced activation of whole-cell KATP currents in these cells (Trapp & Ballanyi, 1995). The slope conductance of the linear part of the calculated I-V curves (Fig. 2B) revealed an average single-channel conductance of 72·7 ± 4·9 pS (n = 3). As measured at 0 mV, channel activation occurred in bursts with a single open time of 2·2 ms and an open probability of 0·051, as calculated from continuous recordings with a duration of 20-40 s. Under these conditions, mean close time was 41 ms with a fast component of 0·8 ms (Fig. 2C).
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Figure 2. Voltage dependence and open-close time characteristics of KATP channel activity A, cell-attached recording of cyanide (CN-)-induced KATP channel currents at different potentials. B, the slope conductance (dotted line) of the linear part of the current-voltage relation revealed a single-channel conductance of 75 pS. C, plots of the open-closed time distribution as analysed during a 32 s continuous recording during CN- application at 0 mV. Whereas the left histogram shows a monoexponential open time distribution, the closed times distribution in the right histogram had a more complex pattern, indicating the existence of more than one closed state.
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Whole-cell recording
In 3 of 11 DVNs recorded in the whole-cell configuration, anoxic exposure for 10-12 min led, after a delay of 1-2 min, to a sustained hyperpolarization and a decrease of Rm resulting in block of spontaneous spike discharge (Fig. 3A). In three different cells, reduction of spike frequency occurred without development of a major hyperpolarization or Rm decrease after more than 6 min of anoxia (Fig. 3B) whereas in the remaining five DVNs, neither resting potential nor the frequency or peak amplitude of action potentials was profoundly affected. Whole-cell recordings were also obtained from three cells of the dorsal vagal nucleus that did not show stimulus-evoked or spontaneous spike discharge, or synaptic activity. As tested in two of these presumed glial cells (see below), a 5 min period of anoxia led to a stable depolarization by 5 and 7 mV (Fig. 3C).
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Figure 3. Effects of anoxia on membrane potential of neurons and glia in the dorsal vagal nucleus A, whole-cell recording of a DVN revealed a sustained hyperpolarization and decrease of membrane resistance (measured by regular application of hyperpolarizing current), which led to block of spontaneous spike discharge (overshooting action potentials were truncated). B, in this neuron, anoxia led, after about 10 min, to a decrease in the frequency of spontaneous spike activity without occurrence of a major hyperpolarization or decrease of membrane resistance. C, in this glial cell, anoxia led to a stable and reversible depolarization.
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Molecular biology
In the three DVNs that hyperpolarized during anoxia, in one neuron in which spike discharge was suppressed without occurrence of a major hyperpolarization and in a further DVN that did not respond to oxygen depletion, cytoplasm was harvested through the patch electrode with the gigaseal formation maintained. The material was then processed for aRNA/cDNA amplification. Subsequent PCR analysis using primers for the Kir channel subunits Kir6.1 and Kir6.2, and the SUR receptor subtypes SUR1 and SUR2 and 1/50 of the total volume of the amplified cDNA product in each reaction yielded a high level of SUR1 receptor fragments and also clearly detectable levels of Kir6.2 subunits.
All primers used were tested for functionality and sensitivity with 0·1 ng of original cloned cDNA and 50 ng of rat tissue DNA as template. As a control for the analysed cell type primer combinations for the neurofilament middle protein (NF-M), as well as glial fibrillary acidic protein (GFAP), were employed to specifically amplify cDNA fragments from DVNs and glial cells, respectively (Fig. 4). Moreover, adequate H2O controls for all primer combinations were performed and found negative. As shown on the agarose gel in Fig. 4A, the primer pair that was designed to amplify a core fragment of Kir6.1 amplified a strong 865 bp band from the Kir6.1 vector template and from rat brain cDNA, but not from the cDNA templates of any of the single cells (DVN1-3 and glia). Also, the primer combination that successfully amplified a 603 bp SUR2 fragment from both rat brain and atrial cDNA failed to yield detectable amplification products from the single cell source (Fig. 4D). In contrast, clearly detectable fragments were observed using Kir6.2 (Fig. 4B, 553 bp) and SUR 1 (Fig. 4C, 539 bp) primers with cDNA templates obtained from all DVN. When subcloned, grown to large scale and sequenced on both strands, the fragments from DVNs exhibited complete base pair identity to the cDNA sequences obtained previously from rat cDNA libraries (see Methods). Thus, PCR analysis of T7-polymerase-amplified cell-specific RNA with specific primer pairs for SUR1/2 and Kir6.1/2 suggests that a SUR1 and Kir6.2 translate to form a major subtype of KATP channel in the DVNs.
A corresponding analysis of the three recorded glial cells showed a dominant expression of Kir6.2 and a rather faint signal for SUR1, whereas no mRNA for SUR2 or Kir6.1 was detected (Glia ; Fig. 4). These cells are likely to be glia, since GFAP was clearly expressed (Kettenmann & Ransom, 1995). On the other hand, a fragment of NF-M could be amplified from the three DVNs (Fig. 4E).
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Figure 4. PCR analysis of amplified aRNA from single cells of the dorsal vagal nucleus to identify transcripts of Kir6 and SUR isoforms
The 1·5 % agarose gels show bands of DNA fragments amplified with primer pairs specific for Kir6.1 (A), Kir6.2 (B), SUR1 (C), SUR2 (D), as well as the neurofilament middle protein (NF-M) and glial fibrillary acidic protein (GFAP; E). Next to H2O controls for each primer combination (first lane next to molecular marker), templates were of three different DVNs (DVN1-3), a glial cell (Glia), rat brain cDNA (50 ng), and 0·1 ng plasmid clone (except in D where 50 ng of heart atrial cDNA have been used). Primers were tested to amplify fragments of 865 bp (Kir6.1), 553 bp (Kir6.2), 539 bp (SUR1), and 603 bp (SUR2). For primer sequences, see Methods. The bottom gel (E) demonstrates amplification of a 873 bp NF-M fragment from DVN1-3 as well as a 523 bp fragment from the glial cell. Fifty nanograms of rat brain cDNA were used in the last two lanes as control for both sets of primers. The molecular weight marker is a
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DISCUSSION |
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TopAbstractIntroductionMethodsResultsDiscussionReferences |
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Our previous studies showed that in DVNs, both anoxia and glucose depletion evoke a hyperpolarization (Ballanyi et al. 1996) which is due to a sustained K+ outward current (Trapp & Ballanyi, 1995). The observation that the sulphonylurea tolbutamide and glibenclamide blocked and that diazoxide mimicked these responses (Trapp & Ballanyi, 1995; Ballanyi et al. 1996) suggested opening of KATP channels as the underlying mechanism. In the present study, it was found that block of aerobic metabolism with CN- led to activation of tolbutamide-sensitive single-channel currents with a reversal potential close to the presumed K+ equilibrium potential (see also Trapp & Ballanyi, 1995; Ballanyi et al. 1996). The similarity of single-channel conductance and open-closed probabilities with those of KATP channels in other tissues (Ashcroft & Ashcroft, 1990; Schwanstecher & Panten, 1994; Terzic, Tung & Kurachi, 1994) substantiate the assumption that KATP channels are functionally active in DVNs.
This view is supported further by the results obtained from the molecular single cell analysis in the present study. We have chosen the elaborate technique of aRNA amplification which, in contrast to conventional single-cell RT-PCR techniques, allows simultaneous mRNA profiling of a series of genes from a single cell, since it became evident from studies on non-neuronal tissues that KATP channels are constituted of different combinations of at least two different SUR and Kir channel subunits (Aguilar-Bryan et al. 1995; Inagaki et al. 1995, 1996; Tucker et al. 1997). We found in all five DVNs analysed that SUR1 is dominantly expressed and mRNA for Kir6.2 is also clearly detectable, whereas there is no evidence for RNA transcripts of SUR2 or Kir6.1. This particular pattern of combined expression of SUR1 and Kir6.2 appears to be neuron specific, since an almost reciprocal distribution of a dominant signal for Kir6.2 and, in this case, a rather faint signal for SUR1 (without occurrence of SUR2 or Kir6.1) was found in GFAP-positive glial cells. Preliminary results indicate that an almost identical pattern of Kir6.2 and SUR1 is expressed also in glial cells from other brain regions (A. Karschin, unpublished observations). The finding of a high expression level of Kir6.2 in the glia is not surprising, since K+ inward rectifiers are a characteristic feature of mammalian glia (Kettenmann & Ransom, 1995). At present, we cannot decide whether the (low) expression of SUR1 in the glia in combination with Kir6.2 provides functional KATP channels. Due to the high background K+ conductance (Kettenmann & Ransom, 1995), single-channel measurements must be performed for such an analysis.
The results suggest that the structure of KATP channels in the medullary neurons is identical to that revealed for pancreatic 
cells (Inagaki et al. 1995) and, according to a preliminary report, also for a subpopulation of substantia nigra neurons (Liß, Bruns & Röper, 1997). This view gains support from the similarities of single-channel properties of the vagal and the cell KATP channels (Ashcroft & Ashcroft, 1990; Inagaki et al. 1995). In contrast, KATP channels in heart and skeletal muscle appear to have a different structure (SUR2 plus Kir6.2; Inagaki et al. 1996) and single-channel properties (Ashcroft & Ashcroft, 1990; Terzic et al. 1994; Inagaki et al. 1996).
It still remains undetermined as to what degree SUR2 receptor isoforms may participate in the formation of KATP channels in the mammalian brain. Moderate SUR2 mRNA levels have been detected in Northern blots (Inagaki et al. 1996) and can be PCR-amplified from single GABAergic neurons in the mouse substantia nigra (J. Röper, personal communication). However, a recent in situ hybridization study that underlines the superior role of Kir6.2 and SUR1 in the formation of the major neuronal KATP channel, failed to detect significant levels of SUR2 mRNA in the rodent brain (Karschin, Ecke, Ashcroft & Karschin, 1997). This suggests a low copy number of SUR2 transcripts or alternatively SUR2 expression in few cells or a sparse cell population in the brain. The lack of amplified SUR2 templates in the present study, using antisense RNA amplification allowing for simultaneous mRNA profiling of a series of genes from a single cell, also covers the recently identified SUR2B splice variant that was primarily found in heart, liver, skeletal muscle, urinary bladder, but at low levels also in the brain. Pharmacological properties determined from functionally reconstituted Kir6.2-SUR2B channels indicate that this novel SUR isoform may underlie typical KATP channels of the smooth muscle (Isomoto et al. 1996).
Another open question that remains to be resolved in future studies is the lack of occurrence of an anoxic hyperpolarization in all DVNs (see also Trapp & Ballanyi, 1995; Ballanyi et al. 1996) despite the apparent ubiquituous expression of SUR1 and Kir6.2 in these cells. It is known that the activity state of these metabolism-gated K+ channels is determined by a highly complex set of cellular constituents (see Terzic et al. 1994). For example, in a subpopulation of DVN, activation of a sustained sulphonylurea-sensitive K+ outward current is revealed within several minutes after establishing the whole-cell configuration using ATP-containing patch electrodes in oxygenated and glucose-containing solution (Trapp et al. 1994). Recent results suggest that such spontaneous activation of KATP channels occurs predominantly in DVNs from brainstem slices which are studied several hours after isolation from the intact animal (K. Ballanyi, J. Brockhaus & A. Kulik, unpublished observations). On the other hand, it was found that in slice preparations from individual rats almost every DVN is hyperpolarized by anoxia via activation of KATP channels, whereas in other slices (from rats of the same strain and age) rather few DVNs in the same region of the dorsal vagal nucleus respond with hyperpolarization (K. Ballanyi, J. Brockhaus & A. Kulik, unpublished observations). This indicates that almost the entire population of DVNs in rats is equipped with functional KATP channels that correspond with expression of SUR1 and Kir6.2 in all DVNs analysed. In individual preparations, for example, metabolic stress by yet unknown intracellular constituents or by changes in the redox state during the isolation procedure might lead to a block of these channels in some cells in vitro. It will be a major challenge to determine which of these factors critically determine the activity state of neuronal KATP channels.
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REFERENCES |
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TopAbstractIntroductionMethodsResultsDiscussionReferences |
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Aguilar-Bryan, L., Nichols, C. G., Wechsler, S. W., Clement, J. P. IV, Boyd, A. E. III, González, G., Herrera-Sosa, H., Nguy, K., Bryan, J. & Nelson, D. A. (1995). Cloning of the cell high affinity sulfonylurea receptor: a regulator of insulin secretion. Science 268, 423-426.
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Acknowledgements
We are indebted to Drs U. Panten, C. Schwanstecher and M. Schwanstecher for advice in single-channel recording techniques and valuable discussions. The study was supported by the DFG. We wish to thank A. A. Grützner, D. Reuter, G. Dowe, H. Wegener and C. P. Adam for technical help, Drs S. Seino, F. Ashcroft, and G. Bell for providing cDNAs of Kir6.1, Kir6.2 and SUR1, respectively, and Dr J. Eberwine for advice on the aRNA amplification procedure.
Corresponding author
K. Ballanyi: II. Physiologisches Institut, Universität Göttingen, Humboldtallee 23, D-37073 Göttingen, Germany.
Email: kb{at}neuro-physiol.med.uni-goettingen.de
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 A. Kulik, S. Trapp, and K. Ballanyi Ischemia But Not Anoxia Evokes Vesicular and Ca2+-Independent Glutamate Release In the Dorsal Vagal Complex In Vitro J Neurophysiol, May 1, 2000; 83(5): 2905 - 2915. [Abstract] [Full Text] [PDF]
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Hind III-
X174Hae III digest; fragment sizes (bp) are indicated on the right. Note that only for Kir6.2 and SUR1 can bands of fragments amplified from single cells DVN1-3 and Glia be detected.