- Email: [email protected]
Finish
NAKED DNA: METHODS transfection in various regions of the tissue. There is high transfection in the peripheral layers of the papilla. Transfection was also seen throughout the tissue. Conclusions: Our study indicates that the transfection of benign kidney cells by holmium laser which is routinely used in endourology is a promising new gene transfer strategy. The ex vivo trials showed that different tissues of the kidney are susceptible for transfection by Ho:YAG laser warranting further studies to optimize transfection. This could prove to be a useful technique in delivering gene therapy to the kidney with minimal invasiveness.
177. A Putative Nuclease Associated with Renal Brush Border Membranes Is Required for Transport of Double Stranded DNA by the Nucleic Acid Conducting Channel Avelino Teixeira,1 Edgar Leal-Pinto,1 Basil Hanss,1 Paul E. Klotman.1 1 Nephrology/Medicine, Mt. Sinai School of Medicine, New York, NY, United States. We have previously described a nucleic acid conducting channel complex (NACh) found in rat proximal tubule brush border membranes (BBM). The BBM were isolated on a 15% Percoll gradient followed by solubilization with CHAPS and the complex purified by a series of chromatography steps. On reconstituting purified NACh in a planar lipid bilayer in a symmetrical buffered solution no current was observed. On addition of 5uM oligonucleotide (ODN, 20-mer deoxythymidine) to each side of the bilayer, current was observed as revealed by clear transitions between closed and opened states (gating) and the channel has been shown to conduct ODN. However, the purified NACh complex does not conduct double stranded DNA (dsDNA). Furthermore dsDNA has been shown to block the activity of the purified NACh actively gating in the presence of ODN. By fusing washed BBM from a 15% Percoll gradient to planar lipid bilayers we have demonstrated that NACh is present in the native membranes and that NACh in native membranes interacts with ODN in a manner indistinguishable from the purified channel complex. In this work we describe the presence of a Mg2+ dependent nuclease associated with washed Percoll purified BBM as observed in an incubation assay. Furthermore the nucleic acid channel activity of Percoll purified BBM reconstituted in a bilayer and actively gating in the presence of ODN is not blocked by dsDNA in the presence of Mg2+. We hypothesized that one strand of the double strand was being degraded by the nuclease activity allowing the remaining strand to be transported as a single strand. To address this question, a 130 bp double stranded DNA of known sequence was added to the trans (ground) chamber of the bilayer apparatus in which reconstituted BBM were actively gating in the presence of ODN. At intervals samples were taken from the opposite chamber and the removed volume replaced with fresh buffer. Samples were also taken at the start of the experiment prior to addition of BBM and prior to addition of the dsDNA. Samples were desalted and concentrated to be used in their entirety in a PCR reaction using primers homologous to the ends of the 130bp dsDNA. DNA of the correct size was recovered from the PCR indicating that at least one of the strands had been conducted by the channel in this period. The PCR of the control samples taken prior to addition of dsDNA were negative. The presence of this nuclease we believe has important implications for the in vivo functioning of this channel and may provide a mechanism for translocation of double stranded DNA across plasma membranes.
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178. Purification of Supercoiled Plasmid DNA for Gene Therapy Joachim Stadler,1 Raf Lemmens,2 Yamuna Dasarathy.3 Amersham Biosciences Europe GmbH, Freiburg, Germany; 2 R&D Separations, Amersham Biosciences AB, Uppsala, Sweden; 3 Amersham Biosciences, Piscataway, NJ, United States.
1
Purification of supercoiled plasmid DNA from other isoforms continues to be a challenge in the field of gene therapy and vaccines. We have formulated a three step chromatographic protocol that will purify supercoiled plasmid DNA from other isoforms. We also have demonstrated that our procedure eliminates contaminants such as genomic DNA, RNA, endotoxins and proteins. This protocol does not involve the addition of RNase and the purity of the end product - supercoiled plasmid DNA - makes it ideal for gene therapy. In the first step, the initial clarified bacterial lysate (concentrated using hollow fiber ultra filtration membranes, in the case of low copy plasmids) is pumped onto a Sepharose® 6 Fast Flow column to remove most of the RNA by group separation. In the second step, the plasmid DNA collected from Sepharose 6 Fast Flow column is pumped onto a PlasmidSelect® column. Under specific buffer conditions, this column purifies selectively supercoiled plasmid DNA by thiophilic aromatic adsorption and removes proteins, endotoxins and the remaining RNA. In the third step, the supercoiled plasmid DNA eluted from the PlasmidSelect column is further purified and concentrated by SOURCE® 30Q column. In conclusion, the process described has the following features: - by using group separation, contaminating RNA can be removed without the use of RNase - by using a thiolic aromatic ligand, complete separation of open circular from supercoiled plasmid DNA can be accomplished - by using an anion exchange medium, the levels of endotoxins and traces of RNA and genomic DNA can be further decreased - by combining three chromatographic steps based on different selectivity and mechanism, an efficient reduction in the levels of contaminants can be achieved - fully scalable for easy transfer to GMP compliant production - Regulatory support package facilitates easy GMP compliance The protocol is made attractive by its flexibility to be generic, scalable, GMP compliant and can be automated using an ÄKTAexplorer 100 system.
179. Large Scale GMP Production, Formulation, and Fill/Finish Magda Marquet,1 Roy Musil,1 Rick Hancock.1 Plasmid Production, Althea Technologies, Inc., San Diego, CA, United States. 1
Large-scale GMP production of plasmid DNA has emerged as one of the challenges industry faces as the clinical aspects of gene therapy continue to grow. While the production quantities for clinical phase I and phase II material can still be met by employing modified laboratory scale methods, phase III and commercial manufacturing require truly scaleable methods. Another trend we are witnessing as products proceed in the clinical pipeline is the tightening of specifications. For example, as doses increase in clinical trials, endotoxin levels must be lowered accordingly. In the same vein, thorough plasmid characterization becomes a crucial aspect of the manufacturing development process. An often overlooked part of the entire development process is the detailed characterization of the formulation and final fill parameters. Numerous clinical formulations now include plasmid DNA with several excipients and adjuvants to assure greater efficacy. Further adding to the complexity of final formulation is discerning the interaction of the drug product with the final filling container. A number of different raw materials may be included in the composition of a final container (boro-silicate Molecular Therapy Vol. 7, No. 5, May 2003, Part 2 of 2 Parts
Copyright © The American Society of Gene Therapy
NAKED DNA: METHODS glass, silanized rubber, poly-carbonate plastics etc.) all of which may impact the properties, including stability, of the final drug product. We will present some of the challenges presented by production, formulation, and filling as well as some alternative solutions to these challenges.
180. Use of WAVE® Denaturing HPLC to Screen for Subtle Mutations in Plasmid DNA: Potential Application for Genetic Stability Evaluation Terrie Martin,1 J. Adams,2 Luman Wing,2 Adrian Vilalta.1 1 Gene Optimization and Design, Vical Inc, San Diego, CA, United States; 2Transgenomic Inc, San Diego, CA, United States. Use of plasmid DNA for gene delivery has shown great promise for both vaccine and therapeutic protein applications. One of the many advantages to plasmid DNA is that it lends itself to be well characterized as a pharmaceutical agent. One of the most important characteristics of any gene delivery agent is the sequence of the nucleic acid; any mutation/insertion in the sequence needs to be evaluated. A statistically significant evaluation of the stability of the plasmid’s DNA sequence during bacterial fermentation requires the sequencing of a large number of plasmid clones. An alternative screening method would be useful in both reducing the cost of the evaluation as well as increasing the statistical power of the evaluation. Clones found suspect by the screening method would then be further analyzed by conventional DNA sequencing. To this end, we have evaluated the WAVE® denaturing HPLC (Transgenomic, Inc.) methodology to detect mutations in plasmid DNA, including point mutations, clusters of point mutations as well as short deletions. We have found conditions that allow for reproducible detection of all of these mutations. Mutations found by this screening method were confirmed by conventional DNA sequencing. We conclude that the WAVE® technology could be used as a rapid and reliable screen to analyze a large number of plasmid clones as part of a genetic stability evaluation scheme.
181. The Role of Deoxyribonuclease II during Transfection Dasein Howell,1 Michael A. Barry.1,2,3 Department of Immunology, Baylor College of Medicine, Houston, TX; 2Center for Cell and Gene Therapy, and Department of Molecular and Human Genetics, Baylor College of Medicine, Houston, TX; 3Department of Bioengineering, Rice University, Houston, TX. 1
In order to understand the variables that influence cellular transfection, it is necessary to know the basic biology of the progression of the DNA to the nucleus. This information can then be used to improve transfection reagents and protocols for both in vitro and in vivo applications. The stability of the introduced DNA is one factor that can influence transfection efficiency. The potential for degradation of the DNA is evident due to the existence of extracellular and intracellular nucleases. We have investigated deoxyribonuclease II (DNase II), an acidic endonuclease active in the lysosome, to determine if it plays a negative role during transfection. This hypothesis relies on the endocytic model of DNA uptake. If endosomes containing the transfected DNA fuse with lysosomes, the DNA can theoretically be degraded by DNase II. This would establish DNase II as a potentially important barrier to transfection. Although this idea is generally accepted based on studies using non-specific nuclease inhibitors, data that directly implicate DNase II is lacking. To investigate the degradative potential of DNase II, we used two in vitro cell culture systems. The first system relied on tetracycline-inducible DNase II gene expression in HeLa cells that caused a 16-fold increase of DNase II activity levels over endogenous levels. Control cells were engineered to induce Lac Molecular Therapy Vol. 7, No. 5, May 2003, Part 2 of 2 Parts Copyright © The American Society of Gene Therapy
Z gene expression. Experiments comparing the transfection efficiency of un-induced versus induced cells sought to determine if higher levels of DNase II activity correlated with decreased levels of transfection. Cells were transfected by calcium-phosphate/DNA co-precipitation method with variable amounts of a plasmid encoding the luciferase gene, and measurement of luciferase activity was used to assess transfection efficiency. Data from these experiments showed a 2-fold decrease of luciferase activity in cells that had induced DNase II but not Lac Z. This effect was not markedly influenced by the amount of DNA used, nor were there any cytotoxic effects due to DNase II induction. The second in vitro system used to investigate our hypothesis was based on murine embryonic fibroblasts derived from DNase II wildtype and transgenic knockout mice. Experiments utilizing these cells were designed to determine if the cells that lacked DNase II had a higher transfection efficiency than wildtype cells. These fibroblasts were cultured in vitro and transfected with a luciferase plasmid using several methods. Levels of luciferase activity in the transfected knockout cells were found to be approximately 10-fold higher than those in wildtype cells. Overall, these data, together with the results from the inducible HeLa system, directly support our hypothesis that DNase II is an enzymatic barrier to transfection.
182. The Nucleic Acid Conducting Channel Is Present in Native Renal Brush Border Membranes Edgar Leal-Pinto,1 Basil Hanss,1 Avelino Teixeira,1 Paul E. Klotman.1 1 Division of Nephrology, Department of Medicine, Mt. Sinai School of Medicine, New York, NY. We previously described a cell surface channel that is highly selective for nucleic acid. This channel was purified to homogeneity by solubilizing renal brush border membranes with CHAPS and separation by a four-step FPLC protocol and it was characterized by reconstitution in planar lipid bilayers. We have established that this channel consists of at least two subunits; a pore-forming subunit that is blocked by heparan sulfate and a regulatory subunit that is blocked by L-malate (PNAS 99:1707-1712, 2002). In the current studies, experiments were performed to determine if a similar channel is present in native brush border membranes (BBM). To test this, we purified BBM as we previously described (PNAS 95:19211926, 1998) with no purification beyond isolation of vesicles (i.e., no solubilization or FPLC separation). In 10 experiments in which BBM were reconstituted in planar lipid bilayer, bathed in symmetrical buffered solution (200 mM CsCl, 5 mM BaCl2, 1 mM GdCl3, 1 mM CaCl2, pH = 7.4), no current was observed. However, upon the addition of symmetrical oligodeoxynucleotide (ODN; 20mer deoxythymidine, 5 mM), channel activity was present as evidenced by clear transitions between closed and open states. The slope conductance was 9 ± 0.9 pS and no significant rectification or voltage dependence was observed. Establishment of a 10-fold gradient for ODN resulted in a shift of the reversal potential (Erev) to +14±0.1 mV, a value closer to the equilibrium potential for ODN. No significant change in Erev was observed when similar gradients were created for the other ions present in the solution. Changes in Ca 2+ concentration on the trans side had a profound effect on open probability changing from < 1% in zero Ca 2+ to approximately 100 % in 1 mM Ca 2+. Alteration in cis Ca 2+ concentration was without effect. Addition of L-malate (1 mM) to only the cis side or heparan sulfate (20 mg/ml) to only the trans side reduced open probability more than 90%. These data demonstrate that the characteristics of the nucleic acid channel in brush border membranes are identical to those previously reported for purified protein consisting of a heparan sulfate sensitive component and an L-malate sensitive component.
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177. A Putative Nuclease Associated with Renal Brush Border Membranes Is Required for Transport of Double Stranded DNA by the Nucleic Acid Conducting Channel Avelino Teixeira,1 Edgar Leal-Pinto,1 Basil Hanss,1 Paul E. Klotman.1 1 Nephrology/Medicine, Mt. Sinai School of Medicine, New York, NY, United States. We have previously described a nucleic acid conducting channel complex (NACh) found in rat proximal tubule brush border membranes (BBM). The BBM were isolated on a 15% Percoll gradient followed by solubilization with CHAPS and the complex purified by a series of chromatography steps. On reconstituting purified NACh in a planar lipid bilayer in a symmetrical buffered solution no current was observed. On addition of 5uM oligonucleotide (ODN, 20-mer deoxythymidine) to each side of the bilayer, current was observed as revealed by clear transitions between closed and opened states (gating) and the channel has been shown to conduct ODN. However, the purified NACh complex does not conduct double stranded DNA (dsDNA). Furthermore dsDNA has been shown to block the activity of the purified NACh actively gating in the presence of ODN. By fusing washed BBM from a 15% Percoll gradient to planar lipid bilayers we have demonstrated that NACh is present in the native membranes and that NACh in native membranes interacts with ODN in a manner indistinguishable from the purified channel complex. In this work we describe the presence of a Mg2+ dependent nuclease associated with washed Percoll purified BBM as observed in an incubation assay. Furthermore the nucleic acid channel activity of Percoll purified BBM reconstituted in a bilayer and actively gating in the presence of ODN is not blocked by dsDNA in the presence of Mg2+. We hypothesized that one strand of the double strand was being degraded by the nuclease activity allowing the remaining strand to be transported as a single strand. To address this question, a 130 bp double stranded DNA of known sequence was added to the trans (ground) chamber of the bilayer apparatus in which reconstituted BBM were actively gating in the presence of ODN. At intervals samples were taken from the opposite chamber and the removed volume replaced with fresh buffer. Samples were also taken at the start of the experiment prior to addition of BBM and prior to addition of the dsDNA. Samples were desalted and concentrated to be used in their entirety in a PCR reaction using primers homologous to the ends of the 130bp dsDNA. DNA of the correct size was recovered from the PCR indicating that at least one of the strands had been conducted by the channel in this period. The PCR of the control samples taken prior to addition of dsDNA were negative. The presence of this nuclease we believe has important implications for the in vivo functioning of this channel and may provide a mechanism for translocation of double stranded DNA across plasma membranes.
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178. Purification of Supercoiled Plasmid DNA for Gene Therapy Joachim Stadler,1 Raf Lemmens,2 Yamuna Dasarathy.3 Amersham Biosciences Europe GmbH, Freiburg, Germany; 2 R&D Separations, Amersham Biosciences AB, Uppsala, Sweden; 3 Amersham Biosciences, Piscataway, NJ, United States.
1
Purification of supercoiled plasmid DNA from other isoforms continues to be a challenge in the field of gene therapy and vaccines. We have formulated a three step chromatographic protocol that will purify supercoiled plasmid DNA from other isoforms. We also have demonstrated that our procedure eliminates contaminants such as genomic DNA, RNA, endotoxins and proteins. This protocol does not involve the addition of RNase and the purity of the end product - supercoiled plasmid DNA - makes it ideal for gene therapy. In the first step, the initial clarified bacterial lysate (concentrated using hollow fiber ultra filtration membranes, in the case of low copy plasmids) is pumped onto a Sepharose® 6 Fast Flow column to remove most of the RNA by group separation. In the second step, the plasmid DNA collected from Sepharose 6 Fast Flow column is pumped onto a PlasmidSelect® column. Under specific buffer conditions, this column purifies selectively supercoiled plasmid DNA by thiophilic aromatic adsorption and removes proteins, endotoxins and the remaining RNA. In the third step, the supercoiled plasmid DNA eluted from the PlasmidSelect column is further purified and concentrated by SOURCE® 30Q column. In conclusion, the process described has the following features: - by using group separation, contaminating RNA can be removed without the use of RNase - by using a thiolic aromatic ligand, complete separation of open circular from supercoiled plasmid DNA can be accomplished - by using an anion exchange medium, the levels of endotoxins and traces of RNA and genomic DNA can be further decreased - by combining three chromatographic steps based on different selectivity and mechanism, an efficient reduction in the levels of contaminants can be achieved - fully scalable for easy transfer to GMP compliant production - Regulatory support package facilitates easy GMP compliance The protocol is made attractive by its flexibility to be generic, scalable, GMP compliant and can be automated using an ÄKTAexplorer 100 system.
179. Large Scale GMP Production, Formulation, and Fill/Finish Magda Marquet,1 Roy Musil,1 Rick Hancock.1 Plasmid Production, Althea Technologies, Inc., San Diego, CA, United States. 1
Large-scale GMP production of plasmid DNA has emerged as one of the challenges industry faces as the clinical aspects of gene therapy continue to grow. While the production quantities for clinical phase I and phase II material can still be met by employing modified laboratory scale methods, phase III and commercial manufacturing require truly scaleable methods. Another trend we are witnessing as products proceed in the clinical pipeline is the tightening of specifications. For example, as doses increase in clinical trials, endotoxin levels must be lowered accordingly. In the same vein, thorough plasmid characterization becomes a crucial aspect of the manufacturing development process. An often overlooked part of the entire development process is the detailed characterization of the formulation and final fill parameters. Numerous clinical formulations now include plasmid DNA with several excipients and adjuvants to assure greater efficacy. Further adding to the complexity of final formulation is discerning the interaction of the drug product with the final filling container. A number of different raw materials may be included in the composition of a final container (boro-silicate Molecular Therapy Vol. 7, No. 5, May 2003, Part 2 of 2 Parts
Copyright © The American Society of Gene Therapy
NAKED DNA: METHODS glass, silanized rubber, poly-carbonate plastics etc.) all of which may impact the properties, including stability, of the final drug product. We will present some of the challenges presented by production, formulation, and filling as well as some alternative solutions to these challenges.
180. Use of WAVE® Denaturing HPLC to Screen for Subtle Mutations in Plasmid DNA: Potential Application for Genetic Stability Evaluation Terrie Martin,1 J. Adams,2 Luman Wing,2 Adrian Vilalta.1 1 Gene Optimization and Design, Vical Inc, San Diego, CA, United States; 2Transgenomic Inc, San Diego, CA, United States. Use of plasmid DNA for gene delivery has shown great promise for both vaccine and therapeutic protein applications. One of the many advantages to plasmid DNA is that it lends itself to be well characterized as a pharmaceutical agent. One of the most important characteristics of any gene delivery agent is the sequence of the nucleic acid; any mutation/insertion in the sequence needs to be evaluated. A statistically significant evaluation of the stability of the plasmid’s DNA sequence during bacterial fermentation requires the sequencing of a large number of plasmid clones. An alternative screening method would be useful in both reducing the cost of the evaluation as well as increasing the statistical power of the evaluation. Clones found suspect by the screening method would then be further analyzed by conventional DNA sequencing. To this end, we have evaluated the WAVE® denaturing HPLC (Transgenomic, Inc.) methodology to detect mutations in plasmid DNA, including point mutations, clusters of point mutations as well as short deletions. We have found conditions that allow for reproducible detection of all of these mutations. Mutations found by this screening method were confirmed by conventional DNA sequencing. We conclude that the WAVE® technology could be used as a rapid and reliable screen to analyze a large number of plasmid clones as part of a genetic stability evaluation scheme.
181. The Role of Deoxyribonuclease II during Transfection Dasein Howell,1 Michael A. Barry.1,2,3 Department of Immunology, Baylor College of Medicine, Houston, TX; 2Center for Cell and Gene Therapy, and Department of Molecular and Human Genetics, Baylor College of Medicine, Houston, TX; 3Department of Bioengineering, Rice University, Houston, TX. 1
In order to understand the variables that influence cellular transfection, it is necessary to know the basic biology of the progression of the DNA to the nucleus. This information can then be used to improve transfection reagents and protocols for both in vitro and in vivo applications. The stability of the introduced DNA is one factor that can influence transfection efficiency. The potential for degradation of the DNA is evident due to the existence of extracellular and intracellular nucleases. We have investigated deoxyribonuclease II (DNase II), an acidic endonuclease active in the lysosome, to determine if it plays a negative role during transfection. This hypothesis relies on the endocytic model of DNA uptake. If endosomes containing the transfected DNA fuse with lysosomes, the DNA can theoretically be degraded by DNase II. This would establish DNase II as a potentially important barrier to transfection. Although this idea is generally accepted based on studies using non-specific nuclease inhibitors, data that directly implicate DNase II is lacking. To investigate the degradative potential of DNase II, we used two in vitro cell culture systems. The first system relied on tetracycline-inducible DNase II gene expression in HeLa cells that caused a 16-fold increase of DNase II activity levels over endogenous levels. Control cells were engineered to induce Lac Molecular Therapy Vol. 7, No. 5, May 2003, Part 2 of 2 Parts Copyright © The American Society of Gene Therapy
Z gene expression. Experiments comparing the transfection efficiency of un-induced versus induced cells sought to determine if higher levels of DNase II activity correlated with decreased levels of transfection. Cells were transfected by calcium-phosphate/DNA co-precipitation method with variable amounts of a plasmid encoding the luciferase gene, and measurement of luciferase activity was used to assess transfection efficiency. Data from these experiments showed a 2-fold decrease of luciferase activity in cells that had induced DNase II but not Lac Z. This effect was not markedly influenced by the amount of DNA used, nor were there any cytotoxic effects due to DNase II induction. The second in vitro system used to investigate our hypothesis was based on murine embryonic fibroblasts derived from DNase II wildtype and transgenic knockout mice. Experiments utilizing these cells were designed to determine if the cells that lacked DNase II had a higher transfection efficiency than wildtype cells. These fibroblasts were cultured in vitro and transfected with a luciferase plasmid using several methods. Levels of luciferase activity in the transfected knockout cells were found to be approximately 10-fold higher than those in wildtype cells. Overall, these data, together with the results from the inducible HeLa system, directly support our hypothesis that DNase II is an enzymatic barrier to transfection.
182. The Nucleic Acid Conducting Channel Is Present in Native Renal Brush Border Membranes Edgar Leal-Pinto,1 Basil Hanss,1 Avelino Teixeira,1 Paul E. Klotman.1 1 Division of Nephrology, Department of Medicine, Mt. Sinai School of Medicine, New York, NY. We previously described a cell surface channel that is highly selective for nucleic acid. This channel was purified to homogeneity by solubilizing renal brush border membranes with CHAPS and separation by a four-step FPLC protocol and it was characterized by reconstitution in planar lipid bilayers. We have established that this channel consists of at least two subunits; a pore-forming subunit that is blocked by heparan sulfate and a regulatory subunit that is blocked by L-malate (PNAS 99:1707-1712, 2002). In the current studies, experiments were performed to determine if a similar channel is present in native brush border membranes (BBM). To test this, we purified BBM as we previously described (PNAS 95:19211926, 1998) with no purification beyond isolation of vesicles (i.e., no solubilization or FPLC separation). In 10 experiments in which BBM were reconstituted in planar lipid bilayer, bathed in symmetrical buffered solution (200 mM CsCl, 5 mM BaCl2, 1 mM GdCl3, 1 mM CaCl2, pH = 7.4), no current was observed. However, upon the addition of symmetrical oligodeoxynucleotide (ODN; 20mer deoxythymidine, 5 mM), channel activity was present as evidenced by clear transitions between closed and open states. The slope conductance was 9 ± 0.9 pS and no significant rectification or voltage dependence was observed. Establishment of a 10-fold gradient for ODN resulted in a shift of the reversal potential (Erev) to +14±0.1 mV, a value closer to the equilibrium potential for ODN. No significant change in Erev was observed when similar gradients were created for the other ions present in the solution. Changes in Ca 2+ concentration on the trans side had a profound effect on open probability changing from < 1% in zero Ca 2+ to approximately 100 % in 1 mM Ca 2+. Alteration in cis Ca 2+ concentration was without effect. Addition of L-malate (1 mM) to only the cis side or heparan sulfate (20 mg/ml) to only the trans side reduced open probability more than 90%. These data demonstrate that the characteristics of the nucleic acid channel in brush border membranes are identical to those previously reported for purified protein consisting of a heparan sulfate sensitive component and an L-malate sensitive component.
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