Verification of Kv 1.1, Kv 1.2 and Kv 1.4 alfa-subunit Mutant Constructs
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Arnulfo Torres
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Introduction
ABSTRACT
Ion channels control neural signaling, hormone secretion, cell volume regulation, as well as salt and water flow across epithelia. These physiological functions are related with the number of cell surface channels (Hill, B. 1992). Recent studies suggest the role of ER export signals in controlling the amount of surface potassium channels. One signal, FCYENE, has been found to increase the cell surface expression of some potassium channels(Ma, D et al. 2000). By in vitro translation, this study verifies the mutation of voltage-dependent potassium channels (Kv) Kv 1.1, Kv 1.2 and Kv 1.4 alfa-subunits. All subunits were cloned using pGEM-T vector system; in addition, Kv 1.1 was cloned in pcDNA3. The sequence FCYENE has been added to the carboxy-terminus of the three aforementioned channels. The corresponding protein bands visualized via SDS-PAGE correlate with the expected molecular weight of each alfa-subunit.
INTRODUCTION
Potassium channels are found in virtually all cells. Some of them are activated by voltage change. Kv are normally closed during the resting potential of the cell, but open upon membrane depolarization. They are also involved in the repolarization of the action potential, and thus, in the electrical excitability of nerve and muscle fibers, including cardiac muscle. Kv are constructed of pore-forming alfa-subunits that may associate with various types of alfa-subunits. (Ashcroft F, 2000). This present study concerns the Kv 1.1, Kv 1.2 and Kv 1.4 alfa-subunits.
Although a majority of studies suggest that ER export is limited primarily by quality control (Rothman, J. 1987, Hurtley, S. and Helenius, A. 1989), export signals could play an important role in the concentration of secreted and membrane proteins. The putative export signal sequence, FCYENE, has been found to enhance the export of some potassium channels to the cell surface. In the case of Kv 1.2, the addition of FCYENE to the carboxy-terminus is correlated with major surface expression in transfected COS 7 cells. (Ma, D et al. 2000).
The dynamic expression of the three wild-type channels is different, possessing the following hierarchy of expression: Kv1.4 >> Kv1.2>>Kv1.1. Therefore, a comparison among their mutants will aid in clarification of the role of FCYENE as an export signal. In vitro translation permits analysis, via SDS-PAGE, of the core protein without any post-translational modification. This data can then be used as a point of reference for later studies, in which these channels are post-translationally modified.
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Figures
Figure 1-The new mutant PCR product contains the FCYENE sequence
Figure 2-PCR screening with the specific primer for FCYENE. The screened colonies with this method contain the mutation before the stop codon
Figure 3-In the lane containing the luciferase control, the smear is caused by too much protein loaded on the gel. The 61 kDa band observed in the lanes for Kv1.1(pcDNA3) and Kv1.4 are the result of leakage from the luciferase control lane.Background labeling of the 42 kDa band was anticipated, due to the use of a different [35S]-methionine, other than Redivue. Variations in the amount of loaded protein between the luciferase control and the mutant channels would have resulted in a better picture.
DISCUSSION
PCR is applicable to a variety of analyses. PCR amplification permits mutagenesis of cDNA templates via adjustment of the thermocycling parameters, as well as the variation of the initial concentration of the DNA template or the concentration of primers. Suggested parameters, such as a low cycle number, and high concentration of DNA template are reported to yield greater accuracy in obtaining the desired mutation. In this study, although a high cycle number was used, the results correlated with the expected size of the DNA and core proteins.
Another factor that provides some advantages, as well as disadvantages, is the use of different plasmids in mutagenesis. An advantage of the pGEM-T system is that it does not require restriction enzyme digestion for ligation. Also, the insertional inactivation of beta-galactosidase gene permits easier screening of transformants. On the other hand, to obtain the proper orientation of the insert, it is necessary to perform a secondary screen by PCR. In contrast, the pcDNA3
plasmid permits directional cloning. However, since pcDNA3 lacks insertional inactivation, colonies must be chosen randomly for screening, and then confirmed by PCR. Thus, in this study, it was relatively easy and efficient to find all the mutants in pGEM-T, while only one mutant (Kv 1.1) was recovered in pcDNA3.
Although Taq polymerase can introduce undesired mutations, the results of in vitro translation in these experiments demonstrates that mutations (e.g. insertion of a nucleotide creating a stop codon) due to the infidelity of Taq polymerase were not accidentally inserted into the sequence.
REFERENCES
Ashcroft, Frances M. (2000). Ion Channels and Disease. Academic Press.
Hille, B. (1992) Ionic Channels of Excitable Membranes. Sinauer Associates, Sunderland MA, (ed).
Hurtley, S, M and Helenius, A (1989) Protein oligomerization in the endoplasmic reticulum.
Annu Rev Cell Biol. 1989;5:277.
Ma, D., Zerangue, N., Lin Y., Collins, A., Yu, M., Jan, Y. and Jan L.(2001) Role of ER Export Signals in Controlling Surface Potassium Channel Numbers. Science 291: 316.
Rothman, J.E. (1987) Protein sorting by selective retention in the endoplasmic reticulum and Golgi stack. Cell. 1987 50: 521.
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Acknowledgments
am especially grateful to Rocco Coli, Sabrina Volpi, and Ira Daly for their patience and countless hours of assistance. I also would like to thank Dr. Thornhill for providing the wildtype cDNA constructs and for introducing me into the wonderful world of potassium channels. Thanks also to Dr. Rubin for the use of his lab and his help to make this project.
