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J. Biol. Chem., Vol. 282, Issue 33, 24294-24301, August 17, 2007

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Patch Clamp and Phenotypic Analyses of a Prokaryotic Cyclic Nucleotide-gated K⁺ Channel Using Escherichia coli as a Host^*

Mario Meng-Chiang Kuo, Yoshiro Saimi, Ching Kung^¶, and Senyon Choe¹

From the Structural Biology Laboratory, The Salk Institute, La Jolla, California 92037 and Laboratory of Molecular Biology and ^¶Department of Genetics, University of Wisconsin-Madison, Madison, Wisconsin 53706

Prokaryotic ion channels have been valuable in providing structural models for understanding ion filtration and channel-gating mechanisms. However, their functional examinations have remained rare and usually been carried out by incorporating purified channel protein into artificial lipid membranes. Here we demonstrate the utilization of Escherichia coli to host the functional analyses by examining a putative cyclic nucleotide-gated K⁺ channel cloned from Magnetospirillum magnetotacticum, MmaK. When expressed in wild-type E. coli cells, MmaK renders the host sensitive to millimolar concentrations of externally applied K⁺, indicating MmaK forms a functional K⁺ conduit in the E. coli membrane in vivo. After enlarging these cells into giant spheroplasts, macro- and microscopic MmaK currents are readily detected in excised E. coli membrane patches by a patch clamp. We show that MmaK is indeed gated by submicromolar cAMP and 10-fold higher concentration of cGMP and manifests as an inwardly rectified, K⁺-specific current with a 10.8 pS unitary conductance at -100 mV. Additionally, MmaK is inactivated by slightly acidic pH only from the cytoplasmic side. Our in vitro biophysical characterizations of MmaK correlate with its in vivo phenotype in E. coli, implicating its critical role as an intracellular cAMP and pH sensor for modulating bacterial membrane potential. Exemplified by MmaK functional studies, we establish that E. coli and its giant spheroplast provide a convenient and versatile system to express foreign channels for biophysical analyses that can be further dovetailed with microbial genetics.

Received for publication, May 1, 2007 , and in revised form, June 21, 2007.

^* This work was supported by National Institutes of Health Grants GM74821 (to S. C.), GM054867 (to Y. S.), and GM047856 (to C. K.) and the Vilas Trust of the University of Wisconsin-Madison. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

¹ To whom correspondence should be addressed: 10010 N. Torry Pines Rd., La Jolla, CA 92037. Fax: 858-452-3683; E-mail: choe{at}salk.edu.

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