KcsA: It’s a Potassium Channel

Ion conduction and selectivity properties of KcsA, a bacterial ion channel of known structure, were studied in a planar lipid bilayer system at the single-channel level. Selectivity sequences for permeant ions were determined by symmetrical solution conductance (K Rb , NH 4 , Tl Cs , Na , Li ) and by reversal potentials under bi-ionic or mixed-ion conditions (Tl K Rb NH 4 Na , Li ). Determination of reversal potentials with submillivolt accuracy shows that K is over 150-fold more permeant than Na . Variation of conductance with concentration under symmetrical salt conditions is complex, with at least two ion-binding processes revealing themselves: a high affinity process below 20 mM and a low affinity process over the range 100–1,000 mM. These properties are analogous to those seen in many eukaryotic K channels, and they establish KcsA as a faithful structural model for ion permeation in eukaryotic K channels. key words: ion conductivity • selectivity I N T R O D U C T I O N Investigations of K channel mechanisms have been marked by functional feast and structural famine. For nearly 50 yr, K channels in eukaryotic cells have been subjected to detailed electrophysiological analysis for two basic purposes: (1) to understand their varied biological roles in ion transport and membrane electrical behavior, and (2) to reveal their underlying molecular characteristics. The electrical properties of K channels have been unusually informative toward both of these ends, and indeed accumulated work from many labs at the purely functional level has led to a remarkably detailed molecular picture of K channels. The high resolution structure of KcsA, a prokaryotic K channel, now provides a direct structural rationale for the functional behaviors long studied in eukaryotic K channels (Doyle et al., 1998). Therefore, it is ironic that the electrophysiological properties of KcsA itself have been only sparsely described, and at rather low functional resolution. To assess the validity of KcsA as a structural reference for permeation and selectivity in the ubiquitous K channel family, a close examination of ion conduction in this bacterial channel is needed. Two features of eukaryotic K channels are considered universal: (1) high selectivity among monovalent cations and (2) multiple ion occupancy in the pore. These channels permit permeation by K , Rb , NH 4 , and Tl (Hille, 2001) while excluding Na by 1,000fold (Yellen, 1984; Neyton and Miller, 1988a; Heginbotham and MacKinnon, 1993). For the KcsA structure to be considered mechanistically explanatory for eukaryotic K channels, high selectivity should be observed in this prokaryotic channel as well. However, the few published reports on ion conduction in KcsA offer sharply contradictory views of its pore. On one hand, KcsA channels reconstituted into liposomes showed familiar properties: strong selection for K and Rb over Na and specific block by Ba 2 (Cuello et al., 1998; Heginbotham et al., 1998), but these experiments used low resolution radioactive fluxes. In contrast, several direct single-channel studies (Schrempf et al., 1995; Meuser et al., 1999; Splitt et al., 2000) reported robust conduction by Na , Li , and Mg 2 along with K , a characteristic never described for any eukaryotic K channel. These reports led to three alarming conclusions: (1) that in its conducting conformation, the permeation pathway of KcsA is 6 Å wide at its narrowest point; (2) that K permeates with a full hydration shell; and (3) that the high resolution structure of KcsA is misleading as a model for ion conduction in K channels. In this study, we examine ion-permeation properties of KcsA with a classical, high resolution electrophysiological approach. Purified KcsA protein was reconstituted into a chemically defined planar lipid bilayer system, and single-channel properties were observed under varying conditions of ion concentration, ion type, and voltage. We show that KcsA is readily permeable to K and its close analogues Rb , NH 4 , and Tl , whereas permeability for Na , Li , and Cs is undetectable. The experiments show that in its ion-conduction properties KcsA behaves according to familiar principles long established in eukaryotic K channels. The online version of this article contains supplemental material. The present address of L. Heginbotham is Department of Molecular Biophysics and Biochemistry, Yale University New Haven, CT 06520. Address correspondence to Christopher Miller, Department of Biochemistry, Howard Hughes Medical Institute, Brandeis University, Waltham, MA 02454. Fax: (781) 736-2365; E-mail: cmiller@ brandeis.edu on A uust 0, 2017 jgp.rress.org D ow nladed fom http://doi.org/1 .1085/jgp.118.3.303 Supplemental material can be found at: 304 Ionic Selectivity in KcsA M A T E R I A L S A N D M E T H O D S