Clustering and Coupled Gating Modulate the Activity in KcsA, a Potassium Channel Model

Abstract

Different patterns of channel activity have been detected by patch clamping excised membrane patches from reconstituted giant liposomes containing purified KcsA, a potassium channel from prokaryotes. The more frequent pattern has a characteristic low channel opening probability and exhibits many other features reported for KcsA reconstituted into planar lipid bilayers, including a moderate voltage dependence, blockade by Na ؉ , and a strict dependence on acidic pH for channel opening. The predominant gating event in this low channel opening probability pattern corresponds to the positive coupling of two KcsA channels. However, other activity patterns have been detected as well, which are characterized by a high channel opening probability (HOP patterns), positive coupling of mostly five concerted channels, and profound changes in other KcsA features, including a different voltage dependence, channel opening at neutral pH, and lack of Na During the last decades, the use of high resolution electrophysiological techniques to study ion channels has provided a large amount of information on functional aspects of these important membrane proteins. Such a detailed information on channel function, however, has not been accompanied by structural knowledge until recently, when several structurally simpler homologues of mammalian ion channels found in extremophyle bacteria or Archaea and remarkably resistant to harsh experimental conditions, have been purified, crystallized and their structure solved at high resolution by x-ray diffraction methods (1-4). A K ϩ channel from the soil bacteria Streptomyces lividans named KcsA 4 (1), a homotetramer made up of identical 160-amino acid subunits, was the first of such structures to be solved (5, 6), and, although the x-ray structure corresponds to a closed channel conformation, it has contributed much to our current understanding of ion selectivity and permeation. Ironically, there was little or no functional information on KcsA by the time its structure was solved, and then several groups undertook the task of characterizing its single channel properties, which has been surrounded by controversy. For instance, Schrempf's group, discoverers of KcsA in S. lividans, reported a strong dependence of channel opening on acidic pH, multiple conductance states with opening probabilities near 0.5, and unusual permeabilities to Na ϩ , Li ϩ , Ca 2ϩ , or Mg 2ϩ , along with K ϩ (7-9). In contrast, Miller's group (10, 11) using purified KcsA reconstituted into planar lipid bilayers found a single conductance state with a much lower opening probability, as well as orthodox ion selectivity and other properties to validate KcsA as a bona fide K ϩ channel and as a faithful structural model for these molecules. The above discrepancies were never fully explained but, still, it became generally accepted that KcsA behaves as a moderately voltage-dependent, K ϩ -selective channel with a characteristic low opening probability and the peculiar property of opening only in response to very acidic pH condit...

Supramolecular Organization and Functional Implications of K+ Channel Clusters in Membranes

“…By combining DNP‐ssNMR experiments with MD simulations, we demonstrated that K + channel gating and clustering are correlated in membranes, which hints at a relationship between KcsA localization and function. Thereby, our study solves a striking conflict6b in the growing KcsA cluster literature, and strongly supports previous reports that KcsA channels can open in a concerted manner 6a. Intriguingly, our study discloses that the membrane‐associated M0 helix is a key element for cluster formation, and likely also for interchannel communication.…”

“…Hence, our DNP‐ssNMR experiments in native bacterial lipids revealed that cluster formation is a reversible, dynamic process that is correlated with the gating cycle of KcsA. Moreover, our study unambiguously demonstrates that open channels form clusters, which explains previous results from Molina and co‐workers 6a. Furthermore, our study reveals that the opening of the activating gate strongly stimulates cluster formation.…”

“…It is currently not clear if or how clustering modulates KcsA function, and contradicting studies have been published in recent years. Molina and co‐workers showed by electrophysiological measurements in liposomes that clustered channels open together in a concerted manner, and that clustering strongly increases channel open probability 6a. However, in stark contrast to the studies by Molina and co‐workers, AFM experiments, using truncated channels in zwitterionic lipids exposed to a mica surface, came to the conclusion that open channels do not form clusters 6b.…”

Probing the Mechanism of Exocytosis at the Hair Cell Ribbon Synapse

“…The compound Poisson model explored here offers a framework also for future analysis and simulations of exocytosis at ribbon synapses. It will as well be useful for describing other physiological stochastic processes with coordinated elementary events such as coupled gating of ion channels (Lehnart et al, 2003;Molina et al, 2006).…”

Molecular and Functional Characterization of a Na+-K+ Transporter from the Trk Family in the Ectomycorrhizal Fungus Hebeloma cylindrosporum

“…The deduced HcTrk1 amino acid sequence displays 12 hydrophobic domains, like other members of the Trk family initially thought to possess 12 transmembrane segments (19,(41)(42)(43). Latterly, based on multiple sequence alignments and residue conservation, Durell et al (14) have proposed that members of the Trk superfamily, which comprises Trk and KtrAB from prokaryotes, Trk from yeast, and HKT from plants, have evolved from minimal bacterial K ϩ channel proteins displaying two transmembrane segments, M1 and M2, surrounding a hydrophobic pore P, forming the so-called MPM structure (44). Trk transporters present four sequential MPM motifs, named A to D (Fig.…”