Purification and initial characterization of Plasmodium falciparum K+ channels, PfKch1 and PfKch2 produced in Saccharomyces cerevisiae.
ABSTRACT: Resistance towards known antimalarial drugs poses a significant problem, urging for novel drugs that target vital proteins in the malaria parasite Plasmodium falciparum. However, recombinant production of malaria proteins is notoriously difficult. To address this, we have investigated two putative K+ channels, PfKch1 and PfKch2, identified in the P. falciparum genome. We show that PfKch1 and PfKch2 and a C-terminally truncated version of PfKch1 (PfKch11-1094) could indeed be functionally expressed in vivo, since a K+-uptake deficient Saccharomyces cerevisiae strain was complemented by the P. falciparum cDNAs. PfKch11-1094-GFP and GFP-PfKch2 fusion proteins were overexpressed in yeast, purified and reconstituted in lipid bilayers to determine their electrophysiological activity. Single channel conductance amounted to 16?±?1 pS for PfKch11-1094-GFP and 28?±?2 pS for GFP-PfKch2. We predicted regulator of K+-conductance (RCK) domains in the C-terminals of both channels, and we accordingly measured channel activity in the presence of Ca2+.
Project description:Regulator of K(+) conductance (RCK) domains control the activity of a variety of K(+) transporters and channels, including the human large conductance Ca(2+)-activated K(+) channel that is important for blood pressure regulation and control of neuronal firing, and MthK, a prokaryotic Ca(2+)-gated K(+) channel that has yielded structural insight toward mechanisms of RCK domain-controlled channel gating. In MthK, a gating ring of eight RCK domains regulates channel activation by Ca(2+). Here, using electrophysiology and X-ray crystallography, we show that each RCK domain contributes to three different regulatory Ca(2+)-binding sites, two of which are located at the interfaces between adjacent RCK domains. The additional Ca(2+)-binding sites, resulting in a stoichiometry of 24 Ca(2+) ions per channel, is consistent with the steep relation between [Ca(2+)] and MthK channel activity. Comparison of Ca(2+)-bound and unliganded RCK domains suggests a physical mechanism for Ca(2+)-dependent conformational changes that underlie gating in this class of channels.
Project description:Regulator of conduction of K+ (RCK) domains are ubiquitous regulators of channel and transporter activity in prokaryotes and eukaryotes. In humans, RCK domains form an integral component of large-conductance calcium-activated K channels (BK channels), key modulators of nerve, muscle, and endocrine cell function. In this review, we explore how the study of RCK domains in bacterial and human channels has contributed to our understanding of the structural basis of channel function. This knowledge will be critical in identifying mechanisms that underlie BK channelopathies that lead to epilepsy and other diseases, as well as regions of the channel that might be successfully targeted to treat such diseases.
Project description:Large conductance voltage and Ca(2+)-dependent K(+) channels (BK(Ca)) are activated by both membrane depolarization and intracellular Ca(2+). Recent studies on bacterial channels have proposed that a Ca(2+)-induced conformational change within specialized regulators of K(+) conductance (RCK) domains is responsible for channel gating. Each pore-forming alpha subunit of the homotetrameric BK(Ca) channel is expected to contain two intracellular RCK domains. The first RCK domain in BK(Ca) channels (RCK1) has been shown to contain residues critical for Ca(2+) sensitivity, possibly participating in the formation of a Ca(2+)-binding site. The location and structure of the second RCK domain in the BK(Ca) channel (RCK2) is still being examined, and the presence of a high-affinity Ca(2+)-binding site within this region is not yet established. Here, we present a structure-based alignment of the C terminus of BK(Ca) and prokaryotic RCK domains that reveal the location of a second RCK domain in human BK(Ca) channels (hSloRCK2). hSloRCK2 includes a high-affinity Ca(2+)-binding site (Ca bowl) and contains similar secondary structural elements as the bacterial RCK domains. Using CD spectroscopy, we provide evidence that hSloRCK2 undergoes a Ca(2+)-induced change in conformation, associated with an alpha-to-beta structural transition. We also show that the Ca bowl is an essential element for the Ca(2+)-induced rearrangement of hSloRCK2. We speculate that the molecular rearrangements of RCK2 likely underlie the Ca(2+)-dependent gating mechanism of BK(Ca) channels. A structural model of the heterodimeric complex of hSloRCK1 and hSloRCK2 domains is discussed.
Project description:Gap junction channels and hemichannels formed by concatenated connexins were analyzed. Monomeric (hCx26, hCx46), homodimeric (hCx46-hCx46, hCx26-hCx26), and heterodimeric (hCx26-hCx46, hCx46-hCx26) constructs, coupled to GFP, were expressed in HeLa cells. Confocal microscopy showed that the tandems formed gap junction plaques with a reduced plaque area compared to monomeric hCx26 or hCx46. Dye transfer experiments showed that concatenation allows metabolic transfer. Expressed in Xenopus oocytes, the inside-out patch-clamp configuration showed single channels with a conductance of about 46 pS and 39 pS for hemichannels composed of hCx46 and hCx26 monomers, respectively, when chloride was replaced by gluconate on both membrane sides. The conductance was reduced for hCx46-hCx46 and hCx26-hCx26 homodimers, probably due to the concatenation. Heteromerized hemichannels, depending on the connexin-order, were characterized by substates at 26 pS and 16 pS for hCx46-hCx26 and 31 pS and 20 pS for hCx26-hCx46. Because of the linker between the connexins, the properties of the formed hemichannels and gap junction channels (e.g., single channel conductance) may not represent the properties of hetero-oligomerized channels. However, should the removal of the linker be successful, this method could be used to analyze the electrical and metabolic selectivity of such channels and the physiological consequences for a tissue.
Project description:Calcium-dependent gating of large-conductance calcium-activated potassium (BK(Ca)) channels is mediated by the intracellular carboxyl terminus, which contains two domains of regulator of K(+) conductance (RCK). In mammalian BK(Ca) channels, the two RCK domains are separated by a protein segment of 101 residues that is poorly conserved in evolution and predicted to have no regular secondary structures. We investigated the functional importance of this loop using a series of deletion mutations. We found that the length, rather than the specific sequence at the central region of the segment, is critical for the functionality of the channel. As the length of the loop is progressively shorted, the conductance-voltage relationship gradually shifts toward more positive voltages with a minimum length of 70 amino acids, in an apparent response to increased tension within the loop. Thus, the functional activity of the BK(Ca) channel can be modulated by altering the tension of this loop region.
Project description:Large conductance, voltage- and Ca2+-activated K+ (BK(Ca)) channels regulate blood vessel tone, synaptic transmission, and hearing owing to dual activation by membrane depolarization and intracellular Ca2+. Similar to an archeon Ca2+-activated K+ channel, MthK, each of four alpha subunits of BK(Ca) may contain two cytosolic RCK domains and eight of which may form a gating ring. The structure of the MthK channel suggests that the RCK domains reorient with one another upon Ca2+ binding to change the gating ring conformation and open the activation gate. Here we report that the conformational changes of the NH2 terminus of RCK1 (AC region) modulate BK(Ca) gating. Such modulation depends on Ca2+ occupancy and activation states, but is not directly related to the Ca2+ binding sites. These results demonstrate that AC region is important in the allosteric coupling between Ca2+ binding and channel opening. Thus, the conformational changes of the AC region within each RCK domain is likely to be an important step in addition to the reorientation of RCK domains leading to the opening of the BK(Ca) activation gate. Our observations are consistent with a mechanism for Ca2+-dependent activation of BK(Ca) channels such that the AC region inhibits channel activation when the channel is at the closed state in the absence of Ca2+; Ca2+ binding and depolarization relieve this inhibition.
Project description:High-conductance voltage- and Ca2+-activated K+ (BK) channels encode negative feedback regulation of membrane voltage and Ca2+ signaling, playing a central role in numerous physiological processes. We determined the x-ray structure of the human BK Ca2+ gating apparatus at a resolution of 3.0 angstroms and deduced its tetrameric assembly by solving a 6 angstrom resolution structure of a Na+-activated homolog. Two tandem C-terminal regulator of K+ conductance (RCK) domains from each of four channel subunits form a 350-kilodalton gating ring at the intracellular membrane surface. A sequence of aspartic amino acids that is known as the Ca2+ bowl, and is located within the second of the tandem RCK domains, creates four Ca2+ binding sites on the outer perimeter of the gating ring at the "assembly interface" between RCK domains. Functionally important mutations cluster near the Ca2+ bowl, near the "flexible interface" between RCK domains, and on the surface of the gating ring that faces the voltage sensors. The structure suggests that the Ca2+ gating ring, in addition to regulating the pore directly, may also modulate the voltage sensor.
Project description:To date, the biological role of prokaryotic K(+) channels remains unknown. Helicobacter pylori contains a gene encoding a putative K(+) channel (HpKchA) of the two-transmembrane RCK (regulation of K(+) conductance) domain family, but lacks known bacterial K(+) uptake systems. A H. pylori DeltahpKchA mutant presented a strong growth defect at low K(+) concentration, which was compensated by KCl addition. The role of the separate RCK domain was investigated in H. pylori by mutagenesis of its internal start codon, which led to a K(+)-dependent intermediate growth phenotype, consistent with RCK activating channel function. Tagging HpKchA C-terminally, we detected a 1:1 stoichiometry of the full-length HpKchA and the separate RCK domain. We constructed single amino-acid exchanges within the unusual selectivity filter of HpKchA (ATGFGA) in H. pylori and observed complete loss (G74A), a slight defect (G76A or F75G) or wild-type (A77D) channel function. HpKchA was essential for colonization of the murine stomach. These data show, for the first time, a biological function for a prokaryotic K(+) channel, as a K(+) uptake system, essential for the persistence of H. pylori in the gastric environment.
Project description:Previously, we characterized a Shaker-related family of voltage-gated potassium channels (RCK) in rat brain. Now, we describe a second family of voltage-gated potassium channels in the rat nervous system. This family is related to the Drosophila Shaw gene and has been dubbed Raw. In contrast to the RCK potassium channel family the Raw family utilizes extensive alternative splicing for expressing potassium channel subunits with variant C-termini. These alternative C-termini do not appear to influence the electrophysiological and pharmacological properties as studied in the Xenopus oocyte expression system. In situ hybridizations to sections of rat brain indicate that members of the Raw family are expressed in distinct areas of the central nervous system. Probably, Raw channels are expressed predominantly as homomultimers. Immunocytochemical experiments with antibodies against Raw3 and RCK4 proteins which form two distinct A-type potassium channels indicate that in hippocampus the two channels are expressed both in different neurons and in the same ones. In general, properties of Raw potassium channels appeared to be similar to RCK channels. However, Raw outward currents, in contrast to RCK currents, exhibit an intense rectification at test potentials higher than +20 to +40 mV. RCK and Raw channel subunits did not measurably coassemble into RCK/Raw heteromultimers after coinjecting RCK and Raw cRNA into Xenopus oocytes. These results suggest that members of the RCK and the Raw potassium channel families express potassium channels which form independent outward current systems. Combining the results of in situ hybridizations, immunocytochemical staining and expression of the cloned potassium channels in Xenopus oocytes demonstrates that unrestrained mixing of potassium channel subunits to form hybrid channels does not occur in the rat central nervous system. A single neuron is able to express multiple, independently assembled potassium channels.
Project description:Functional mechanoelectrical transduction (MET) channels of cochlear hair cells require the presence of transmembrane channel-like protein isoforms TMC1 or TMC2. We show that TMCs are required for normal stereociliary bundle development and distinctively influence channel properties. TMC1-dependent channels have larger single-channel conductance and in outer hair cells (OHCs) support a tonotopic apex-to-base conductance gradient. Each MET channel complex exhibits multiple conductance states in ~50?pS increments, basal MET channels having more large-conductance levels. Using mice expressing fluorescently tagged TMCs, we show a three-fold increase in number of TMC1 molecules per stereocilium tip from cochlear apex to base, mirroring the channel conductance gradient in OHCs. Single-molecule photobleaching indicates the number of TMC1 molecules per MET complex changes from ~8 at the apex to ~20 at base. The results suggest there are varying numbers of channels per MET complex, each requiring multiple TMC1 molecules, and together operating in a coordinated or cooperative manner.