Miguel Holmgren - US grants

The long term objective of this project is an understanding, at a molecular level, of how the Na/K pump works. This virtually ubiquitous enzyme uses the energy from the hydrolysis of ATP to transport Na+ and K+ ions against their electrochemical gradient. By doing so, the Na/K pump maintains the K gradient that generates the transmembrane resting potential and the Na gradient which is used to produce electrical signals as well as to drive the uphill transport across the cell membrane of other substances. These ionic gradients are also responsible for cell volume regulation. Clinically, the Na/K pump is important because it is the receptor of digoxin, a widely prescribed cardiac steroid used to control some cardiac arrhythmias. In each pump cycle, three Na+ ions and two K+ ions are translocated through the protein. When ions are moved through the protein, electrical charge is translocated which can be measured and used to learn some properties of the movement of ions through the protein. To achieve the objective, voltage-induced steady and transient currents elicited by Na/K pumps will be measured in two preparations: the cloned rat brain pump expressed in a mammalian cell line and the endogenous pump from squid giant axons. For the recombinant rat brain pump, the possibility to manipulate the protein itself will be used to learn about the structure and function of the Na/K pump. Site-directed mutagenesis of selected residues to cysteine, and subsequent chemical modification, will be used to map the exposed surface of the protein. We will also exploit the interaction of palytoxin with the Na/K pump. Palytoxin is a marine toxin that is believed to transform the pump into a "channel", so that ions then move at rates 10,000 times faster than through the normal pumps. In the squid giant axon preparation, the access to internal and external solutions as well as the very fast voltage-clamp will be exploited to learn about the mechanism of ion translocation, particularly for Na+ ions. The three specific aims for this project are (i) to understand the ion-channel-like behavior by the Na/K pump, (ii) to determine the exposed surface of the pathways used by Na+ and K+ ions as they move through the protein, and (iii) to pursue the biophysical properties of the Na+ ion translocation mechanism through the Na/K pump.

Voltage-activated potassium (KV) channels are potassium selective integral membrane proteins formed by the assembly of four homologous subunits. In response to a membrane depolarization, KV channels open allowing K+ to permeate. In some members of KV channels, sustain depolarization leads to inactivation caused by an N terminus gate. In shaker KV channels, the first 20 amino acids at the NH2 terminus of the protein are essential in enabling it to act as a gate. The tip of the NH2 terminus interacts with residues in the intracellular cavity of KV channels, blocking the permeation of K+. By nature of their tetrameric architecture, inactivating KV channels have four N terminus gates and a set of four sites of action in the intracellular cavity. Yet, N-type inactivation is produced by the binding of only one N terminus gate. Is the site of action in the pore specific to the subunit to which the bound N terminus belongs? To study the interactions between the N terminus gate and the site of action we first constructed a Shaker concatemer KV channel having only one free N terminus gate. The three subunits concatenated at the N terminus have the well-known Δ6-46 inactivation removing deletion. In addition, all subunits contained a mutation to remove C-type inactivation (T449V), which will greatly simplify our kinetic analysis. In this concatemer, we observed a fourfold reduction in the association rate relative to Shaker homotetramers, consistent with the presence of only one N terminus gate relative to four (Figure 1 left, center). In Shaker homotetramers, the tip of the N terminus gate acts on position 470 at the intracellular cavity of the channel. Mutating this residue from isoleucine to valine (I470V) has a dramatic effect on the extent of N-type inactivation. This effect was reproduced in our concatemer by mutating I470V in all subunits. We reasoned that if the N terminus gate interacts with only a single subunit inside the pore to produce inactivation, then a single I470V mutation at this interacting subunit should produce a reduction in inactivation comparable to that observed when all four subunits are mutated to I470V. We are presently testing this hypothesis. We also study the gating mechanisms of transporters, like the Na/K-ATPase. This enzyme, a member of the P-type family (named for their phosphorylated intermediates), harnesses the energy from the hydrolysis of one ATP to alternately export 3Na ions and import 2K ions against their electrochemical gradients. By performing this active transport, the Na/K pump plays an essential role in the homeostasis of intracellular Na and K that is crucial to sustaining cell excitability, volume, and Na-dependent secondary transport. On the basis of biochemical data accumulated during the decade following its discovery, the Na/K-ATPase was proposed to alternately transport Na and K ions according to a model known as the Post-Albers scheme. As ions are transported through the Na/K pump, they become temporarily occluded within the protein, inaccessible from either side, before being released. By restricting Na/K pumps to only the reversible transitions associated with deocclusion and extracellular release of Na+, it is possible to detect pre-steady state electrical signals accompanying those transitions. The signals arise because Na+ traverse a fraction of the membrane potential as they enter or leave their binding sites deep within the pump. At a fixed membrane potential and external sodium concentration, the populations of pumps with empty binding sites, and those with bound or occluded Na, reach a steady-state distribution. A sudden change of membrane voltage then shifts the Na-binding equilibrium, and initiates a redistribution of the pump populations towards a new steady state. The consequent change in Na-binding-site occupancy causes Na to travel between the extracellular environment and the pump interior. In so doing they generate a current. As the system approaches a new steady distribution, fewer Na move, and the current declines. The electrical signals therefore appear as transient currents. Using the squid giant axon preparation, which exploits axial current delivery to generate very fast membrane voltage steps, we previously identified three phases of relaxation in transient pump currents (Holmgren et al., 2000): fast (comparable to the voltage-jump time course), medium-speed (tm 0.2-0.5 ms), and slow (ts 1-10 ms). We suggested that each phase reflects a distinct Na-binding event (or release, depending on the direction of the voltage change) with its associated conformational transition (occlusion or deocclusion). In other words, the Na/K-ATPase undergoes dynamic rearrangements that open external gates to allow bound Na access to the extracellular environment immediately prior to release. We would like to understand how these gates operate, the precise dynamic relationships between the three events that release individual Na+ from the Na/K-ATPase, the thermodynamic principles that govern these conformational changes, the structural movements underlying these events, as well as the type of structural dynamics associated with them.

RNA editing of ion channels and receptors has important consequences to their function. RNA editing is a co-transcriptional modification of pre-mRNA performed by an enzyme that converts adenosine into inosine (A to I). Inosine is interpreted as guanosine by the translational machinery. Thereby, RNA editing provides a substantial expansion of the genetic pool of an organism. As might be intuitively expected, A to I conversions often target critical positions of the encoded protein, where changes in function are essential for the physiology of a cell. Over the past six years, we have become increasingly interested in understanding how nature functionally tunes membrane proteins by RNA editing. RNA editing allows multiple protein products from a single gene. This increase in genomic capability does not appear to be random, rather it targets regions of a protein that are functionally important. The classical example is in the GluRB subunit of glutamate-gated ion channels, important for fast excitatory synaptic transmission in the central nervous system. Editing underlies the conversion of glutamine to arginine in the channelfs pore (Sommer et al., 1991), which renders the receptor impermeable to calcium ions (Kohler et al., 1993). Although editing seems to be common among membrane proteins (Hoopengardner et al., 2003), the functional consequences of editing had been explored in only a few examples (Kohler et al., 1993;Burns et al., 1997;Patton et al., 1997;Wang et al., 2000;Berg et al., 2001;Rosenthal and Bezanilla, 2002;Bhalla et al., 2004). We have joined efforts with the laboratory of Josh Rosenthal to approach the subject of RNA editing of membrane proteins in a comprehensive manner. We have already characterized the functional consequences of an I to V conversion within the intracellular cavity of the human KV1.1 channel (Bhalla et al., 2004). In this case, RNA editing targets fast inactivation, an additional gating mechanism of KV1.1 channels. This study also provided the foundation to ask previously inaccessible questions about the interactions between the inactivation particle and the ion conduction pore. Because fast inactivation occurs by the direct occlusion of the permeation pathway by an inactivating particle (Hoshi et al., 1990;Zagotta et al., 1990;Demo and Yellen, 1991;Zhou et al., 2001a), we are asking which amino acids from this particle interact with the core of the channel, and what is the specific mechanisms by which fast inactivation is altered by the I to V conversion . Our preliminary observations suggest that the end of the N-terminus can actually enter deep into the intracellular cavity and interact in close proximity with the edited position. Transporters are essential for ion channel function because they provide and maintain the ionic gradient that allows ions to diffuse through channels. Recently, by comparing genomic and cDNAs sequences, new targets of RNA editing have been identified, among them, proteins involved in ion homeostasis (Stapleton et al., 2006). Interestingly, there is no report in the literature of how RNA editing might alter the function of any transporter. We are taking advantage of the apparent high levels of editing in squid (Patton et al., 1997;Rosenthal and Bezanilla, 2002) to examine RNA editing in transporters, initially focusing our attention on the Na+/K+ pump, a transporter that I have studied over the past 15 years, and the Na+/Ca2+ exchanger. At present, we have cloned the full-length cDNA and genomic DNA for both transporters from squid, and we have found at least four potential RNA editing sites in the Na+/K+ pump and six in the Na+/Ca2+ exchanger. We have been successful in expressing the genomic and edited constructs of these transporters in Xenopus oocytes, which will allow us to characterize the functional consequences of the editing events. We have already uncovered potentially interesting functional consequences in both transporters. In the Na+/K+ pump, RNA editing seems to produce an increase in the apparent affinity for intracellular ATP and changes in the binding/release and occlusion/deoclusion kinetics of external Na+. In the exchanger, recovery from intracellular Na+ inactivation appears to be altered by editing. Our goal is to develop a mechanistic understanding of how editing influence the function of these transporters, hopefully to the level of details we are acquiring in KV1.1 channels.

RNA editing of ion channels and receptors has important consequences to their function. RNA editing is a co-transcriptional modification of pre-mRNA performed by an enzyme that converts adenosine into inosine (A to I). Inosine is interpreted as guanosine by the translational machinery. Thereby, RNA editing provides a substantial expansion of the genetic pool of an organism. As might be intuitively expected, A to I conversions often target critical positions of the encoded protein, where changes in function are essential for the physiology of a cell. Over the past six years, we have become increasingly interested in understanding how nature functionally tunes membrane proteins by RNA editing. RNA editing allows multiple protein products from a single gene. This increase in genomic capability does not appear to be random, rather it targets regions of a protein that are functionally important. The classical example is in the GluRB subunit of glutamate-gated ion channels, important for fast excitatory synaptic transmission in the central nervous system. Editing underlies the conversion of glutamine to arginine in the channelfs pore (Sommer et al., 1991), which renders the receptor impermeable to calcium ions (Kohler et al., 1993). Although editing seems to be common among membrane proteins (Hoopengardner et al., 2003), the functional consequences of editing had been explored in only a few examples (Kohler et al., 1993; Burns et al., 1997; Patton et al., 1997; Wang et al., 2000; Berg et al., 2001; Rosenthal and Bezanilla, 2002; Bhalla et al., 2004). We have joined efforts with the laboratory of Josh Rosenthal to approach the subject of RNA editing of membrane proteins in a comprehensive manner. In excitable cells, precisely synchronized ionic currents generate the electric potentials that are the currency of communication. For the system to operate, the timing of turning the currents off is as important as the timing of turning them on. Accordingly, ion channels have developed intricate systems to switch off that override the signals to stay on, a process known as inactivation. In rodent and human KV1.1 channels, RNA editing recodes a highly conserved isoleucine to a valine (I400) (Hoopengardner et al., 2003). This conversion is regulated in different regions of the nervous system. Structural (Long et al., 2005) and functional (Liu et al., 1997) data show that I400 is in the lining of the permeation pathway, in a region known as the channelfs intracellular cavity. I400V selectively targets the process of fast inactivation (Bhalla et al., 2004), allowing edited channels to recover from inactivation about 20 times faster than their unedited counterparts. For a neuron, this change in function would greatly influence action potential shape, signal propagation and the firing pattern (Connor and Stevens, 1971; Aldrich et al., 1979; Debanne et al., 1997; Hoffman et al., 1997; Giese et al., 1998; Johnston et al., 1999). In this study we investigated the precise mechanism by which I400V alters fast inactivation. The robust functional consequence of editing on inactivation is conserved in phylogenetically diverse KV channels, and mimicking I400V in Shaker KV channels (I470V) produces an increase in the speed of recovery from inactivation comparable to that seen in the human isoform (Bhalla et al., 2004). Using an intracellular cysteine-less construct of shaker, we studied the mechanistic details underlying the I400V phenotype by changing the chemistry at this site by mutating it to a cysteine, and attaching different chemical moieties. Our results are consistent with the simple idea that nature uses RNA editing to increase the unbinding kinetics of the inactivation particle. It accomplishes this by selectively reducing the hydrophobic interaction between the tip of the N-terminus and its receptor, the edited codon within the intracellular cavity. Further, our experiments suggest that to obstruct permeation the inactivation particle must penetrate deeply into the intracellular cavity in an extended conformation. We have recently began three new venues in our studies of RNA editing. First, we are performing illumina sequences of different species of cephalopods to understand the extent of RNA editing and how conserved are the sites among different species. Second, we would like to understand how RNA editing occurs at the single cell level. We are performing preliminary experiments using an adapted FISH methodology to detect single nucleotide variants at the single mRNA molecule level. Third, have begun studying the kinetics of editing by the enzyme ADAR2 using in vitro assays.