Brian Roland Larsen

Contributions of the Na+/K+-ATPase, NKCC1, and Kir4.1 to hippocampal K+ clearance and volume responses

Network activity in the brain is associated with a transient increase in extracellular K+ concentration. The excess K+ is removed from the extracellular space by mechanisms proposed to involve Kir4.1‐mediated spatial buffering, the Na+/K+/2Cl− cotransporter 1 (NKCC1), and/or Na+/K+‐ATPase activity. Their individual contribution to [K+]o management has been of extended controversy. This study aimed, by several complementary approaches, to delineate the transport characteristics of Kir4.1, NKCC1, and Na+/K+‐ATPase and to resolve their involvement in clearance of extracellular K+ transients. Primary cultures of rat astrocytes displayed robust NKCC1 activity with [K+]o increases above basal levels. Increased [K+]o produced NKCC1‐mediated swelling of cultured astrocytes and NKCC1 could thereby potentially act as a mechanism of K+ clearance while concomitantly mediate the associated shrinkage of the extracellular space. In rat hippocampal slices, inhibition of NKCC1 failed to affect the rate of K+ removal from the extracellular space while Kir4.1 enacted its spatial buffering only during a local [K+]o increase. In contrast, inhibition of the different isoforms of Na+/K+‐ATPase reduced post‐stimulus clearance of K+ transients. The astrocyte‐characteristic α2β2 subunit composition of Na+/K+‐ATPase, when expressed in Xenopus oocytes, displayed a K+ affinity and voltage‐sensitivity that would render this subunit composition specifically geared for controlling [K+]o during neuronal activity. In rat hippocampal slices, simultaneous measurements of the extracellular space volume revealed that neither Kir4.1, NKCC1, nor Na+/K+‐ATPase accounted for the stimulus‐induced shrinkage of the extracellular space. Thus, NKCC1 plays no role in activity‐induced extracellular K+ recovery in native hippocampal tissue while Kir4.1 and Na+/K+‐ATPase serve temporally distinct roles. GLIA 2014;62:608–622

Managing Brain Extracellular K+ during Neuronal Activity: The Physiological Role of the Na+/K+-ATPase Subunit Isoforms

During neuronal activity in the brain, extracellular K+ rises and is subsequently removed to prevent a widespread depolarization. One of the key players in regulating extracellular K+ is the Na+/K+-ATPase, although the relative involvement and physiological impact of the different subunit isoform compositions of the Na+/K+-ATPase remain unresolved. The various cell types in the brain serve a certain temporal contribution in the face of network activity; astrocytes respond directly to the immediate release of K+ from neurons, whereas the neurons themselves become the primary K+ absorbers as activity ends. The kinetic characteristics of the catalytic α subunit isoforms of the Na+/K+-ATPase are, partly, determined by the accessory β subunit with which they combine. The isoform combinations expressed by astrocytes and neurons, respectively, appear to be in line with the kinetic characteristics required to fulfill their distinct physiological roles in clearance of K+ from the extracellular space in the face of neuronal activity. Understanding the nature, impact and effects of the various Na+/K+-ATPase isoform combinations in K+ management in the central nervous system might reveal insights into pathological conditions such as epilepsy, migraine, and spreading depolarization following cerebral ischemia. In addition, particular neurological diseases occur as a result of mutations in the α2- (familial hemiplegic migraine type 2) and α3 isoforms (rapid-onset dystonia parkinsonism/alternating hemiplegia of childhood). This review addresses aspects of the Na+/K+-ATPase in the regulation of extracellular K+ in the central nervous system as well as the related pathophysiology. Understanding the physiological setting in non-pathological tissue would provide a better understanding of the pathological events occurring during disease.

Kir4.1-mediated spatial buffering of K+: Experimental challenges in determination of its temporal and quantitative contribution to K+ clearance in the brain

Neuronal activity results in release of K+ into the extracellular space of the central nervous system. If the excess K+ is allowed to accumulate, neuronal firing will be compromised by the ensuing neuronal membrane depolarization. The surrounding glial cells are involved in clearing K+ from the extracellular space by molecular mechanism(s), the identity of which have been a matter of controversy for over half a century. Kir4.1-mediated spatial buffering of K+ has been promoted as a major contributor to K+ removal although its quantitative and temporal contribution has remained undefined. We discuss the biophysical and experimental challenges regarding determination of the contribution of Kir4.1 to extracellular K+ management during neuronal activity. It is concluded that 1) the geometry of the experimental preparation is crucial for detection of Kir4.1-mediated spatial buffering and 2) Kir4.1 enacts spatial buffering of K+ during but not after neuronal activity.

Hepatocyte growth factor activator inhibitor-2 prevents shedding of matriptase.

Hepatocyte growth factor activator inhibitor-2 (HAI-2) is an inhibitor of many proteases in vitro, including the membrane-bound serine protease, matriptase. Studies of knock-out mice have shown that HAI-2 is essential for placental development only in mice expressing matriptase, suggesting that HAI-2 is important for regulation of matriptase. Previous studies have shown that recombinant expression of matriptase was unsuccessful unless co-expressed with another HAI, HAI-1. In the present study we show that when human matriptase is recombinantly expressed alone in the canine cell line MDCK, then human matriptase mRNA can be detected and the human matriptase ectodomain is shed to the media, suggesting that matriptase expressed alone is rapidly transported through the secretory pathway and shed. Whereas matriptase expressed together with HAI-1 or HAI-2 accumulates on the plasma membrane where it is activated, as judged by cleavage at Arg614 and increased peptidolytic activity of the cell extracts. Mutagenesis of Kunitz domain 1 but not Kunitz domain 2 abolished this function of HAI-2. HAI-2 seems to carry out its function intracellularly as this is where the vast majority of HAI-2 is located and since HAI-2 could not be detected on the basolateral plasma membrane where matriptase resides. However, minor amounts of HAI-2 not undergoing endocytosis could be detected on the apical plasma membrane. Our results suggest that Kunitz domain 1 of HAI-2 cause matriptase to accumulate in a membrane-bound form on the basolateral plasma membrane.

Regulation and Function of AQP4 in the Central Nervous System.

Aquaporin 4 (AQP4) is the predominant water channel in the mammalian brain and is mainly expressed in the perivascular glial endfeet at the brain–blood interface. Based on studies on AQP4−/− mice, AQP4 has been assigned physiological roles in stimulus-induced K+ clearance, paravascular fluid flow, and brain edema formation. Conflicting data have been presented on the role of AQP4 in K+ clearance and associated extracellular space shrinkage and on the stroke-induced alterations of AQP4 expression levels during edema formation, raising questions about the functional importance of AQP4 in these (patho)physiological aspects. Phosphorylation-dependent gating of AQP4 has been proposed as a regulatory mechanism for AQP4-mediated osmotic water transport. This paradigm was, however, recently challenged by experimental evidence and molecular dynamics simulations. Regulatory patterns and physiological roles for AQP4 thus remain to be fully explored.

AQP4 plasma membrane trafficking or channel gating is not significantly modulated by phosphorylation at COOH-terminal serine residues

Aquaporin 4 (AQP4) is the predominant water channel in the mammalian brain and is mainly expressed in the perivascular glial endfeet at the brain-blood interface. AQP4 serves as a water entry site during brain edema formation, and regulation of AQP4 may therefore be of therapeutic interest. Phosphorylation of aquaporins can regulate plasma membrane localization and, possibly, the unit water permeability via gating of the AQP channel itself. In vivo phosphorylation of six serine residues in the COOH terminus of AQP4 has been detected by mass spectrometry: Ser(276), Ser(285), Ser(315), Ser(316), Ser(321), and Ser(322). To address the role of these phosphorylation sites for AQP4 function, serine-to-alanine mutants were created to abolish the phosphorylation sites. All mutants were detected at the plasma membrane of transfected C6 cells, with the fraction of the total cellular AQP4 expressed at the plasma membrane of transfected C6 cells being similar between the wild-type (WT) and mutant forms of AQP4. Activation of protein kinases A, C, and G in primary astrocytic cultures did not affect the plasma membrane abundance of AQP4. The unit water permeability was determined for the mutant AQP4s upon heterologous expression in Xenopus laevis oocytes (along with serine-to-aspartate mutants of the same residues to mimic a phosphorylation). None of the mutant AQP4 constructs displayed alterations in the unit water permeability. Thus phosphorylation of six different serine residues in the COOH terminus of AQP4 appears not to be required for proper plasma membrane localization of AQP4 or to act as a molecular switch to gate the water channel.

Glutamate transporter activity promotes enhanced Na+/K+-ATPase-mediated extracellular K+ management during neuronal activity

Management of glutamate and K+ in brain extracellular space is of critical importance to neuronal function. The astrocytic α2β2 Na+/K+‐ATPase isoform combination is activated by the K+ transients occurring during neuronal activity. In the present study, we report that glutamate transporter‐mediated astrocytic Na+ transients stimulate the Na+/K+‐ATPase and thus the clearance of extracellular K+. Specifically, the astrocytic α2β1 Na+/K+‐ATPase subunit combination displays an apparent Na+ affinity primed to react to physiological changes in intracellular Na+. Accordingly, we demonstrate a distinct physiological role in K+ management for each of the two astrocytic Na+/K+‐ATPase β‐subunits.

Activity-dependent astrocyte swelling is mediated by pH-regulating mechanisms: LARSEN and MACAULAY

During neuronal activity in the mammalian brain, the K+ released into the synaptic space is initially buffered by the astrocytic compartment. In parallel, the extracellular space (ECS) shrinks, presumably due to astrocytic cell swelling. With the Na+/K+/2Cl− cotransporter and the Kir4.1/AQP4 complex not required for the astrocytic cell swelling in the hippocampus, the molecular mechanisms underlying the activity‐dependent ECS shrinkage have remained unresolved. To identify these molecular mechanisms, we employed ion‐sensitive microelectrodes to measure changes in ECS, [K+]o and [H+]o/pHo during electrical stimulation of rat hippocampal slices. Transporters and receptors responding directly to the K+ and glutamate released into the extracellular space (the K+/Cl− cotransporter, KCC, glutamate transporters and G protein‐coupled receptors) did not modulate the extracellular space dynamics. The HCO3– ‐transporting mechanism, which in astrocytes mainly constitutes the electrogenic Na+/ HCO3– cotransporter 1 (NBCe1), is activated by the K+‐mediated depolarization of the astrocytic membrane. Inhibition of this transporter reduced the ECS shrinkage by ∼25% without affecting the K+ transients, pointing to NBCe1 as a key contributor to the stimulus‐induced astrocytic cell swelling. Inhibition of the monocarboxylate cotransporters (MCT), like‐wise, reduced the ECS shrinkage by ∼25% without compromising the K+ transients. Isosmotic reduction of extracellular Cl− revealed a requirement for this ion in parts of the ECS shrinkage. Taken together, the stimulus‐evoked astrocytic cell swelling does not appear to occur as a direct effect of the K+ clearance, as earlier proposed, but partly via the pH‐regulating transport mechanisms activated by the K+‐induced astrocytic depolarization and the activity‐dependent metabolism.

The α2β2 isoform combination dominates the astrocytic Na+/K+-ATPase activity and is rendered nonfunctional by the α2.G301R familial hemiplegic migraine type 2-associated mutation

Synaptic activity results in transient elevations in extracellular K+, clearance of which is critical for sustained function of the nervous system. The K+ clearance is, in part, accomplished by the neighboring astrocytes by mechanisms involving the Na+/K+‐ATPase. The Na+/K+‐ATPase consists of an α and a β subunit, each with several isoforms present in the central nervous system, of which the α2β2 and α2β1 isoform combinations are kinetically geared for astrocytic K+ clearance. While transcript analysis data designate α2β2 as predominantly astrocytic, the relative quantitative protein distribution and isoform pairing remain unknown. As cultured astrocytes altered their isoform expression in vitro, we isolated a pure astrocytic fraction from rat brain by a novel immunomagnetic separation approach in order to determine the expression levels of α and β isoforms by immunoblotting. In order to compare the abundance of isoforms in astrocytic samples, semi‐quantification was carried out with polyhistidine‐tagged Na+/K+‐ATPase subunit isoforms expressed in Xenopus laevis oocytes as standards to obtain an efficiency factor for each antibody. Proximity ligation assay illustrated that α2 paired efficiently with both β1 and β2 and the semi‐quantification of the astrocytic fraction indicated that the astrocytic Na+/K+‐ATPase is dominated by α2, paired with β1 or β2 (in a 1:9 ratio). We demonstrate that while the familial hemiplegic migraine‐associated α2.G301R mutant was not functionally expressed at the plasma membrane in a heterologous expression system, α2+/G301R mice displayed normal protein levels of α2 and glutamate transporters and that the one functional allele suffices to manage the general K+ dynamics.

Sensing and regulation of cell volume – we know so much and yet understand so little: TRPV4 as a sensor of volume changes but possibly without a volume-regulatory role?

ABSTRACT Cellular volume changes lead to initiation of cell volume regulatory events, the molecular identity of which remains unresolved. We here discuss experimental challenges associated with investigation of volume regulation during application of large, non-physiological osmotic gradients. The TRPV4 ion channel responds to volume increase irrespectively of the molecular mechanism underlying cell swelling, and is thus considered a sensor of volume changes. Evidence pointing towards the involvement of TRPV4 in subsequent volume regulatory mechanisms is intriguing, yet far from conclusive. We here present an experimental setting with astrocytic cell swelling in the absence of externally applied osmotic gradients, and the lack of evidence for involvement of TRPV4 in this regulatory volume response. Our aim with these new data and the preceding discussion is to stimulate further experimental effort in this area of research to clarify the role of TRPV4 and other channels and transporters in regulatory volume responses.