Two Ways To Convert a Low-Affinity Potassium Channel to High Affinity: Control of Bacillus subtilis KtrCD by Glutamate

ABSTRACT Potassium and glutamate are the major cation and anion, respectively, in every living cell. Due to the high concentrations of both ions, the cytoplasm of all cells can be regarded as a potassium glutamate solution. This implies that the concentrations of both ions need to be balanced. While the control of potassium uptake by glutamate is well established for eukaryotic cells, much less is known about the mechanisms that link potassium homeostasis to glutamate availability in bacteria. Here, we have discovered that the availability of glutamate strongly decreases the minimal external potassium concentration required for the highly abundant Bacillus subtilis potassium channel KtrCD to accumulate potassium. In contrast, the inducible KtrAB and KimA potassium uptake systems have high apparent affinities for potassium even in the absence of glutamate. Experiments with mutant strains revealed that the KtrD subunit responds to the presence of glutamate. For full activity, KtrD synergistically requires the presence of the regulatory subunit KtrC and of glutamate. The analysis of suppressor mutants of a strain that has KtrCD as the only potassium uptake system and that experiences severe potassium starvation identified a mutation in the ion selectivity filter of KtrD (Gly282 to Val) that similarly results in a strongly glutamate-independent increase of the apparent affinity for potassium. Thus, this work has identified two conditions that increase the apparent affinity of KtrCD for potassium, i.e., external glutamate and the acquisition of a single point mutation in KtrD. IMPORTANCE In each living cell, potassium is required for maintaining the intracellular pH and for the activity of essential enzymes. Like most other bacteria, Bacillus subtilis possesses multiple low- and high-affinity potassium uptake systems. Their activity is regulated by the second messenger cyclic di-AMP. Moreover, the pools of the most abundant ions potassium and glutamate must be balanced. We report two conditions under which the low-affinity potassium channel KtrCD is able to mediate potassium uptake at low external potassium concentrations: physiologically, the presence of glutamate results in a severely increased potassium uptake. Moreover, this is achieved by a mutation affecting the selectivity filter of the KtrD channel. These results highlight the integration between potassium and glutamate homeostasis in bacteria.

Merits and Limitations of Studying Neuronal Depolarization-Dependent Processes Using Elevated External Potassium

Elevated extracellular potassium chloride is widely used to achieve membrane depolarization of cultured neurons. This technique has illuminated mechanisms of calcium influx through L-type voltage sensitive calcium channels, activity-regulated signaling, downstream transcriptional events, and many other intracellular responses to depolarization. However, there is enormous variability in these treatments, including durations from seconds to days and concentrations from 3mM to 150 mM KCl. Differential effects of these variable protocols on neuronal activity and transcriptional programs are underexplored. Furthermore, potassium chloride treatments in vitro are criticized for being poor representatives of in vivo phenomena and are questioned for their effects on cell viability. In this review, we discuss the intracellular consequences of elevated extracellular potassium chloride treatment in vitro, the variability of such treatments in the literature, the strengths and limitations of this tool, and relevance of these studies to brain functions and dysfunctions.

Growth Inhibition by External Potassium of Escherichia coli Lacking PtsN (EIIANtr) Is Caused by Potassium Limitation Mediated by YcgO

ABSTRACTThe absence of PtsN, the terminal phosphoacceptor of the phosphotransferase system comprising PtsP-PtsO-PtsN, inEscherichia coliconfers a potassium-sensitive (Ks) phenotype as the external K+concentration ([K+]e) is increased above 5 mM. A growth-inhibitory increase in intracellular K+content, resulting from hyperactivated Trk-mediated K+uptake, is thought to cause this Ks. We provide evidence that the Ksof the ΔptsNmutant is associated with K+limitation. Accordingly, the moderate Ksdisplayed by the ΔptsNmutant was exacerbated in the absence of the Trk and Kup K+uptake transporters and was associated with reduced cellular K+content. Conversely, overproduction of multiple K+uptake proteins suppressed the Ks. Expression of PtsN variants bearing the H73A, H73D, and H73E substitutions of the phosphorylation site histidine of PtsN complemented the Ks. Absence of the predicted inner membrane protein YcgO (also called CvrA) suppressed the Ks, which was correlated with elevated cellular K+content in the ΔptsNmutant, but the ΔptsNmutation did not alter YcgO levels. Heterologous overexpression ofycgOalso led to Ksthat was associated with reduced cellular K+content, exacerbated by the absence of Trk and Kup and alleviated by overproduction of Kup. Our findings are compatible with a model that postulates that Ksin the ΔptsNmutant occurs due to K+limitation resulting from activation of K+efflux mediated by YcgO, which may be additionally stimulated by [K+]e, implicating a role for PtsN (possibly its dephosphorylated form) as an inhibitor of YcgO activity.IMPORTANCEThis study examines the physiological link between the phosphotransferase system comprising PtsP-PtsO-PtsN and K+ion metabolism inE. coli. Studies on the physiological defect that renders anE. colimutant lacking PtsN to be growth inhibited by external K+indicate that growth impairment results from cellular K+limitation that is mediated by YcgO, a predicted inner membrane protein. Additional observations suggest that dephospho-PtsN may inhibit and external K+may stimulate K+limitation mediated by YcgO. It is speculated that YcgO-mediated K+limitation may be an output of a response to certain stresses, which by modulating the phosphotransfer capacity of the PtsP-PtsO-PtsN phosphorelay leads to growth cessation and stress tolerance.

Identification of a key residue in Kv7.1 potassium channel essential for sensing external potassium ions

Kv7.1 voltage-gated K+ (Kv) channels are present in the apical membranes of marginal cells of the stria vascularis of the inner ear, where they mediate K+ efflux into the scala media (cochlear duct) of the cochlea. As such, they are exposed to the K+-rich (∼150 mM of external K+ (K+e)) environment of the endolymph. Previous studies have shown that Kv7.1 currents are substantially suppressed by high K+e (independent of the effects of altering the electrochemical gradient). However, the molecular basis for this inhibition, which is believed to involve stabilization of an inactivated state, remains unclear. Using sequence alignment of S5-pore linkers of several Kv channels, we identified a key residue, E290, found in only a few Kv channels including Kv7.1. We used substituted cysteine accessibility methods and patch-clamp analysis to provide evidence that the ability of Kv7.1 to sense K+e depends on E290, and that the charge at this position is essential for Kv7.1’s K+e sensitivity. We propose that Kv7.1 may use this feedback mechanism to maintain the magnitude of the endocochlear potential, which boosts the driving force to generate the receptor potential of hair cells. The implications of our findings transcend the auditory system; mutations at this position also result in long QT syndrome in the heart.

Comparative Analysis ofkdpandktrMutants Reveals Distinct Roles of the Potassium Transporters in the Model Cyanobacterium Synechocystis sp. Strain PCC 6803

Photoautotrophic bacteria have developed mechanisms to maintain K+homeostasis under conditions of changing ionic concentrations in the environment.Synechocystissp. strain PCC 6803 contains genes encoding a well-characterized Ktr-type K+uptake transporter (Ktr) and a putative ATP-dependent transporter specific for K+(Kdp). The contributions of each of these K+transport systems to cellular K+homeostasis have not yet been defined conclusively. To verify the functionality of Kdp,kdpgenes were expressed inEscherichia coli, where Kdp conferred K+uptake, albeit with lower rates than were conferred by Ktr. An on-chip microfluidic device enabled monitoring of the biphasic initial volume recovery of singleSynechocystiscells after hyperosmotic shock. Here, Ktr functioned as the primary K+uptake system during the first recovery phase, whereas Kdp did not contribute significantly. The expression of thekdpoperon inSynechocystiswas induced by extracellular K+depletion. Correspondingly, Kdp-mediated K+uptake supportedSynechocystiscell growth with trace amounts of external potassium. This induction ofkdpexpression depended on two adjacent genes,hik20andrre19, encoding a putative two-component system. The circadian expression ofkdpandktrpeaked at subjective dawn, which may support the acquisition of K+required for the regular diurnal photosynthetic metabolism. These results indicate that Kdp contributes to the maintenance of a basal intracellular K+concentration under conditions of limited K+in natural environments, whereas Ktr mediates fast potassium movements in the presence of greater K+availability. Through their distinct activities, both Ktr and Kdp coordinate the responses ofSynechocystisto changes in K+levels under fluctuating environmental conditions.