Coordination of K+Transporters in Neurospora: TRK1 Is Scarce and Constitutive, while HAK1 Is Abundant and Highly Regulated

Abstract

ABSTRACTFungi, plants, and bacteria accumulate potassium via two distinct molecular machines not directly coupled to ATP hydrolysis. The first, designated TRK, HKT, or KTR, has eight transmembrane helices and is folded like known potassium channels, while the second, designated HAK, KT, or KUP, has 12 transmembrane helices and resembles MFS class proteins. One of each type functions in the model organismNeurospora crassa, where both are readily accessible for biochemical, genetic, and electrophysiological characterization. We have now determined the operating balance between Trk1p and Hak1p under several important conditions, including potassium limitation and carbon starvation. Growth measurements, epitope tagging, and quantitative Western blotting have shown the geneHAK1to be much more highly regulated than isTRK1. This conclusion follows from three experimental results: (i) Trk1p is expressed constitutively but at low levels, and it is barely sensitive to extracellular [K+] and/or the coexpression ofHAK1; (ii) Hak1p is abundant but is markedly depressed by elevated extracellular concentrations of K+and by coexpression ofTRK1; and (iii) Carbon starvation slowly enhances Hak1p expression and depresses Trk1p expression, yielding steady-state Hak1p:Trk1p ratios of ∼500:1,viz., 10- to 50-fold larger than that in K+- and carbon-replete cells. Additionally, it appears that both potassium transporters can adjust kinetically to sustained low-K+stress by means of progressively increasing transporter affinity for extracellular K+. The underlying observations are (iv) that K+influx via Trk1p remains nearly constant at ∼9 mM/h when extracellular K+is progressively depleted below 0.05 mM and (v) that K+influx via Hak1p remains at ∼3 mM/h when extracellular K+is depleted below 0.1 mM.