Structural basis for ion selectivity in TMEM175 K+ channels

Abstract

The TMEM175 family constitutes recently discovered K+channels that are important for autophagosome turnover and lysosomal pH regulation and are associated with the early onset of Parkinson Disease. TMEM175 channels lack a P-loop selectivity filter, a hallmark of all known K+ channels, raising the question how selectivity is achieved. Here, we report the X-ray structure of a closed bacterial TMEM175 channel in complex with a nanobody fusion-protein disclosing bound K+ ions. Our analysis revealed that a highly conserved layer of threonine residues in the pore conveys a basal K+ selectivity. An additional layer comprising two serines in human TMEM175 increases selectivity further and renders this channel sensitive to 4-aminopyridine and Zn2+. Our findings suggest that large hydrophobic side chains occlude the pore, forming a physical gate, and that channel opening by iris-like motions simultaneously relocates the gate and exposes the otherwise concealed selectivity filter to the pore lumen.

Translocation of TMEM175 Lysosomal Potassium Channel to the Plasma Membrane by Dynasore Compounds

“…The IC 50 78.7 ± 0.5 µM ( n = 5, Figure 3 B) is close to the previously reported 35 µM value, which was measured in 150 mM K + at +100 mV by patch clamping endosomes dilated with Rab5-Q79L overexpression, i.e., with opposite current direction and 4-AP applied on the other (cytoplasmic) side of the membrane [ 10 ]. We observed slightly voltage-dependent inhibition of mouse TMEM175 by 4-AP ( Figure 3 C,D), in contrast to the previously reported voltage-independent inhibition of human TMEM175 in HEK293T cells [ 13 ]. TMEM175 current was more profoundly inhibited by 4-AP at +60 mV than at −60 mV, suggesting that the positively charged inhibitor binds into the pore from the cytoplasmic direction.…”

“…Although human TMEM175 is highly selective for potassium, about 20–40-fold over Na + , it does not contain the P loop [ 10 ]. Atomic resolution structures of human TMEM175 provided insight into the alternative mechanisms of K + selectivity [ 11 , 12 , 13 ]. Mammalian TMEM175 subunits contain two repeats of six-transmembrane-segment domains in a single polypeptide chain (altogether 12 TMS/subunit, Figure 4 A), and the channel functions as the homodimer of subunits (24 TMS/channel).…”

“…The protein complex possesses a transmembrane ion-conduction pore in its central axis, formed by the amino acid side chains of TM1 and TM7 pore-lining helices. Highly conserved rings of isoleucine [ 11 , 12 ], or threonine and serine residues [ 13 ] around the axis of the pore, have been reported to be the structural determinants of K + selectivity.…”

“…TMEM175 has recently been identified as the ubiquitous K + channel of endosomal and lysosomal membranes [ 10 ]. Despite its importance, only a few studies have been reported about the electrophysiological properties of TMEM175 [ 10 , 11 , 12 , 13 , 14 ], in part because of the problematic accessibility of the native channel in the intracellular organelles. TMEM175 has an unusual ion selectivity profile, as determined in patch clamp measurements of endosomes enlarged by Rab5-Q79L transfection.…”

“…TMEM175 is inhibited by 4-aminopyridine (4-AP), an inhibitor of voltage-gated potassium channels, however, 4-AP is also effective on the maintained K + current of TMEM175 at negative membrane potential values [ 10 ]. Two groups also reported K + currents of approximately 200 pA, measured in HEK293 cells overexpressing mammalian TMEM175, by the whole cell patch clamp method [ 11 , 13 ]. These TMEM175 currents exceeded the endogenous K + currents of non-expressing cells, and allowed the examination of channel function, however, did not reach the nA range, which is characteristic for the expression of other K + channel types normally targeted to the plasma membrane.…”