Removal of phospho-head groups of membrane lipids immobilizes voltage sensors of K+ channels

Abstract

A fundamental question regarding the gating mechanism of voltage-activated K + (Kv) channels is how five positively charged voltage-sensing residues in the fourth transmembrane (TM) segment 1, 2 are energetically stabilized, as they operate in a low-dielectric cell membrane. The simplest solution would be to pair them with negative charges 3 . However, too few negatively charged channel residues are positioned for such a role 4,5 . Recent studies suggest that some of the channel's positively charged residues are exposed to cell membrane phospholipids and interact with their head groups [5][6][7][8][9] . A key question nonetheless remains: Is the phospho-head of membrane lipids necessary for proper function of the voltage sensor itself? Here, we find that a given type of Kv channel may interact with several species of phospholipid, and that enzymatic removal of their negatively charged phospho-head creates an insuperable energy barrier for the positively charged voltage sensor to move through the initial gating step(s), thus immobilizing it, and also raises the energy barrier for the downstream step(s).Kv2.1 channels, expressed in Xenopus oocytes, interact with sphingomyelin 8 present mainly in the outer leaf of plasma membranes. To investigate the importance of phospho-head groups of membrane lipids in Kv channel gating we employed bacterial sphingomyelinases C and D (SMases C and D) 10,11 . Both enzymes specifically hydrolyze sphingomyelin but in different ways (Fig. 1a): SMase D removes only choline and leaves the lipid ceramide-1-phosphate behind in the membrane whereas SMase C removes phosphocholine, leaving ceramide behind 12,13 . A comparison of the effects of these two enzymes on the channels will thus help reveal the functional significance of the phosphodiester group in voltage gating.SMase D of Corynebacterium pseudotuberculosis 11 shifts the conductance-voltage (G-V) relation of Kv2.1 by about -30 mV 8 (Fig. 1d), allowing channels to be activated at a negative voltage where they otherwise remain largely deactivated (Fig. 1c). The effect is maximal within 2 minutes and persists for at least 24 hours ( Fig. 1c, d; cells cannot regenerate sphingomyelin from ceramide-1-phosphate). It does not require direct exposure of channels to SMase D as it also occurred in Kv2.1-expressing oocytes that had been treated with SMase D and then thoroughly washed prior to injection with Kv2.1 cRNA (Fig. 1e).* Please address correspondence to: Dr. Zhe Lu, University of Pennsylvania, Department of Physiology, D302A Richards Building, 3700 Hamilton Walk, Philadelphia, PA 19104, Tel: 215-573-7711, FAX: 215-573-1940, zhelu@mail.med.upenn Additional exposure of such oocytes to SMase D, as expected, caused no further shift. Thus, SMase D acts through its lipase activity, not by direct binding to the channel. Our previous study 8 supports an electrostatic mechanism whereby removal of the positively charged choline favors the activated state of the positively charged voltage sensors.Unlike SMase D, SMase C removes the nega...

Amphipathic antenna of an inward rectifier K + channel responds to changes in the inner membrane leaflet

“…These charges are crucial sites for PIP 2 -binding in the Kir channels (16,36,37). In the crystal structure of Kir2.2, the IF helix (residues 61-69) forms an α-helix that corresponds to the C-terminal half of M0 (numbers [11][12][13][14][15][16][17][18], and R65 in the IF helix (corresponding to K14 in M0) interacts with the tether helix in the cytoplasmic domain. On the other hand, the KcsA channel has the least bulky CPD among the K channels, and the open-stabilizing effect of M0 is directed toward the charged lipids on the inner leaflet.…”

“…Data have been reported on the functionally modifying effects of membrane lipids on channel proteins that result in the gating equilibrium being altered (6,(9)(10)(11)(12)(13). In voltage-gated channels, the voltage-sensor domain (VSD) primarily senses changes in the membrane electric field, but this sensing is modulated by the lipid composition (9,11,13).…”

“…In voltage-gated channels, the voltage-sensor domain (VSD) primarily senses changes in the membrane electric field, but this sensing is modulated by the lipid composition (9,11,13). For the non-voltage-gated (the twotransmembrane-helix inward-rectifier type) channels, such as the KcsA potassium channel from Streptomyces lividans, the underlying mechanism of the effect exerted by lipids is still undergoing extensive investigation, even though the structural information on the channel proteins cocrystallized with lipid molecules has already been reported (14)(15)(16)).…”

Functional diversity of potassium channel voltage-sensing domains

“…These charge-charge interactions are necessary to reduce the energy of charged residues in a low dielectric constant environment such as the lipid bilayer. Apart from the intra protein salt bridges, positive charges are stabilized by interactions with phospholipid head groups 68,69 and hydration waters present in the inner and outer vestibules. 28 It is worth mentioning that other voltage-gated potassium channels such as the cardiac potassium channel hERG, the sperm SpIH inward rectifier, a member of the HCN type of channels and the Slo maxi-K channels, seem to share the same mechanism of activation of the VSD domain described above, [70][71][72] possibly implying that the VSDs from these channels preserve the canonical structure described previously for Kv channels.…”

bSUM: A bead-supported unilamellar membrane system facilitating unidirectional insertion of membrane proteins into giant vesicles

“…the proteinaceous parts are solely responsible for voltagedependent function. Recent studies, however, recognize that lipids play critically important roles in the controlled opening or closing (gating) of these ion channels (Ramu et al, 2006;Schmidt et al, 2006;Schmidt and MacKinnon, 2008;Xu et al, 2008;Zheng et al, 2011;Jiang and Gonen, 2012;Brohawn et al, 2014;Hite et al, 2014). Compelling evidence from these studies suggests that lipids surrounding a voltage-gated potassium (Kv) channel exert strong gating effects on the channels by directly affecting the energetics behind the conformational changes of the voltage-sensor domains.…”