Neutralization of a single arginine residue gates open a two-pore domain, alkali-activated K ⁺ channel

Abstract

Potassium channels share a common selectivity filter that determines the conduction characteristics of the pore. Diversity in K ؉ channels is given by how they are gated open. TASK-2, TALK-1, and TALK-2 are two-pore region (2P) KCNK K ؉ channels gated open by extracellular alkalinization. We have explored the mechanism for this alkalinization-dependent gating using molecular simulation and site-directed mutagenesis followed by functional assay. We show that the side chain of a single arginine residue (R224) near the pore senses pH in TASK-2 with an unusual pK a of 8.0, a shift likely due to its hydrophobic environment. R224 would block the channel through an electrostatic effect on the pore, a situation relieved by its deprotonation by alkalinization. A lysine residue in TALK-2 fulfills the same role but with a largely unchanged pK a, which correlates with an environment that stabilizes its positive charge. In addition to suggesting unified alkaline pH-gating mechanisms within the TALK subfamily of channels, our results illustrate in a physiological context the principle that hydrophobic environment can drastically modulate the pK a of charged amino acids within a protein.KCNK channels ͉ molecular simulation ͉ TALK-2 ͉ TASK-2 A ll K ϩ channels contain a highly conserved sequence, the P domain, which forms the selectivity filter and generally six transmembrane ␣-helices. The K ϩ channel pore is formed by four identical subunits, each comprising a P-domain and two of the six transmembrane ␣-helices encircling the ion conduction pathway with a 4-fold symmetry. Structures attached to the pore-forming domains are able to transduce signals, such as changes in transmembrane voltage and intra-or extracellular messages, into gating of the pore (1, 2). Potassium channels of the KCNK superfamily (3, 4) are remarkable in that they possess two P-domains and four ␣-helices in each subunit, form dimers, and are mostly open (''leak channels'') at resting potential. Potassium-selective leaks are fundamental to the function of various cells including nerve, muscle, and epithelia. There are 16 mammalian members to the KCNK family and their gating is variously regulated by free fatty acids, membrane tension, G protein-generated signaling, and extracellular pH. Among KCNK channels gated by extracellular pH, TASK-1 and TASK-3 form a subfamily (TASK) of channels blocked by extracellular protons (5-8). A second subfamily (TALK) of KCNK channels comprises TASK-2, TALK-1, and TALK-2 ¶ , all activated by extracellular alkalinization. TASK-2 participates in ion fluxes necessary for cell volume regulation (10, 11), and its physiological and possible pathological importance has also been highlighted by studies in a TASK-2 knockout mouse (12) that revealed a metabolic acidosis and hypotension caused by renal loss of HCO K ϩ channels of the TASK subfamily are blocked by protons by titration of a histidine (N in TALK channels) residue in the first P domain (6-8), making them responsive to pH in the physiological range. The pH-sensing mechanism ...

An Extracellular Ion Pathway Plays a Central Role in the Cooperative Gating of a K2P K+ Channel by Extracellular pH*

“…The presence of side portals with predominantly negative walls in TASK-3 is crucial in providing a restricted space for the gating process we postulate. The effect of local environment in the modulation of the pK a of key residues in both enzymes and ion channels is known to play a role in their catalysis/gating mechanisms (22,40). The mechanism for cooperativity of channel gating proposed here, a perturbation in environment caused by the transported ion itself leading to a shift in pK a of a gating residue, is, to our knowledge, novel, as is the proposed role of the newly discovered extracellular cap structure of K 2P channels in the process.…”

“…Standard whole-cell patch clamp recordings were performed as described elsewhere (22,23). Currents were measured at several potentials as indicated.…”

Grafting voltage and pharmacological sensitivity in potassium channels

“…The gating of the Kv channel pore involves both the lower bundle-crossing gate at the cytoplasmic entrance to the channel and the upper gate at the selectivity filter close to the outer mouth of the channel [54,55]. By contrast, the gating of K2P channels was thought to occur through their upper gate [56][57][58][59]. However, except for the upper gate, recent evidence shows that K2P channels may also possess a lower bundle-crossing gate, similar to Kv channels [59][60][61].…”

B lymphocytes taken to task: a role for a background conductance K2P K+ channel in B cells. Focus on “Expression of TASK-2 and its upregulation by B cell receptor stimulation in WEHI-231 mouse immature B cells”

“…Two subunits associate to form a dimer with bilateral symmetry and pseudo-fourfold symmetry around the pore as shown in the bottom diagram (seen from the extracellular side looking into the membrane): P1 and P2 domains contribute to the pore with identical P-domains facing each other diagonally across the permeation pathway (7). B: dependence of TASK-2 activity on extracellular pH (17). The box shows a range of Ϯ 0.3 pH units around physiological pH.…”

“…TASK-2 belongs to the TALK group of K 2P channels comprising TASK-2, TALK-1, and TALK-2, which share the characteristic of being activated by extracellular alkalinization (17,19). Figure 1B shows the extracellular pH dependence of TASK-2 activity.…”