Calcium ions open a selectivity filter gate during activation of the MthK potassium channel

Abstract

Ion channel opening and closing is fundamental to cellular signaling and homeostasis. Gates that control K+ channel activity were found both at an intracellular pore constriction and within the selectivity filter near the extracellular side but the specific location of the gate that opens Ca2+-activated K+ channels has remained elusive. Using the Methanobacterium thermoautotrophicum homolog (MthK) and a stopped-flow fluorometric assay for fast channel activation, we show that intracellular quaternary ammonium blockers bind to closed MthK channels. Since the blockers are known to bind inside a central channel cavity, past the intracellular entryway, the gate must be within the selectivity filter. Furthermore, the blockers access the closed channel slower than the open channel, suggesting that the intracellular entryway narrows upon pore closure, without preventing access of either the blockers or the smaller K+. Thus, Ca2+-dependent gating in MthK occurs at the selectivity filter with coupled movement of the intracellular helices.

Structural and Functional Characterization of a Calcium-Activated Cation Channel From Tsukamurella Paurometabola

“…However, while the sequence similarity of NaKTs and MthK is high (Fig. 1a), the latter does not possess the proline (Pro63) that forms part of the S Ca binding site observed in NaKTs and the filter gating observed in the MthK channel is believed to be allosterically triggered by the binding of Ca 2 þ to the cytoplasmic RCK domains at the intracellular side 26 . A recent study based on chimeric channels that replaced the selectivity filter of the bacterial non-selective cation channel NaK, TVGDGNFS, with a cyclic-nucleotide gated channel filter sequence, TIGETPPP also displayed Ca 2 þ block (B14 mM) 27 .…”

“…A Ca 2 þ -dependent gating located within the selectivity filter was also shown to control the K þ conduction in the MthK channel 26 . However, while the sequence similarity of NaKTs and MthK is high (Fig.…”

Stopped-Flow Fluorometric Ion Flux Assay for Ligand-Gated Ion Channel Studies

“…A variant, based on Tl + selective fluorescent indicators, later was employed and commercialized for cell-based flux assays [9]. The method also has been adapted for use in gramicidin channel assays [10–12], and more recently was employed using updated methods for biophysical studies on purified, reconstituted K + channels [13–15]. Thus, although we here describe the assay as it was used for K + selective channels, the assay will work equally well for any cation-selective ion channel that is permeable to Tl + .…”

“…The stretched exponential function was extensively tested and validated for this purpose during the development of the stopped-flow method for gramicidin activity assays [10] and has worked equally well in the analysis of Tl + flux through K + channels [13–15]. Given the large number of experiments typically performed, including repeated mixing reactions for a specific sample and experimental condition, multiple experimental conditions of interest, and ultimately, multiple sample preparations to confirm experimental reproducibility, it is convenient to develop a semi-automated analysis procedure using a software package such as Matlab® (Mathworks).…”

“…This non-physical property of the stretched exponential can be side-stepped by using a “modified stretched exponential” function that derives from a simple transformation along the time axis [25]. Generally, this approach has not been necessary in the analysis of these experiments, but has been used to compare “modified” stretched exponential rates at 0 ms in specific cases [27,15]. …”