Cobalt ion interaction with TMEM16A calcium-activated chloride channel: Inhibition and potentiation

Abstract

TMEM16A, a Ca 2+-sensitive Clchannel, plays key roles in many physiological functions related to Cltransport across lipid membranes. Activation of this channel is mediated via binding intracellular Ca 2+ to the channel with a relatively high apparent affinity, roughly in the sub-μM to low μM concentration range. Recently available high-resolution structures of TMEM16 molecules reveal that the high-affinity Ca 2+ activation sites are formed by several acidic amino acids, using their negatively charged sidechain carboxylates to coordinate the bound Ca 2+. In this study, we examine the interaction of TMEM16A with a divalent cation, Co 2+ , which by itself cannot activate current in TMEM16A. This divalent cation, however, has two effects when applied intracellularly. It inhibits the Ca 2+-induced TMEM16A current by competing with Ca 2+ for the aforementioned high-affinity activation sites. In addition, Co 2 + also potentiates the Ca 2+-induced current with a low affinity. This potentiation effect requires high concentration (mM) of Co 2+ , similar to our previous findings that high concentrations (mM) of intracellular Ca 2+ ([Ca 2+ ] i) can induce more TMEM16A current after the Ca 2 +-activation sites are saturated by tens of μM [Ca 2+ ] i. The degrees of potentiation by Co 2+ and Ca 2+ also roughly correlate with each other. Interestingly, mutating a pore residue of TMEM16A, Y589, alters the degree of potentiation in that the smaller the sidechain of the replaced residue, the larger the potentiation induced by divalent cations. We suggest that the Co 2+ potentiation and the Ca 2+ potentiation share a similar mechanism by increasing Clflux through the channel pore, perhaps due to an increase of positive pore potential after the binding of divalent cations to phospholipids in the pore. A smaller sidechain of a pore residue may allow the pore to accommodate more phospholipids, thus enhancing the current potentiation caused by high concentrations of divalent cations.

Binding of Amyloid β(1–42)-Calmodulin Complexes to Plasma Membrane Lipid Rafts in Cerebellar Granule Neurons Alters Resting Cytosolic Calcium Homeostasis

“…Of note, the inner filter effect due to the absorbance of 5 mM Co 2+ in the wavelength range 470–550 nm, calculated as indicated in the Materials and Methods section, can only account for less than 5% quenching of this fluorescence. Co 2+ is a well calcium channel blocker at millimolar concentration, and has also been shown to bind to Ca 2+ sites in proteins (see for example [ 53 , 54 ]). Co 2+ has a R 0 value of 1.2 nm as the FRET acceptor of the green fluorescence of fluorescein [ 55 ].…”

Divalent Cation Modulation of Ion Permeation in TMEM16 Proteins

“…We have previously shown that intracellular Co 2+ (up to 20 mM) by itself does not activate current in WT 16A . However, intracellular Co 2+ potentiates the Ca 2+ -induced WT 16A current, followed by an inhibition effect [ 38 ]. Co 2+ inhibition on the WT 16A current (see Figure 1 A) is easier to understand—Co 2+ competes with intracellular Ca 2+ for the high-affinity Ca 2+ -activation sites [ 38 ].…”

“…However, intracellular Co 2+ potentiates the Ca 2+ -induced WT 16A current, followed by an inhibition effect [ 38 ]. Co 2+ inhibition on the WT 16A current (see Figure 1 A) is easier to understand—Co 2+ competes with intracellular Ca 2+ for the high-affinity Ca 2+ -activation sites [ 38 ]. The current potentiation by Co 2+ , however, is a different mechanism because the potentiation occurs before the inhibition.…”

“…We have reported that a high concentration (hundreds of µM or above) of intracellular Co 2+ ([Co 2+ ] i ) potentiates the TMEM16A current induced by saturating [Ca 2+ ] i [ 38 ]. This Co 2+ potentiation effect (see Figure 1 A) appeared to be similar to the current potentiation by mM [Ca 2+ ] i after the activation of TMEM16A is saturated (see Figure 1 B).…”

“…This Co 2+ potentiation effect (see Figure 1 A) appeared to be similar to the current potentiation by mM [Ca 2+ ] i after the activation of TMEM16A is saturated (see Figure 1 B). We suggested that Ca 2+ and Co 2+ may bind to the pore region of TMEM16A to potentiate Cl - currents [ 38 ]. In the present study, we further examined this current potentiation mechanism.…”