Minimal Structural Rearrangement of the Cytoplasmic Pore during Activation of the 5-HT3A Receptor

Sandip Panicker,Hans Cruz,Christine Arrabit,Ka Fai Suen,Paul A Slesinger

doi:10.1074/jbc.m403545200

Abstract

Ligand-gated ion channel receptors mediate the response of fast neurotransmitters by opening in less than a millisecond. Here, we investigated the activation mechanism of a serotonin-gated receptor (5-HT(3A)) by systematically introducing cysteine substitutions throughout the pore-lining M1-M2 loop and M2 transmembrane domain. We hypothesized that multiple cysteines in the narrowest region of the pore, which together can form a high affinity binding site for metal cations, would reveal changes in pore structure during gating. Using cadmium (Cd2+) as a probe, two cysteine substitutions in the cytoplasmic selectivity filter, S2'C and, to a lesser extent, G-2'C, showed high affinity inhibition with Cd2+ when applied extracellularly in the open state. Cd2+ inhibition in S2'C was attenuated if applied in the presence of an open-channel inhibitor and showed voltage-dependent recovery, indicating a direct effect of Cd2+ in the pore. When applied intracellularly, Cd2+ appeared to bind S2'C receptors in the closed state. The ability of cysteine side chains at the 2' and -2' positions to coordinate Cd2+ in both the native open and closed states of the channel suggests that the cytoplasmic selectivity filter of 5-HT(3A) receptors maintains a narrow pore during channel gating.

Highlights

Ligand-gated ion channel (LGIC)1 receptors are responsible for rapid chemical transmission between neurons in the nervous system [1]
In this study we investigated the conformational changes that underlie opening of ligand-gated ion channels by taking advantage of the physiochemical properties of Cd2ϩ and cysteines engineered into the 5-HT3A receptor
By introducing a high affinity metal divalent binding site in a narrow region of the pore of the 5-HT3A receptor, we showed that both native open and closed states of the receptor can coordinate Cd2ϩ

Summary

EXPERIMENTAL PROCEDURES

Molecular Biology—Cysteine mutants of 5-HT3A were constructed previously [9]. Four mutants (L8ЈC, G10ЈC, Y11ЈC, F14ЈC) were not tested because they yielded little or no currents when expressed alone (see Ref. 9). Oocytes were perfused continuously with a solution containing 96 mM NaCl (Aldrich), 2 mM KCl, 4.69 mM MgCl2 (free Mg2ϩ ϭ 3.3 mM), 0.5 mM EGTA, and 5 mM HEPES (pH 7.6 with NaOH) while was voltage clamped at Ϫ80 mV. The effect of extracellular Cd2ϩ administration on receptors expressed in HEK-293T cells was studied by first establishing a base-line current response from receptors by exposure to a 2-s 5-HT pulse followed by a 58-s wash (1 sweep) to allow for recovery from desensitization. For 1 ␮M Cd2ϩ experiments (Fig. 4), 5-HT was applied initially for 0.75 s followed by 2 s of 5-HT and Cd2ϩ and a 58-s wash This was repeated until maximal inhibition was achieved. For voltage-dependent recovery experiments (Fig. 6), maximal Cd2ϩ inhibition was elicited by applying 2-s pulses of 5-HT plus Cd2ϩ (200 ␮M) in 5 successive sweeps.

RESULTS

When plotted as a function of cumulative time exposed to

DISCUSSION