Regulation of Neuronal M-Channel Gating in an Isoform-Specific Manner: Functional Interplay between Calmodulin and Syntaxin 1A

Abstract

Whereas neuronal M-type Kϩ channels composed of KCNQ2 and KCNQ3 subunits regulate firing properties of neurons, presynaptic KCNQ2 subunits were demonstrated to regulate neurotransmitter release by directly influencing presynaptic function. Two interaction partners of M-channels, syntaxin 1A and calmodulin, are known to act presynaptically, syntaxin serving as a major protein component of the membrane fusion machinery and calmodulin serving as regulator of several processes related to neurotransmitter release. Notably, both partners specifically modulate KCNQ2 but not KCNQ3 subunits, suggesting selective presynaptic targeting to directly regulate exocytosis without interference in neuronal firing properties. Here, having first demonstrated in Xenopus oocytes, using analysis of single-channel biophysics, that both modulators downregulate the open probability of KCNQ2 but not KCNQ3 homomers, we sought to resolve the channel structural determinants that confer the isoform-specific gating downregulation and to get insights into the molecular events underlying this mechanism. We show, using optical, biochemical, electrophysiological, and molecular biology analyses, the existence of constitutive interactions between the N and C termini in homomeric KCNQ2 and KCNQ3 channels in living cells. Furthermore, rearrangement in the relative orientation of the KCNQ2 termini that accompanies reduction in single-channel open probability is induced by both regulators, strongly suggesting that closer N-C termini proximity underlies gating downregulation. Different structural determinants, identified at the N and C termini of KCNQ3, prevent the effects by syntaxin 1A and calmodulin, respectively. Moreover, we show that the syntaxin 1A and calmodulin effects can be additive or blocked at different concentration ranges of calmodulin, bearing physiological significance with regard to presynaptic exocytosis.

Regulation of neuronal KCNQ2 channel by Src: dual rearrangement of cytosolic termini underlies bidirectional gating regulation

“…2A) or Q3 (Fig. 2B), with or without Src, at a saturating voltage of 0 mV, using the cell-attached configuration of the patch-clamp technique, as previously described (Etzioni et al, 2011). Surprisingly, the Po values of Q2 and Q3 homomers were oppositely affected by Src: Q2 Po increased ( Fig.…”

“…We set out to further explore this using chimeric Q2-Q3 subunits with exchanged N-terminal and C-terminal sequences. First, we tested the effect of Src on Q2 (helixA) , a Q2 subunit in which helix A, the membrane-proximal C-terminal helix that contains the C-terminal tyrosine, was exchanged with the corresponding helix A of Q3 (Etzioni et al, 2011). As expected in light of the very high similarity between the two helices (Fig.…”

“…Indeed, channel gating modulators have been shown to alter N-terminus-C-terminus interactions (Lippiat et al, 2002;Tsuboi et al, 2004;Lvov et al, 2009;Etzioni et al, 2011). Specifically, constitutive N-terminus-C-terminus interactions have been characterized in Q2 and Q3, and have been shown in Q2 to be modulated by CaM and syntaxin, accompanying changes in Q2 Po (Etzioni et al, 2011). Thus, we set out to explore whether the Src-induced gating regulation is accompanied by specific changes in the N-terminus-C-terminus interaction within Q2 and Q3 (Fig.…”

“…We previously put forward a mechanism for modulation of M-channel gating that was mediated by calmodulin (CaM) and syntaxin 1A (Syx, also known as STX1A) in Xenopus oocytes. This mechanism was solely targeted at Q2 subunits, downregulating the open probability of Q2 channels through a rearrangement in the relative orientation of their intracellular N-and C-termini (Etzioni et al, 2011). Such subunit specificity suggests selective targeting at presynaptic homomeric Q2 channels to directly regulate exocytosis, without interference with axonal Q2-Q3 heteromeric channels and neuronal firing properties (Regev et al, 2009).…”

Tracking Ca2+-dependent and Ca2+-independent conformational transitions in syntaxin 1A during exocytosis in neuroendocrine cells

“…Next, we analyzed the conformations adopted by CSYS and CSYS-5RK, using the spectral FRET technique in Xenopus oocytes (Etzioni et al, 2011;Zheng et al, 2003). The probes were efficiently targeted to the PM and exhibited a high FRET signal under resting conditions (static FRET; Fig.…”