Abstract
KCNQ2/KCNQ3 channels are the molecular correlates of the neuronal M-channels, which play a major role in the control of neuronal excitability. Notably, they differ from homomeric KCNQ2 channels in their distribution pattern within neurons, with unique expression of KCNQ2 in axons and nerve terminals. Here, combined reciprocal coimmunoprecipitation and two-electrode voltage clamp analyses in Xenopus oocytes revealed a strong association of syntaxin 1A, a major component of the exocytotic SNARE complex, with KCNQ2 homomeric channels resulting in a ∼2-fold reduction in macroscopic conductance and ∼2-fold slower activation kinetics. Remarkably, the interaction of KCNQ2/Q3 heteromeric channels with syntaxin 1A was significantly weaker and KCNQ3 homomeric channels were practically resistant to syntaxin 1A. Analysis of different KCNQ2 and KCNQ3 chimeras and deletion mutants combined with in-vitro binding analysis pinpointed a crucial C-terminal syntaxin 1A-association domain in KCNQ2. Pull-down and coimmunoprecipitation analyses in hippocampal and cortical synaptosomes demonstrated a physical interaction of brain KCNQ2 with syntaxin 1A, and confocal immunofluorescence microscopy showed high colocalization of KCNQ2 and syntaxin 1A at presynaptic varicosities. The selective interaction of syntaxin 1A with KCNQ2, combined with a numerical simulation of syntaxin 1A's impact in a firing-neuron model, suggest that syntaxin 1A's interaction is targeted at regulating KCNQ2 channels to fine-tune presynaptic transmitter release, without interfering with the function of KCNQ2/3 channels in neuronal firing frequency adaptation.
Highlights
The voltage-dependent M-type potassium current (M-current) is a subthreshold, slowly activating and noninactivating voltagegated potassium current that is thought to stabilize membrane potential and control neuronal excitability by limiting repetitive firing [1,2,3,4].The heterotetrameric KCNQ2/KCNQ3 channel complex, which belongs to the KCNQ family of voltage-dependent K+ channels, has been identified as the main molecular correlate of the M-channel [5,6,7]
In which we characterized syntaxin 1A’s interactions with Kv channels [29,30,31,46,47], we studied the interaction of syntaxin 1A with KCNQ2, KCNQ2/3 and KCNQ3 channels in the heterologous expression system of Xenopus oocytes, where biochemical and electrophysiological analyses can be performed simultaneously
Quantification over several similar experiments of the intensity ratios of coprecipitated syntaxin 1A to the different channel subunits, coexpressed in the same cells, showed that the amount of syntaxin 1A associated with KCNQ2 was,two, fiveand threefold larger than that with KCNQ2/3, KCNQ3 and KCNQ1, respectively (Fig. 1C)
Summary
The voltage-dependent M-type potassium current (M-current) is a subthreshold, slowly activating and noninactivating voltagegated potassium current that is thought to stabilize membrane potential and control neuronal excitability by limiting repetitive firing [1,2,3,4].The heterotetrameric KCNQ2/KCNQ3 channel complex, which belongs to the KCNQ family of voltage-dependent K+ channels, has been identified as the main molecular correlate of the M-channel [5,6,7]. The voltage-dependent M-type potassium current (M-current) is a subthreshold, slowly activating and noninactivating voltagegated potassium current that is thought to stabilize membrane potential and control neuronal excitability by limiting repetitive firing [1,2,3,4]. KCNQ2, but not KNCQ3, subunits are expressed presynaptically on axons and nerve terminals, where they might regulate action potential propagation or neurotransmitter release [8,10]. Modulation of the M-current has profound effects on brain excitability. Inhibition of M-channels by muscarinic agonist and other neurotransmitters enhances action-potential firing in central and autonomic neurons [2,11,12]. Recent studies have shown that calmodulin (CaM) binds to the KCNQ2 and KCNQ3 C termini and may function as an auxiliary channel subunit [17,20,21]
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