Dual Phosphorylations Underlie Modulation of Unitary KCNQ K+ Channels by Src Tyrosine Kinase

Abstract

Src tyrosine kinase suppresses KCNQ (M-type) K ؉ channels in a subunit-specific manner representing a mode of modulation distinct from that involving G protein-coupled receptors. We probed the molecular and biophysical mechanisms of this modulation using mutagenesis, biochemistry, and both whole-cell and single channel modes of patch clamp recording. Immunoprecipitation assays showed that Src associates with KCNQ2-5 subunits but phosphorylates only KCNQ3-5. Using KCNQ3 as a background, we found that mutation of a tyrosine in the amino terminus (Tyr-67) or one in the carboxyl terminus (Tyr-349) abolished Src-dependent modulation of heterologously expressed KCNQ2/3 heteromultimers. The tyrosine phosphorylation was much weaker for either the KCNQ3-Y67F or KCNQ3-Y349F mutants and wholly absent in the KCNQ3-Y67F/Y349F double mutant. Biotinylation assays showed that Src activity does not alter the membrane abundance of channels in the plasma membrane. In recordings from cell-attached patches containing a single KCNQ2/3 channel, we found that Src inhibits the open probability of the channels. Kinetic analysis was consistent with the channels having two discrete open times and three closed times. Src activity reduced the durations of the longest open time and lengthened the longest closed time of the channels. The implications for the mechanisms of channel regulation by the dual phosphorylations on both channel termini are discussed.Regulation of neuronal activity by tyrosine kinases has emerged as an important theme in neurobiology. In addition to their well documented role in neuronal proliferation and trophic responses are actions on several different types of ion channels that link alterations in electrical activity to neuronal plasticity. Examples are non-receptor tyrosine kinase modulation of Shaker family K ϩ channels (1-7) and growth factor receptor regulation of voltage-gated Ca 2ϩ channels (8 -10). The family of KCNQ genes make K ϩ channels composed of KCNQ1-5 subunits, which are responsible for M-type K ϩ currents in the nervous and auditory systems (11-14) and slow delayed rectifier K ϩ currents in the heart (15, 16). KCNQ1-5 channels have recently been incorporated into the Kv nomenclature as Kv7.1-5 (17). Compared with other Kv channels, KCNQ channels have an extended intracellular carboxyl terminus that seems to be the target of many modulatory signals. Examples are modulation by Ca 2ϩ (18), using calmodulin as the channel Ca 2ϩ sensor (19), and regulation by plasma membrane phosphoinositides (20 -22) perhaps in concert with protein kinase C (23).Src is the prototypical member of the family of non-receptor tyrosine kinases that also includes Fyn, Hck, Lck, and Yes. These proteins often act via two mechanisms: by binding to substrate via Src homology (SH) 1 2 and SH3 domains and/or by phosphorylation of target tyrosines (24). For example, distinct effects of Src actions by these two mechanisms have been described for Kv1.4 and Kv1.5 K ϩ channels (6). We recently showed subunit-specific modulation of KCNQ c...

Reactive oxygen species are second messengers of neurokinin signaling in peripheral sensory neurons

“…Thus, we have shown clearly that SP-mediated enhancement of M current in DRG is PLC dependent and is inhibited by PTX, both supporting a role for GPCRs. Src also is widely reported to have an inhibitory effect on M channels, an effect mediated by phosphorylation of two tyrosines [e.g., Y67 and Y349 in Kv7.3 (54)(55)(56)]. …”

Identification by mass spectrometry and functional characterization of two phosphorylation sites of KCNQ2/KCNQ3 channels

“…PIP 2 depletion is the likely mechanism mediating the classic muscarinic inhibition of M-current (14-16), but other mechanisms, including phosphorylation, may contribute to channel modulation by other neurotransmitters (16). Indeed, KCNQ1 and KCNQ2 subunits associate with a variety of kinases and phosphatases via A-kinase anchor proteins (17)(18)(19)(20), and mutational studies implicate KCNQ2͞KCNQ3 phosphorylation sites for channel regulation by protein kinase A (PKA), protein kinase C (PKC), and src tyrosine kinase (10,19,21,22). KCNQ2 and KCNQ3 subunits possess large intracellular domains containing many additional potential phosphorylation sites (Fig.…”

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

“…A previous co-immunoprecipitation analysis in CHO cells could detect a prominent association of Src with Q2 that was not less than that observed with Q3; however, Src-dependent tyrosine phosphorylation was only detected in Q3 (Li et al, 2004). However, in view of the reduction of Q2 currents mediated by Src in oocytes (Fig.…”

“…Intriguingly, two tyrosine residues located in opposite termini of the Q3 subunit have been shown to be involved in the Src action (Li et al, 2004), leading us to hypothesize that the modulation by Src might potentially involve crosstalk between the cytosolic termini, similar to the modulation by CaM and Syx. Furthermore, whereas in CHO cells Src has been reported to target Q3 and not Q2 , a study in HEK cells has suggested that Src mediates a tonic increase in Q2 channel function (Jow and Wang, 2000).…”

“…Another M-channels modulator is Src, a non-receptor tyrosine kinase, expressed by virtually all cells, including diverse neuronal cell types, where it is localized in dendrites, axons and nerve terminals (Sugrue et al, 1990). In rat sympathetic neurons, Src has been shown to suppress native M-currents Ebner-Bennatan et al, 2012) and, in CHO cells, Src associates with Q2, Q3, Q4 and Q5 subunits and downregulates the open probability of heteromeric Q2-Q3 channels (Li et al, 2004). However, the Gamper et al study suggested that Src was only able to phosphorylate Q3, Q4 and Q5 subunits.…”